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saccharomyces cerevisiae polarizes cell growth to the bud during cell replication and to the mating projection when cells are induced by pheromones to change their shape to form shmoos . first , the site on the cell surface is selected by intrinsic and extrinsic cues . second , cell polarity is established by the activation of small gtpases with cdc42 as the major player . last , a multiprotein machine is assembled that spools out actin cables to direct post - golgi traffic to the site of polarized cell growth ( drubin and nelson , 1996 ; madden and snyder , 1998 ; pruyne and bretscher , 2000 ; chang and peter , 2003 ) . during budding , membrane traffic is directed by actin cables to the bud , and the septin ring at mother - daughter cell neck region functions as a physical barrier , preventing diffusion of membrane components from the bud to the mother cell ( barral et al . , 2000 ; the biosynthetic transport is directed to the shmoo tip ( pruyne and bretscher , 2000 ) . however , there is no diffusion barrier like the septin ring and most proteins diffuse laterally over the entire cell surface . nevertheless , a specific subset of proteins required for mating is clustered at the tip of the mating projection . we have shown previously that the polarized distribution of membrane components involves raft lipids ( sphingolipids and ergosterol ) and the actin cytoskeleton ( bagnat and simons , 2002 ) . based on these observations , we proposed that lipid rafts are clustered at the tip of the mating projection and that this process is important for the retention of the associated molecules at the mating projection . recently , it was demonstrated that cycles of endocytosis combined with polarized membrane delivery into the mating projection are able to restrict protein localization to the tips of shmoos ( valdez - taubas and pelham , 2003 ) . here , we report that polarized localization of fus1p , a type i transmembrane protein involved in cell fusion ( trueheart and fink , 1989 ) , does not require endocytosis . instead , the protein is retained at the tip of the mating projection through the interaction of its cytosolic tail with a multiprotein scaffolding machinery . additionally , we provide evidence that the lipid bilayer at the tip of the mating projection is more ordered than over the cell body and that sphingolipids are required for this specific lipid organization . to revisit the kinetic recycling model we have analyzed the role of polarized delivery and endocytosis in polarizing fus1p , a type i transmembrane protein involved in cell fusion ( trueheart and fink , 1989 ; nolan et al . , 2006 ) , to the tip of the mating projection . we first analyzed shmoo tip delivery of fus1p and compared it to another marker protein that is distributed all over the plasma membrane of mating cells , mid2p ( fig . mid2p is a cell wall integrity sensor , and similarly to fus1p , it is a type i transmembrane protein ( philip and levin , 2001 ) . 1 h after induction of expression , the marker proteins were delivered to the shmoo tip where both were localized at this point . however , 2 h later mid2p had diffused over the entire plasma membrane , whereas fus1p remained at the tip . we also used snc1p , a yeast v - snare involved in post - golgi plasma membrane transport , as a second marker protein that is tip localized . it has been shown that snc1p polarization was abolished after inhibition of endocytosis ( valdez - taubas and pelham , 2003 ) . in endocytosis - deficient cells , snc1p was no longer polarized but was distributed over the plasma membrane of the shmooing cells . in contrast , the polarization of fus1p to the tip of the mating projection remained normal in end4 cells ( fig . 2 ) . thus , there must be an alternative mechanism that maintains biosynthetically delivered fus1p at the shmoo tip irrespective of ongoing cycles of endocytosis and exocytosis . important also to note is that most mutants that inhibit endocytosis mate with similar efficiency as wild - type cells ( brizzio et al . , 1998 ) . these findings confirm that the kinetic polarization model using endocytosis and polarized exocytosis is involved in local concentration of membrane proteins such as snc1p . however , this is not the only mechanism used by shmooing cells to polarize their mating machinery . localization of gfp - tagged fus1p and mid2p at different time points after induction of expression in shmooing cells . wild - type cells carrying plasmids mbq30 or mbq35 were treated with -factor for 3 h and after that , galactose was added to induce protein expression ( for details see materials and methods ) . polarized distribution of snc1p , but not of fus1p , is dependent on endocytosis . blocked endocytosis in end4 disrupted polarized distribution of snc1p , but did not affect tip localization of fus1p , compared with wild - type cells . the percentage of cells with fluorescence on the plasma membrane limited to the mating projection is given in the bottom right corner of the gfp images . for fus1p in wild - type and end4 we counted 218 and 327 cells , respectively , and for snc1p it was 220 and 100 cells , respectively . in wild - type cells , in addition to the plasma membrane , snc1p was found in intracellular structures due to protein cycling . these structures were not visible in the endocytosis mutant because the protein was trapped on the plasma membrane . fus1p in wild - type cells was found at the plasma membrane and in the vacuole . one reason why fus1p is retained at the mating tip could be due to interaction with the cell wall , as was demonstrated for glycosylphosphatidylinositol - anchored proteins ( de sampaio et al . , 1999 ) . thus , we constructed fusion proteins between fus1p and mid2p where we swapped the extracellular , the transmembrane , and the cytosolic domains of the two proteins ( schematically shown in fig . 3 ) . analysis of their surface distribution demonstrated that the information for mating tip retention was localized to the cytosolic tail ( fig . the mid2 protein carrying the cytoplasmic domain from fus1p was localized to the mating projection . page and western blot analysis confirmed that this protein displayed a pattern of glycosylation , typical for mature mid2p ( lommel et al . , 2004 and unpublished data ) . we then analyzed how this chimeric protein behaved in mutants in which endocytosis was inhibited , both in end4 cells and at the nonpermissive temperature in end4 - 1 ts cells . this chimeric protein behaved like fus1p and maintained its polarization in endocytosis - deficient cells ( fig . 4 a and fig . thus , we concluded that the cytosolic tail of fus1p mediates protein retention at the tip and that interactions with the cell wall can not explain the polarization . the cytosolic tail of fus1p is 416 amino acids long and contains an src kinase homology 3 ( sh3 ) domain close to its cooh terminus , followed by a proline - rich domain , both known to be responsible for protein protein interactions ( tong et al . , 2002 ) . we deleted the sh3 domain from the chimeric protein mid - fus ( used in fig . 4 a ) or fus1p ( not depicted ) and saw no effect on polarization . at this time , a report from nelson et al . ( 2004 ) appeared , in which a detailed analysis of the cytoplasmic domain of fus1p was described . they showed that both domains were important for mating efficiency , but even the double - mutant protein was polarized normally in wild - type cells . because mutations in these domains prevented protein interaction with the scaffolding machinery ( nelson et al . , 2004 ) we considered the possibility that the double mutant of fus1p could be polarized via the endocytic recycling mechanism . to test this possibility we expressed the mutated fus1p in the endocytosis - deficient strain and found that protein polarization was still normal ( fig . 4 b ) . we concluded that additional sites on the cytoplasmic tail of fus1p might contribute to fus1p retention to the mating tip . in a detailed two - hybrid analysis it was demonstrated that the cytosolic tail of fus1p interacts with several key players in mating polarity , including the gtp - bound form of cdc42 , components of the polarisome pea2p and bni1p , fus2p , and ste5p , the scaffold protein for map kinase signaling ( nelson et al . , 2004 ) . we analyzed the mid - fus protein in pea2 , bni1 , fus2 , spa2 , bud6 , fus1 , and ste5 deletion mutants . the localization was normal in all mutants accept in ste5 cells , which did not form shmoos and in bni1 , which exhibits a subtle defect in protein polarization ( fig . we propose that fus1p is directly embedded in a dynamic network of protein protein interactions that is responsible for scaffolding and localization of fus1 to the shmoo tip as part of the mating machinery . fus1p , mid2p , and different chimeric proteins were expressed in wild - type cells treated with -factor . the schematic representation of the expressed fusion proteins is shown on the right and the swapped domains are indicated . internal membrane staining is seen in f and g , suggesting that protein sorting to the cell surface was compromised . however , the fraction of protein that was delivered to the plasma membrane was not polarized . fus1p does not require its sh3 or the proline - rich domain for polarized localization . the chimeric mid(cyt - fussh3 ) protein , which carries a cytoplasmic tail from fus1p without sh3 domain , was equally well polarized in the wild - type and end4 cells ( a ) . the percentage of cells with fluorescence on the plasma membrane limited to the mating projection is indicated ( n = 361 and 138 for the wild - type and end4 cells , respectively ) . similarly , protein carrying point mutations that affect the function of the proline - rich region or the sh3 domain ( fus1p(p422a ) and fus1p - sh3(w473s ) , respectively ) , or the double mutant of fus1p ( fus1p(p422a)-sh(w473s ) ) , was polarized in end4 cells ( b ) . based on the findings ( 1 ) that polarization of the mating machinery to the shmoo tip is inhibited in erg6 and in lcb1 - 100 cells ; ( 2 ) that mutations that severely affect the synthesis of the major raft lipids in yeast reduced mating efficiency ; and ( 3 ) on the polarized distribution of filipin , a molecule that has high affinity for sterols , we postulated previously that raft lipid clustering plays a role in establishing and maintaining mating tip polarization ( bagnat and simons , 2002 ) . because partitioning of filipin does not directly correlate with lipid ordering in the bilayer , we took advantage of a dye that does . laurdan is an environmentally sensitive dye that has a peak of emission shifting from 500 nm in liquid - disordered membranes to 440 nm in ordered membrane domains ( gaus et al . , 2003 ) . we simultaneously recorded the laurdan fluorescence intensity in two channels . by expressing a normalized ratio of the two emission regions the general polarization ( gp ; see materials and methods)laurdan fluorescence provides a relative measure of lipid order in cell membranes . importantly , laurdan does not preferentially partition into a specific lipid phase , nor do gp values depend on the local probe concentration within the membrane ( gaus et al . , 2003 ) . the gp images revealed that the membrane at that mating projection is more condensed and ordered than the domain on the opposite site of the cell ( fig . hence , the membrane at the mating projections displayed the biophysical characteristic that is expected for raft clustering . the coalescence of condensed membrane at the tips of shmoos was also found to occur in the endocytosis - deficient strain but not in the sphingolipid mutant lcb1 - 100 ( fig . 5 ) . these data clearly demonstrated the asymmetric organization of the lipids in the plasma membrane of yeast cells during mating . more detailed analysis is needed to understand the molecular mechanisms responsible for the formation and maintenance of the mating projection . -factor treated wild - type ( a and d ) , lcb1 - 100 ( b and e ) , and end4 ( c and f ) cells were killed with 5 mm nan3 , fixed with 24% paraformaldehyde , labeled with 250 m laurdan for 5 min , and imaged in water . gp values were calculated from the laurdan intensity images and pseudocolored as indicated in a ( low to high gp values , black to yellow ) . gp values of membranes at the mating tip and at the cell body opposite the mating tip were measured for 40 ( d ) , 28 ( e ) , and 43 ( f ) individual cells . . means sd are 0.398 0.118 and 0.191 0.090 ( d ) , 0.256 0.143 and 0.202 0.104 ( e ) , and 0.372 0.075 and 0.256 0.111 ( f ) for mating tip and cell body , respectively . the difference of the gp value at the mating projection between wild - type and sphingolipid mutant cells was statistically significant ( p < 0.001 ) but there was no statistically significant difference between wild - type and end4 cells ( p > 0.05 ) . also , mammalian cells use raft clustering to polarize their cell surfaces during cell migration or cell cell contacting during immune recognition . in migrating neutrophils it was demonstrated that lipid raft clusters are localized to the rear of the cells in an actin - dependent manner ( seveau et al . , 2001 ) . ( 2001 ) showed that two types of raft clusters are assembled at opposite poles , at the leading edge and at the uropod . recently , it was also demonstrated that when the t cell receptor is activated , a condensed raft cluster is formed at the activation site ( gaus et al . each raft - clustering process is specific in that a subset of raft components is included in the assembly , associating and disassociating from the cluster dependent on their raft - partitioning characteristics ( simons and vaz , 2004 ; kusumi et al . , 2005 ) and the kinetics of the protein protein interactions ( harder , 2004 ; douglass and vale , 2005 ) . this mechanism could also drive the surface polarization during yeast mating ( bagnat and simons , 2002 ) . nevertheless , coming together of proteins with a condensed lipid domain at the mating tip could lead to activation of the mating machinery spatially and temporally by specific lipid for instance , the egf receptor has been shown to be activated by interactions with the ganglioside gd1a and the glutamate receptor by raft - cholesterol ( eroglu et al . recently , it was demonstrated that yeast ste5p , a protein that plays a crucial role in pheromone signaling and interacts with the cytoplasmic tail of fus1p , has a phospholipid binding domain that is necessary for protein localization and signaling ( winters et al . , 2005 ) . the generation of cell surface polarity during yeast mating is thus a complex process involving on one hand endocytosis and recycling and on the other hand establishment of the site where the mating machinery is scaffolded and the membrane reorganized . it is our contention that complex membrane processes such as cell surface polarization are driven by protein however , only future work directed specifically toward analysis of these issues will unravel the mechanisms involved . in this study the following yeast strains were used : rh690 - 15d [ wild - type ] ( mata his4 , leu2 , ura3 , lys2 , bar1 ) was obtained from h. riezman ( university of basel , basel , switzerland ) , and rh1965 [ end4 ] ( mata his4 , leu2 , ura3 , lys2 , bar1 , end4leu2 ) and rh268 - 1 [ en4 - 1 ts ] ( mata his4 , leu2 , ura3 , lys2 , bar1 , end4 - 1 ( ts ) ) were obtained from c. walch - solimena ( mpj - cbg , dresden , germany ) . 1302-wt ( by4742 ) , pea2 , bni1 , fus2 , spa2 , bud6 , fus1 and ste5 deletions are in by strains derived from s288c ( mata ; his31 ; leu20 ; met150 ; ura30 ) and were obtained from euroscarf . cells were grown overnight in yeast extract / peptone ( yp ) medium containing 2% raffinose ( ypraf ) as a carbon source at 24c . for the induction of expression from the gal - s promoter , 2% galactose was added . to induce a mating response , 5 m -factor ( sigma - aldrich ) was added and cells were incubated for 3 h at 24c ( or as indicated ) . plasmids used in this study are listed in table i. all constructs created in our lab are based on the centromeric plasmid p416 ( mumberg et al . , 1995 ) and plasmids p4269 , p4580 , and p4667 containing mutants of fus1 under control of its own promoter were obtained from the c. boone lab ( university of toronto , toronto , canada ; nelson et al . , 2004 ) . the plasmid containing gfp - snc1 under control of constitutive tpi promoter was obtained from the h. pelham lab ( mrc laboratory of molecular biology , cambridge , uk ; lewis et al . , 2000 ) . construction of plasmids mbq30 , mbq35 , tpq53 , and tpq55 was described previously ( bagnat and simons , 2002 ; proszynski et al . , 2004 , 2005 ) . plasmids tpq63 , tpq65 , tpq72 , and tpq57 were created by triple ligation method , where two pcr - amplified fragments of dna are introduced into a vector ( for details see proszynski et al . , 2004 ) . to generate tpq63 , a dna fragment coding the extracellular domain of fus1p linked to the transmembrane domain ( tmd ) from mid2p ( amplified from plasmid tpq55 with primers containing xbai and bamhi sites ) and a dna fragment coding the cytoplasmic tail of fus1p fused to gfp ( amplified from tpq53 with primers containing bglii and hindiii sites ) were co - ligated to the mbq1 vector digested with xbai hindiii to create tpq65 , a fragment of dna coding the extracellular domain of mid2p linked to the tmd from fus1p ( amplified from plasmid tpq53 with xbai and bamhi sites added on the primers ) and a dna fragment containing the cytoplasmic tail of mid2p fused to gfp ( amplified from mbq35 with bglii and hindiii sites added on the primers ) were coligated into mbq1 ( xbai hindiii ) . to make tpq72 , a dna fragment coding the extracellular domain and tmd of fus1p ( amplified from plasmid mbq30 with primers containing xbai and bamhi sites ) and a dna fragment containing the cytoplasmic tail of mid2p fused to gfp ( prepared as for tpq65 ) were co - ligated to the xbai hindiii - digested vector mbq1 . plasmid tpq57 was made by co - ligation of a dna fragment coding gfp ( flanked by bamhi hindiii sites ) with a dna fragment coding the truncated ( sh3 ) version of fus1p ( amplified from mbq30 with praimers containing xbai bglii sites ) into the mbq1 vector ( xbai hindiii ) . primers tpq94 and tpq97 were constructed by homologus recombination in rh690 - 15d cells . to generate tpq94 , a dna fragment ( obtained from tpq63 with bglii hindiii digestion ) coding the tmd from mid2 and the cytoplasmic tail from fus1 , followed by the gfp , was cotransformed with mbq35 ( linearized with bamhi ) . to create tpq97 , a dna fragment coding the truncated cytoplasmic tail of fus1p followed by the gfp coding sequence ( pcr amplified from tpq57 ) was cotransformed with the nhei - linearized tpq97 . the successful recombination was verified by observation of fluorescence in microscope and sequencing of the plasmids . images were taken with a microscope ( model bx61 ; olympus ) , a camera ( rt slider spot ; diagnostic instruments , inc . ) , and metamorph software ( molecular devices ) . for laurdan microscopy , cells were treated with 5 mm sodium azide and 4% paraformaldehyde for 10 min at 24c ; 250 m laurdan ( molecular probes ) was added for a further 5 min at 24c and cells were washed twice and imaged in water . laurdan fluorescence was excited at 800 nm with a verdi / mira 900 multi - photon laser system and intensity images were recorded simultaneously in the range of 400445 nm and 445530 nm for the two channels ( bio - rad laboratories ) , respectively . the generalized polarization gp , defined as\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation*}{\mathrm{gp}}=\frac{{\mathrm{i}}_{(400 - 460)}-{\mathrm{i}}_{(470 - 530)}}{{\mathrm{i}}_{{\mathrm{(400 - 460)}}}+_{(470 - 530)}}\end{equation*}\end{document}was calculated for each pixel using the two laurdan intensity images . the gp values were determined at the mating tip or opposite the tip in a region measuring 1.2 0.2 m , and each data point ( or symbol ) in the scatter plots represents derivatives from one individual cell . gp values were corrected using the g - factor obtained for laurdan in dmso for each experiment . means and standard deviation of multiple comparisons were compared with one - way anova with tukey 's post - testing assuming gaussian distributions ( prism ) ( gaus et al . , 2003 ) . in this study the following yeast strains were used : rh690 - 15d [ wild - type ] ( mata his4 , leu2 , ura3 , lys2 , bar1 ) was obtained from h. riezman ( university of basel , basel , switzerland ) , and rh1965 [ end4 ] ( mata his4 , leu2 , ura3 , lys2 , bar1 , end4leu2 ) and rh268 - 1 [ en4 - 1 ts ] ( mata his4 , leu2 , ura3 , lys2 , bar1 , end4 - 1 ( ts ) ) were obtained from c. walch - solimena ( mpj - cbg , dresden , germany ) . 1302-wt ( by4742 ) , pea2 , bni1 , fus2 , spa2 , bud6 , fus1 and ste5 deletions are in by strains derived from s288c ( mata ; his31 ; leu20 ; met150 ; ura30 ) and were obtained from euroscarf . cells were grown overnight in yeast extract / peptone ( yp ) medium containing 2% raffinose ( ypraf ) as a carbon source at 24c . for the induction of expression from the gal - s promoter , 2% galactose was added . to induce a mating response , 5 m -factor ( sigma - aldrich ) was added and cells were incubated for 3 h at 24c ( or as indicated ) . plasmids used in this study are listed in table i. all constructs created in our lab are based on the centromeric plasmid p416 ( mumberg et al . , 1995 ) and plasmids p4269 , p4580 , and p4667 containing mutants of fus1 under control of its own promoter were obtained from the c. boone lab ( university of toronto , toronto , canada ; nelson et al . , 2004 ) . the plasmid containing gfp - snc1 under control of constitutive tpi promoter was obtained from the h. pelham lab ( mrc laboratory of molecular biology , cambridge , uk ; lewis et al . , 2000 ) . construction of plasmids mbq30 , mbq35 , tpq53 , and tpq55 was described previously ( bagnat and simons , 2002 ; proszynski et al . , 2004 , 2005 ) . plasmids tpq63 , tpq65 , tpq72 , and tpq57 were created by triple ligation method , where two pcr - amplified fragments of dna are introduced into a vector ( for details see proszynski et al . , 2004 ) . to generate tpq63 , a dna fragment coding the extracellular domain of fus1p linked to the transmembrane domain ( tmd ) from mid2p ( amplified from plasmid tpq55 with primers containing xbai and bamhi sites ) and a dna fragment coding the cytoplasmic tail of fus1p fused to gfp ( amplified from tpq53 with primers containing bglii and hindiii sites ) were co - ligated to the mbq1 vector digested with xbai hindiii . to create tpq65 , a fragment of dna coding the extracellular domain of mid2p linked to the tmd from fus1p ( amplified from plasmid tpq53 with xbai and bamhi sites added on the primers ) and a dna fragment containing the cytoplasmic tail of mid2p fused to gfp ( amplified from mbq35 with bglii and hindiii sites added on the primers ) were coligated into mbq1 ( xbai hindiii ) . to make tpq72 , a dna fragment coding the extracellular domain and tmd of fus1p ( amplified from plasmid mbq30 with primers containing xbai and bamhi sites ) and a dna fragment containing the cytoplasmic tail of mid2p fused to gfp ( prepared as for tpq65 ) were co - ligated to the xbai hindiii - digested vector mbq1 . plasmid tpq57 was made by co - ligation of a dna fragment coding gfp ( flanked by bamhi hindiii sites ) with a dna fragment coding the truncated ( sh3 ) version of fus1p ( amplified from mbq30 with praimers containing xbai bglii sites ) into the mbq1 vector ( xbai hindiii ) . primers tpq94 and tpq97 were constructed by homologus recombination in rh690 - 15d cells . to generate tpq94 , a dna fragment ( obtained from tpq63 with bglii hindiii digestion ) coding the tmd from mid2 and the cytoplasmic tail from fus1 , followed by the gfp , was cotransformed with mbq35 ( linearized with bamhi ) . to create tpq97 , a dna fragment coding the truncated cytoplasmic tail of fus1p followed by the gfp coding sequence ( pcr amplified from tpq57 ) was cotransformed with the nhei - linearized tpq97 . the successful recombination was verified by observation of fluorescence in microscope and sequencing of the plasmids . images were taken with a microscope ( model bx61 ; olympus ) , a camera ( rt slider spot ; diagnostic instruments , inc . ) , and metamorph software ( molecular devices ) . for laurdan microscopy , cells were treated with 5 mm sodium azide and 4% paraformaldehyde for 10 min at 24c ; 250 m laurdan ( molecular probes ) was added for a further 5 min at 24c and cells were washed twice and imaged in water . laurdan fluorescence was excited at 800 nm with a verdi / mira 900 multi - photon laser system and intensity images were recorded simultaneously in the range of 400445 nm and 445530 nm for the two channels ( bio - rad laboratories ) , respectively . the generalized polarization gp , defined as\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation*}{\mathrm{gp}}=\frac{{\mathrm{i}}_{(400 - 460)}-{\mathrm{i}}_{(470 - 530)}}{{\mathrm{i}}_{{\mathrm{(400 - 460)}}}+_{(470 - 530)}}\end{equation*}\end{document}was calculated for each pixel using the two laurdan intensity images . the gp values were determined at the mating tip or opposite the tip in a region measuring 1.2 0.2 m , and each data point ( or symbol ) in the scatter plots represents derivatives from one individual cell . gp values were corrected using the g - factor obtained for laurdan in dmso for each experiment . means and standard deviation of multiple comparisons were compared with one - way anova with tukey 's post - testing assuming gaussian distributions ( prism ) ( gaus et al . , 2003 ) .

the yeast mating cell provides a simple paradigm for analyzing mechanisms underlying the generation of surface polarity . endocytic recycling and slow diffusion on the plasma membrane were shown to facilitate polarized surface distribution of snc1p ( valdez - taubas , j. , and h.r . pelham . 2003 . curr . biol . 13:16361640 ) . here , we found that polarization of fus1p , a raft - associated type i transmembrane protein involved in cell fusion , does not depend on endocytosis . instead , fus1p localization to the tip of the mating projection was determined by its cytosolic domain , which binds to peripheral proteins involved in mating tip polarization . furthermore , we provide evidence that the lipid bilayer at the mating projection is more condensed than the plasma membrane enclosing the cell body , and that sphingolipids are required for this lipid organization .

Introduction Results and discussion Materials and methods Strains and growth conditions Plasmids Microscopy Online supplemental material Supplementary Material

saccharomyces cerevisiae polarizes cell growth to the bud during cell replication and to the mating projection when cells are induced by pheromones to change their shape to form shmoos . first , the site on the cell surface is selected by intrinsic and extrinsic cues . last , a multiprotein machine is assembled that spools out actin cables to direct post - golgi traffic to the site of polarized cell growth ( drubin and nelson , 1996 ; madden and snyder , 1998 ; pruyne and bretscher , 2000 ; chang and peter , 2003 ) . during budding , membrane traffic is directed by actin cables to the bud , and the septin ring at mother - daughter cell neck region functions as a physical barrier , preventing diffusion of membrane components from the bud to the mother cell ( barral et al . nevertheless , a specific subset of proteins required for mating is clustered at the tip of the mating projection . we have shown previously that the polarized distribution of membrane components involves raft lipids ( sphingolipids and ergosterol ) and the actin cytoskeleton ( bagnat and simons , 2002 ) . based on these observations , we proposed that lipid rafts are clustered at the tip of the mating projection and that this process is important for the retention of the associated molecules at the mating projection . recently , it was demonstrated that cycles of endocytosis combined with polarized membrane delivery into the mating projection are able to restrict protein localization to the tips of shmoos ( valdez - taubas and pelham , 2003 ) . here , we report that polarized localization of fus1p , a type i transmembrane protein involved in cell fusion ( trueheart and fink , 1989 ) , does not require endocytosis . instead , the protein is retained at the tip of the mating projection through the interaction of its cytosolic tail with a multiprotein scaffolding machinery . additionally , we provide evidence that the lipid bilayer at the tip of the mating projection is more ordered than over the cell body and that sphingolipids are required for this specific lipid organization . to revisit the kinetic recycling model we have analyzed the role of polarized delivery and endocytosis in polarizing fus1p , a type i transmembrane protein involved in cell fusion ( trueheart and fink , 1989 ; nolan et al . , 2006 ) , to the tip of the mating projection . we first analyzed shmoo tip delivery of fus1p and compared it to another marker protein that is distributed all over the plasma membrane of mating cells , mid2p ( fig . mid2p is a cell wall integrity sensor , and similarly to fus1p , it is a type i transmembrane protein ( philip and levin , 2001 ) . however , 2 h later mid2p had diffused over the entire plasma membrane , whereas fus1p remained at the tip . we also used snc1p , a yeast v - snare involved in post - golgi plasma membrane transport , as a second marker protein that is tip localized . it has been shown that snc1p polarization was abolished after inhibition of endocytosis ( valdez - taubas and pelham , 2003 ) . in endocytosis - deficient cells , snc1p was no longer polarized but was distributed over the plasma membrane of the shmooing cells . in contrast , the polarization of fus1p to the tip of the mating projection remained normal in end4 cells ( fig . thus , there must be an alternative mechanism that maintains biosynthetically delivered fus1p at the shmoo tip irrespective of ongoing cycles of endocytosis and exocytosis . these findings confirm that the kinetic polarization model using endocytosis and polarized exocytosis is involved in local concentration of membrane proteins such as snc1p . polarized distribution of snc1p , but not of fus1p , is dependent on endocytosis . blocked endocytosis in end4 disrupted polarized distribution of snc1p , but did not affect tip localization of fus1p , compared with wild - type cells . the percentage of cells with fluorescence on the plasma membrane limited to the mating projection is given in the bottom right corner of the gfp images . for fus1p in wild - type and end4 we counted 218 and 327 cells , respectively , and for snc1p it was 220 and 100 cells , respectively . in wild - type cells , in addition to the plasma membrane , snc1p was found in intracellular structures due to protein cycling . these structures were not visible in the endocytosis mutant because the protein was trapped on the plasma membrane . fus1p in wild - type cells was found at the plasma membrane and in the vacuole . one reason why fus1p is retained at the mating tip could be due to interaction with the cell wall , as was demonstrated for glycosylphosphatidylinositol - anchored proteins ( de sampaio et al . thus , we constructed fusion proteins between fus1p and mid2p where we swapped the extracellular , the transmembrane , and the cytosolic domains of the two proteins ( schematically shown in fig . analysis of their surface distribution demonstrated that the information for mating tip retention was localized to the cytosolic tail ( fig . the mid2 protein carrying the cytoplasmic domain from fus1p was localized to the mating projection . we then analyzed how this chimeric protein behaved in mutants in which endocytosis was inhibited , both in end4 cells and at the nonpermissive temperature in end4 - 1 ts cells . thus , we concluded that the cytosolic tail of fus1p mediates protein retention at the tip and that interactions with the cell wall can not explain the polarization . the cytosolic tail of fus1p is 416 amino acids long and contains an src kinase homology 3 ( sh3 ) domain close to its cooh terminus , followed by a proline - rich domain , both known to be responsible for protein protein interactions ( tong et al . at this time , a report from nelson et al . ( 2004 ) appeared , in which a detailed analysis of the cytoplasmic domain of fus1p was described . , 2004 ) we considered the possibility that the double mutant of fus1p could be polarized via the endocytic recycling mechanism . to test this possibility we expressed the mutated fus1p in the endocytosis - deficient strain and found that protein polarization was still normal ( fig . we concluded that additional sites on the cytoplasmic tail of fus1p might contribute to fus1p retention to the mating tip . in a detailed two - hybrid analysis it was demonstrated that the cytosolic tail of fus1p interacts with several key players in mating polarity , including the gtp - bound form of cdc42 , components of the polarisome pea2p and bni1p , fus2p , and ste5p , the scaffold protein for map kinase signaling ( nelson et al . we analyzed the mid - fus protein in pea2 , bni1 , fus2 , spa2 , bud6 , fus1 , and ste5 deletion mutants . we propose that fus1p is directly embedded in a dynamic network of protein protein interactions that is responsible for scaffolding and localization of fus1 to the shmoo tip as part of the mating machinery . fus1p , mid2p , and different chimeric proteins were expressed in wild - type cells treated with -factor . the schematic representation of the expressed fusion proteins is shown on the right and the swapped domains are indicated . internal membrane staining is seen in f and g , suggesting that protein sorting to the cell surface was compromised . however , the fraction of protein that was delivered to the plasma membrane was not polarized . the chimeric mid(cyt - fussh3 ) protein , which carries a cytoplasmic tail from fus1p without sh3 domain , was equally well polarized in the wild - type and end4 cells ( a ) . the percentage of cells with fluorescence on the plasma membrane limited to the mating projection is indicated ( n = 361 and 138 for the wild - type and end4 cells , respectively ) . similarly , protein carrying point mutations that affect the function of the proline - rich region or the sh3 domain ( fus1p(p422a ) and fus1p - sh3(w473s ) , respectively ) , or the double mutant of fus1p ( fus1p(p422a)-sh(w473s ) ) , was polarized in end4 cells ( b ) . based on the findings ( 1 ) that polarization of the mating machinery to the shmoo tip is inhibited in erg6 and in lcb1 - 100 cells ; ( 2 ) that mutations that severely affect the synthesis of the major raft lipids in yeast reduced mating efficiency ; and ( 3 ) on the polarized distribution of filipin , a molecule that has high affinity for sterols , we postulated previously that raft lipid clustering plays a role in establishing and maintaining mating tip polarization ( bagnat and simons , 2002 ) . because partitioning of filipin does not directly correlate with lipid ordering in the bilayer , we took advantage of a dye that does . by expressing a normalized ratio of the two emission regions the general polarization ( gp ; see materials and methods)laurdan fluorescence provides a relative measure of lipid order in cell membranes . importantly , laurdan does not preferentially partition into a specific lipid phase , nor do gp values depend on the local probe concentration within the membrane ( gaus et al . the gp images revealed that the membrane at that mating projection is more condensed and ordered than the domain on the opposite site of the cell ( fig . hence , the membrane at the mating projections displayed the biophysical characteristic that is expected for raft clustering . the coalescence of condensed membrane at the tips of shmoos was also found to occur in the endocytosis - deficient strain but not in the sphingolipid mutant lcb1 - 100 ( fig . these data clearly demonstrated the asymmetric organization of the lipids in the plasma membrane of yeast cells during mating . more detailed analysis is needed to understand the molecular mechanisms responsible for the formation and maintenance of the mating projection . -factor treated wild - type ( a and d ) , lcb1 - 100 ( b and e ) , and end4 ( c and f ) cells were killed with 5 mm nan3 , fixed with 24% paraformaldehyde , labeled with 250 m laurdan for 5 min , and imaged in water . gp values of membranes at the mating tip and at the cell body opposite the mating tip were measured for 40 ( d ) , 28 ( e ) , and 43 ( f ) individual cells . means sd are 0.398 0.118 and 0.191 0.090 ( d ) , 0.256 0.143 and 0.202 0.104 ( e ) , and 0.372 0.075 and 0.256 0.111 ( f ) for mating tip and cell body , respectively . the difference of the gp value at the mating projection between wild - type and sphingolipid mutant cells was statistically significant ( p < 0.001 ) but there was no statistically significant difference between wild - type and end4 cells ( p > 0.05 ) . in migrating neutrophils it was demonstrated that lipid raft clusters are localized to the rear of the cells in an actin - dependent manner ( seveau et al . recently , it was also demonstrated that when the t cell receptor is activated , a condensed raft cluster is formed at the activation site ( gaus et al . , 2005 ) and the kinetics of the protein protein interactions ( harder , 2004 ; douglass and vale , 2005 ) . this mechanism could also drive the surface polarization during yeast mating ( bagnat and simons , 2002 ) . nevertheless , coming together of proteins with a condensed lipid domain at the mating tip could lead to activation of the mating machinery spatially and temporally by specific lipid for instance , the egf receptor has been shown to be activated by interactions with the ganglioside gd1a and the glutamate receptor by raft - cholesterol ( eroglu et al . recently , it was demonstrated that yeast ste5p , a protein that plays a crucial role in pheromone signaling and interacts with the cytoplasmic tail of fus1p , has a phospholipid binding domain that is necessary for protein localization and signaling ( winters et al . the generation of cell surface polarity during yeast mating is thus a complex process involving on one hand endocytosis and recycling and on the other hand establishment of the site where the mating machinery is scaffolded and the membrane reorganized . in this study the following yeast strains were used : rh690 - 15d [ wild - type ] ( mata his4 , leu2 , ura3 , lys2 , bar1 ) was obtained from h. riezman ( university of basel , basel , switzerland ) , and rh1965 [ end4 ] ( mata his4 , leu2 , ura3 , lys2 , bar1 , end4leu2 ) and rh268 - 1 [ en4 - 1 ts ] ( mata his4 , leu2 , ura3 , lys2 , bar1 , end4 - 1 ( ts ) ) were obtained from c. walch - solimena ( mpj - cbg , dresden , germany ) . , 1995 ) and plasmids p4269 , p4580 , and p4667 containing mutants of fus1 under control of its own promoter were obtained from the c. boone lab ( university of toronto , toronto , canada ; nelson et al . to generate tpq63 , a dna fragment coding the extracellular domain of fus1p linked to the transmembrane domain ( tmd ) from mid2p ( amplified from plasmid tpq55 with primers containing xbai and bamhi sites ) and a dna fragment coding the cytoplasmic tail of fus1p fused to gfp ( amplified from tpq53 with primers containing bglii and hindiii sites ) were co - ligated to the mbq1 vector digested with xbai hindiii to create tpq65 , a fragment of dna coding the extracellular domain of mid2p linked to the tmd from fus1p ( amplified from plasmid tpq53 with xbai and bamhi sites added on the primers ) and a dna fragment containing the cytoplasmic tail of mid2p fused to gfp ( amplified from mbq35 with bglii and hindiii sites added on the primers ) were coligated into mbq1 ( xbai hindiii ) . to make tpq72 , a dna fragment coding the extracellular domain and tmd of fus1p ( amplified from plasmid mbq30 with primers containing xbai and bamhi sites ) and a dna fragment containing the cytoplasmic tail of mid2p fused to gfp ( prepared as for tpq65 ) were co - ligated to the xbai hindiii - digested vector mbq1 . to create tpq97 , a dna fragment coding the truncated cytoplasmic tail of fus1p followed by the gfp coding sequence ( pcr amplified from tpq57 ) was cotransformed with the nhei - linearized tpq97 . the successful recombination was verified by observation of fluorescence in microscope and sequencing of the plasmids . images were taken with a microscope ( model bx61 ; olympus ) , a camera ( rt slider spot ; diagnostic instruments , inc . ) , and metamorph software ( molecular devices ) . the gp values were determined at the mating tip or opposite the tip in a region measuring 1.2 0.2 m , and each data point ( or symbol ) in the scatter plots represents derivatives from one individual cell . construction of plasmids mbq30 , mbq35 , tpq53 , and tpq55 was described previously ( bagnat and simons , 2002 ; proszynski et al . plasmids tpq63 , tpq65 , tpq72 , and tpq57 were created by triple ligation method , where two pcr - amplified fragments of dna are introduced into a vector ( for details see proszynski et al . to generate tpq63 , a dna fragment coding the extracellular domain of fus1p linked to the transmembrane domain ( tmd ) from mid2p ( amplified from plasmid tpq55 with primers containing xbai and bamhi sites ) and a dna fragment coding the cytoplasmic tail of fus1p fused to gfp ( amplified from tpq53 with primers containing bglii and hindiii sites ) were co - ligated to the mbq1 vector digested with xbai hindiii . to create tpq65 , a fragment of dna coding the extracellular domain of mid2p linked to the tmd from fus1p ( amplified from plasmid tpq53 with xbai and bamhi sites added on the primers ) and a dna fragment containing the cytoplasmic tail of mid2p fused to gfp ( amplified from mbq35 with bglii and hindiii sites added on the primers ) were coligated into mbq1 ( xbai hindiii ) . to make tpq72 , a dna fragment coding the extracellular domain and tmd of fus1p ( amplified from plasmid mbq30 with primers containing xbai and bamhi sites ) and a dna fragment containing the cytoplasmic tail of mid2p fused to gfp ( prepared as for tpq65 ) were co - ligated to the xbai hindiii - digested vector mbq1 . to generate tpq94 , a dna fragment ( obtained from tpq63 with bglii hindiii digestion ) coding the tmd from mid2 and the cytoplasmic tail from fus1 , followed by the gfp , was cotransformed with mbq35 ( linearized with bamhi ) . to create tpq97 , a dna fragment coding the truncated cytoplasmic tail of fus1p followed by the gfp coding sequence ( pcr amplified from tpq57 ) was cotransformed with the nhei - linearized tpq97 . images were taken with a microscope ( model bx61 ; olympus ) , a camera ( rt slider spot ; diagnostic instruments , inc . ) the gp values were determined at the mating tip or opposite the tip in a region measuring 1.2 0.2 m , and each data point ( or symbol ) in the scatter plots represents derivatives from one individual cell .

aric is a population - based study involving both whites and african americans aged 4564 years in four u.s . communities in north carolina , mississippi , minnesota , and maryland ( 15 ) . between 1987 and 1989 , 15,792 men and women were recruited and attended a baseline exam ( visit 1 ) . human subject research review committees at the involved institutions approved the study , and all participants gave written informed consent . a stratified , nested case - cohort design was used in an ancillary study investigating inflammatory precursors of diabetes . this design has been used to investigate the role of total and high - molecular - weight ( hmw ) adiponectin , leptin , ferritin , free fatty acids , and other plasma biomarkers in the development of diabetes in the aric study ( 4,6,1517 ) . from the baseline cohort , we excluded 2,018 participants with prevalent diabetes , 95 members of ethnic groups other than african americans or whites , 853 participants not returning for any follow - up visit , 26 with no valid diabetes determination at follow - up , 6 with restrictions on stored plasma use , 12 with missing baseline anthropometric data , 2,514 participants in previous aric case - control and case - cohort studies involving cardiovascular disease for whom stored plasma was either previously exhausted or held in reserve , 212 for incomplete fasting ( < 8 h ) , and 316 with missing information for hypertension and inflammation markers . a final sampling frame of 9,740 individuals ( 71% of those in the full cohort without diabetes at baseline ) was established after exclusions . of these , 1,105 participants ( 11.3% of the final sample ) were ascertained to have incident diabetes during follow - up . from the eligible members of this baseline cohort , we selected and measured analytes on ethnicity - stratified ( 50% white , 50% african american ) random samples of both cases of incident diabetes and eligible members of the full cohort ( 1,095 individuals in total ) . some participants within the cohort random sample developed diabetes during follow - up and were part of the case random sample as well . we excluded an additional 11 participants who had missing values for il-18 , leaving a total of 1,084 subjects for analysis . the definitions and methods for baseline measurements ( height , weight , sbp , hypertension , triglycerides [ tgs ] , hdl cholesterol [ hdl - c ] , inflammation markers , and adipocytokines ) have been previously described ( 4,6 ) . a score was computed to indicate low - grade systemic inflammation , ranging from 0 to 6 ; one point was attributed to a value greater than the median of the cohort random sample for each of the six markers of systemic inflammation : il-6 , crp , orosomucoid , sialic acid , white blood cell ( wbc ) count , and fibrinogen ( 4 ) . glucose was measured at baseline and at follow - up visits by standard hexokinase method . smoking was assessed as a binary variable with 0 = never or former smokers and 1 = current smokers . sport and physical activity was assessed with semicontinuous indices created from individual component questions described in detail elsewhere ( 18 ) . the index ranged from 1 ( low ) to 5 ( high ) . for education , we used a four - level categorical variable : 1 , no high school diploma or less ; 2 , high school or some college ; 3 , beyond bachelors ; and 4 , missing data . cases of incident diabetes were defined on the basis of : 1 ) a reported physician diagnosis , 2 ) reported use of antidiabetic medications during the 2 weeks prior to the visit , or 3 ) a fasting ( 8 h ) glucose value of at least 7.0 the date of diabetes incidence was estimated by linear interpolation using glucose values at the ascertaining visit and the previous visit ( 4,6 ) . circulating levels of il-18 were measured in a centralized laboratory in plasma samples that were continuously stored at 70c prior to analysis . all measurements were performed in duplicate using a sandwich elisa according to manufacturer s protocol ( r&d systems , minneapolis , mn ) . weighted analyses were done based on the case - cohort sampling design to allow for inference to the entire cohort . quartile values were used in the models , and test of trend was generated across the quartiles of il-18 . cox proportional hazards models were fitted to investigate the relationship between il-18 and time to onset of diabetes . wald tests of interaction terms were used to test heterogeneity in the association between race and il-18 quartile . all statistical testing was performed using two - sided tests with p = 0.05 as the cutoff for statistical significance . statistical analyses were performed using the sas ( sas institute inc . , cary , nc ) and sudaan ( research triangle institute , research triangle park , nc ) statistical software packages . aric is a population - based study involving both whites and african americans aged 4564 years in four u.s . communities in north carolina , mississippi , minnesota , and maryland ( 15 ) . between 1987 and 1989 , 15,792 men and women were recruited and attended a baseline exam ( visit 1 ) . human subject research review committees at the involved institutions approved the study , and all participants gave written informed consent . a stratified , nested case - cohort design was used in an ancillary study investigating inflammatory precursors of diabetes . this design has been used to investigate the role of total and high - molecular - weight ( hmw ) adiponectin , leptin , ferritin , free fatty acids , and other plasma biomarkers in the development of diabetes in the aric study ( 4,6,1517 ) . from the baseline cohort , we excluded 2,018 participants with prevalent diabetes , 95 members of ethnic groups other than african americans or whites , 853 participants not returning for any follow - up visit , 26 with no valid diabetes determination at follow - up , 6 with restrictions on stored plasma use , 12 with missing baseline anthropometric data , 2,514 participants in previous aric case - control and case - cohort studies involving cardiovascular disease for whom stored plasma was either previously exhausted or held in reserve , 212 for incomplete fasting ( < 8 h ) , and 316 with missing information for hypertension and inflammation markers . a final sampling frame of 9,740 individuals ( 71% of those in the full cohort without diabetes at baseline ) was established after exclusions . of these , 1,105 participants ( 11.3% of the final sample ) were ascertained to have incident diabetes during follow - up . from the eligible members of this baseline cohort , we selected and measured analytes on ethnicity - stratified ( 50% white , 50% african american ) random samples of both cases of incident diabetes and eligible members of the full cohort ( 1,095 individuals in total ) . some participants within the cohort random sample developed diabetes during follow - up and were part of the case random sample as well . we excluded an additional 11 participants who had missing values for il-18 , leaving a total of 1,084 subjects for analysis . the definitions and methods for baseline measurements ( height , weight , sbp , hypertension , triglycerides [ tgs ] , hdl cholesterol [ hdl - c ] , inflammation markers , and adipocytokines ) have been previously described ( 4,6 ) . a score was computed to indicate low - grade systemic inflammation , ranging from 0 to 6 ; one point was attributed to a value greater than the median of the cohort random sample for each of the six markers of systemic inflammation : il-6 , crp , orosomucoid , sialic acid , white blood cell ( wbc ) count , and fibrinogen ( 4 ) . glucose was measured at baseline and at follow - up visits by standard hexokinase method . smoking was assessed as a binary variable with 0 = never or former smokers and 1 = current smokers . sport and physical activity was assessed with semicontinuous indices created from individual component questions described in detail elsewhere ( 18 ) . the index ranged from 1 ( low ) to 5 ( high ) . for education , we used a four - level categorical variable : 1 , no high school diploma or less ; 2 , high school or some college ; 3 , beyond bachelors ; and 4 , missing data . cases of incident diabetes were defined on the basis of : 1 ) a reported physician diagnosis , 2 ) reported use of antidiabetic medications during the 2 weeks prior to the visit , or 3 ) a fasting ( 8 h ) glucose value of at least 7.0 the date of diabetes incidence was estimated by linear interpolation using glucose values at the ascertaining visit and the previous visit ( 4,6 ) . circulating levels of il-18 were measured in a centralized laboratory in plasma samples that were continuously stored at 70c prior to analysis . all measurements were performed in duplicate using a sandwich elisa according to manufacturer s protocol ( r&d systems , minneapolis , mn ) . weighted analyses were done based on the case - cohort sampling design to allow for inference to the entire cohort . quartile values were used in the models , and test of trend was generated across the quartiles of il-18 . cox proportional hazards models were fitted to investigate the relationship between il-18 and time to onset of diabetes . wald tests of interaction terms were used to test heterogeneity in the association between race and il-18 quartile . all statistical testing was performed using two - sided tests with p = 0.05 as the cutoff for statistical significance . statistical analyses were performed using the sas ( sas institute inc . , cary , nc ) and sudaan ( research triangle institute , research triangle park , nc ) statistical software packages . our case - cohort sample of 1,084 aric study participants included 488 ( 45% ) african american and 596 ( 55% ) white individuals . the cohort random sample comprised 630 individuals , and the incident diabetes random sample included 548 individuals . of the participants selected in the cohort random sample , 94 developed diabetes during follow - up , and 71 of them were included in the incident diabetes random sample . thus , there were 23 incident diabetes cases in addition to those in the incident diabetes sample . among cases , 146 were white men , 142 were white women , 68 were african american men , and 192 were african american women . among noncases , table 1 shows overall and ethnicity - specific distribution of the baseline demographic , clinical , and biomarker characteristics in this cohort random sample . supplementary table 1 compares the baseline characteristics of the participants selected in the final sample with those of the entire cohort , stratified by ethnicity . baseline il-18 levels were significantly higher in white compared with african american participants ( median [ interquartile range ] ; 235.6 [ 129.7 ] vs. 190.6 [ 116.4 ] pg / ml ; p < 0.001 ) . this difference was seen in both men ( p < 0.001 ) and women ( p < 0.001 ) ( table 2 ) . a statistically significant interaction between race and il-18 quartile for incident diabetes was found in the study population after adjusting for age , sex , and race ( p = 0.01 ) . however , after additional adjustment for parental history of diabetes , bmi , sbp , and waist - to - hip ratio ( whr ) , the interaction between race and il-18 quartile lost significance ( p = 0.15 ) . baseline characteristics in participants who developed diabetes and those who did not stratified by race / ethnicity distribution of baseline il-18 levels in the cohort random sample by race and sex hrs for developing diabetes by quartiles of il-18 are shown in supplementary table 2 . a statistically significant hr for developing diabetes was seen for study participants in the fourth ( versus first ) quartile of il-18 levels ( hr : 1.5 ; 95% ci : 1.12.1 ) in the crude analysis . the hrs for fourth versus first quartile remained significant even after adjusting for age , sex , study center , and race ( 1.7 ; 1.22.3 ) . the effects were attenuated , and the hr were no longer statistically significant after sequential additional adjustment for parental history of diabetes , bmi , sbp , and whr ( 1.2 ; 0.81.7 ) ; sport index , education level , and smoking status ( 0.9 ; 0.71.7 ) ; glucose , hdl - c , and tgs ( 0.9 , 95% ci : 0.61.4 ) ; inflammation score ( 1.0 ; 0.71.5 ) ; or total and hmw adiponectin ( 1.1 ; 0.81.6 ) ( supplementary table 2 ) . in a similar subanalysis by race ( table 3 ) , risk for developing diabetes in the fourth ( versus first ) quartile of il-18 levels was statistically significant among whites in the crude analysis ( hr : 2.5 ; 95% ci : 1.64.0 ) . this association remained significant after adjustment for age , sex , and study center ( 2.1 ; 1.33.4 ) . however , statistical significance was lost after additional adjustment for clinical variables ( 1.2 ; 0.72.0 ) and when adjustments were made for biochemical and inflammatory markers and adipocytokines . hrs for developing diabetes by quartiles of il-18 stratified by race correlations between il-18 levels and various baseline demographic , clinical , biochemical , inflammatory , and anti - inflammatory variables markedly varied among the whites and the african americans in the cohort random sample ( supplementary table 3 ) . unlike those in african americans , il-18 levels in whites had a significant correlation with age ( p < 0.01 ) and anthropometric characteristics such as waist circumference ( p < 0.001 ) , height ( p = 0.04 ) , whr ( p < 0.001 ) , and bmi ( p < 0.01 ) . similarly , il-18 levels among whites were significantly correlated with sbp ( p < 0.01 ) , fasting glucose ( p < 0.001 ) , and insulin ( p < 0.001 ) . il-18 levels among whites showed a highly significant correlation with hdl - c ( p < 0.001 ) , ldl cholesterol ( ldl - c ; p < 0.001 ) , and tgs ( p < 0.001 ) and moderate correlation with oxidized ldl ( p = 0.04 ) , but no correlation with nonesterified fatty acids ( p = 0.7 ) . in contrast , il-18 levels among african americans had the strongest correlation with nonesterified fatty acids ( p < 0.001 ) and a weaker correlation with hdl - c ( p = 0.05 ) , oxidized ldl ( p = 0.04 ) , and tgs ( p = 0.02 ) . for inflammatory markers , il-18 had a highly statistically significant correlation with il-6 ( p < 0.001 ) , crp ( p < 0.001 ) , intercellular adhesion molecule1 ( p 0.001 ) among whites and a highly significant correlation only with intercellular adhesion molecule1 ( p < 0.001 ) among african americans . although il-18 levels were significantly correlated with total ( p < 0.001 ) and hmw ( p < 0.001 ) adiponectin among whites , no correlation was seen in african americans . this is the first study to look at the association of il-18 levels with incident diabetes in a racially heterogeneous population . two previous studies have looked at this association in predominantly white populations ( 13,14 ) . in our study , higher il-18 levels showed a significant association with incident diabetes in the entire study cohort and in whites , but not in african americans . upon adjustment for clinical risk factors associated with diabetes , including age , parental history of diabetes , bmi , sbp , and whr , this association almost reached statistical significance . further addition of fasting glucose and inflammation score to the models weakened the independent association of il-18 levels with incident diabetes in our study . the findings of our study support the hypothesis that elevated fasting glucose may itself be a regulator of il-18 levels . this is consistent with the other studies showing that persistent hyperglycemia upregulates the synthesis of il-18 and other proinflammatory cytokines ( 19 ) . it has been shown that the degree of lipid peroxidation is directly proportional to blood glucose concentrations ( 20 ) . persistent oxidative stress induces stress - sensitive signaling pathways , such as nuclear factor of light polypeptide gene enhancer in b cells ( nf-b ) . nf-b is a transcription factor that plays a major role in mediating inflammatory responses and in the production of proinflammatory cytokines like il-6 , tnf- , and il-18 . the inflammatory cytokines in turn lead to insulin resistance and overt diabetes ( 21 ) , further increasing the plasma glucose levels . this leads to a vicious cascade phenomenon with a positive - feedback loop , causing difficulty in determining whether fasting glucose is upstream or downstream of il-18 in the pathway for onset of diabetes . it is plausible that both impaired fasting glucose and elevated il-18 levels are drivers of the proinflammatory pathway culminating in diabetes . this hypothesis is further strengthened by our observation that elevated il-18 levels were not associated with incident diabetes independent of inflammatory markers including il-6 , crp , sialic acid , and orosomucoid . this is consistent with previous reports showing that il-18 induces production of tnf- , il-6 , and crp ( 22 ) . in another model in our study , addition of hdl - c and tgs weakened the association of il-18 with incident diabetes . because il-18 is a proinflammatory molecule , further adjustments with lipid parameters associated with inflammation may have resulted in overadjustment and weakening of the independent association of il-18 with incident diabetes . il-18 levels showed a weak association with incident diabetes when adjusted for plasma adiponectin levels in our study . this is consistent with previous studies showing that future risk of diabetes was associated with lower levels of total and hmw adiponectin ( 13 ) . it may imply that though a greater amount of il-18 is produced in tissues outside the adipose tissue ( 23 ) , adiposity may drive the il-18 production in later stages . this is supported by a significant , but weak , correlation of il-18 levels with total and hmw adiponectin levels as well as obesity in the white subgroup of our study . in their study in a white female population , hivert et al . ( 13 ) showed an association of il-18 levels with incident diabetes independent of hmw adiponectin and bmi . the authors , however , did not adjust the models for fasting glucose , inflammatory markers , and lipid parameters , all relevant factors in the occurrence of diabetes . the study also showed that the association of il-18 with diabetes tends to be stronger in women with lower bmi , suggesting that increasing adiposity masks the association of il-18 with diabetes ( 13 ) . our second aim was to evaluate racial differences in il-18 levels and their association with incident diabetes . it is possible that the difference in mean il-18 levels between races is attributable at least in part to genetic and anthropometric differences . in addition , il-18 levels were moderately associated with incident diabetes in whites but not at all in african americans . there was a significant interaction between il-18 and race in their associations with incident diabetes . however , this interaction was attenuated on adjustment for parental history of diabetes , bmi , sbp , and whr . duncan et al . ( 4 ) found an association of low - grade inflammation with diabetes in whites but not in african americans in the aric cohort . it is well known that white cell counts differ in african americans compared with whites ( 24 ) . these findings suggest racial differences in inflammatory burden , triggering factors for inflammatory pathways , and the association of inflammation with diabetes . interestingly , in our study , il-18 levels were correlated strongly with anthropometric measures related to obesity in whites but not in african americans . il-18 levels have been inconsistently associated with bmi and body fat in various studies , raising the question of whether the major source of il-18 is not adipose tissue but muscle and liver . however , the absolute lack of correlation of il-18 with either bmi or whr in african americans in our study is both a novel and interesting finding . moreover , there was no correlation of adiponectin with il-18 levels in african americans , but there was a significant correlation in whites , again raising questions regarding the difference in the source of inflammatory markers and adipocytokines in the two races . similarly , fasting glucose and insulin levels were correlated with il-18 levels in whites but not in african americans , whereas free fatty acids were correlated with il-18 levels in african americans and not in whites . although african americans have long been recognized to have different characteristics of the metabolic syndrome compared with whites ( low tg , high hdl - c , and high glucose ) and increased frequency of incident diabetes , only more recently has there been an appreciation of the differences in inflammation markers associated with cardiometabolic risk . african americans have lower wbc count , il-18 , fetuin - a , cc chemokine ligand5 , and regulated on activation , normal t - cell expressed , and secreted ( rantes ) ( 25 ) than whites , but have higher levels of crp , sialic acid , fibrinogen , c3 , and il-6 than whites in the aric cohort . there is growing evidence that markers of inflammation may have ethnic and genetic differences ( 24,26 ) . such differences are also seen in the association of these inflammatory markers with incident diabetes ( 4 ) . one possible explanation for the racial differences in levels of various chemokines and inflammatory markers may be the polymorphism in the duffy antigen receptor for chemokines ( darc ) ( 27 ) . darc is a blood group alloantigen and serves as a vascular reservoir of proinflammatory cytokines . the difference in the wbc count in african americans compared with whites is due to the common african - derived null variant of the darc gene ( 28 ) . by abolishing the expression of darc on erythrocytes , the duffy null variant may alter the concentration and distribution of chemokines in the blood ( 27 ) . . evolutionary diversity along with associated social and environmental factors may contribute to the variable association of diabetes with insulin resistance , markers of inflammation , and anthropometric parameters in different races ( 29 ) . however , these novel markers of inflammation and their association with incident diabetes need further validation . the study cohort is derived from a large well - studied population based in four different u.s . communities , suitably designed to study racial differences and provide a long prospective follow - up of incident cases of diabetes ( 15 ) . markers of inflammation are usually stable in frozen samples , but this has not been studied over a prolonged period of time . in this study , we used a single baseline measurement of il-18 . whether obtaining multiple measurements over time would have strengthened the association seen in the study elevated il-18 levels are associated with incident diabetes mellitus ( dm ) , but there is a racial heterogeneity in this association . further studies are needed to elucidate the details of this mechanism of dm and the potential interplay of racial differences , anthropometric characteristics , and inflammation . markers of inflammation are usually stable in frozen samples , but this has not been studied over a prolonged period of time . in this study , we used a single baseline measurement of il-18 . whether obtaining multiple measurements over time would have strengthened the association seen in the study elevated il-18 levels are associated with incident diabetes mellitus ( dm ) , but there is a racial heterogeneity in this association . further studies are needed to elucidate the details of this mechanism of dm and the potential interplay of racial differences , anthropometric characteristics , and inflammation .

objectiveelevated plasma interleukin-18 ( il-18 ) has been linked to onset of diabetes mellitus ( dm ) and its complications . however , so far this association has been shown only in predominantly white populations . we examined il-18 levels and their association with incident dm in a racially heterogeneous population.research design and methodsin a nested case - cohort design representing a 9-year follow - up of 9,740 middle - aged , initially healthy , nondiabetic white and african american participants of the atherosclerosis risk in communities study , we selected and measured analytes on race - stratified ( 50% white , 50% african american ) random samples of both cases of incident diabetes ( n = 548 ) and eligible members of the full cohort ( n = 536).resultsbaseline il-18 levels were significantly higher in white participants compared with african american participants ( p < 0.001 ) . although white participants in the fourth ( versus first ) quartile of il-18 levels had a significant hazard ratio ( hr ) for developing dm ( hr : 2.1 , 95% ci : 1.33.4 ) , after adjustment for age , sex , and study center , no difference was seen among african americans ( hr : 1.0 , 95% ci : 0.61.7 ) . unlike those in african americans , il-18 levels in whites had a significant correlation with age ( p < 0.01 ) ; anthropometric characteristics such as waist circumference ( p < 0.001 ) , height ( p = 0.04 ) , waist - to - hip ratio ( p < 0.001 ) , and bmi ( p < 0.01 ) ; and total ( p < 0.001 ) and high - molecular - weight ( p < 0.001 ) adiponectin.conclusionsthere are racial differences in levels of il-18 and the association of il-18 with risk factors and incident type 2 dm . in addition , there seems to be a complex interplay of inflammation and adiposity in the development of dm .

RESEARCH DESIGN AND METHODS Materials Data collection Diagnosis of incident diabetes Measurement of IL-18 levels Statistical analysis RESULTS CONCLUSIONS Limitations Summary

aric is a population - based study involving both whites and african americans aged 4564 years in four u.s . human subject research review committees at the involved institutions approved the study , and all participants gave written informed consent . a stratified , nested case - cohort design was used in an ancillary study investigating inflammatory precursors of diabetes . this design has been used to investigate the role of total and high - molecular - weight ( hmw ) adiponectin , leptin , ferritin , free fatty acids , and other plasma biomarkers in the development of diabetes in the aric study ( 4,6,1517 ) . from the baseline cohort , we excluded 2,018 participants with prevalent diabetes , 95 members of ethnic groups other than african americans or whites , 853 participants not returning for any follow - up visit , 26 with no valid diabetes determination at follow - up , 6 with restrictions on stored plasma use , 12 with missing baseline anthropometric data , 2,514 participants in previous aric case - control and case - cohort studies involving cardiovascular disease for whom stored plasma was either previously exhausted or held in reserve , 212 for incomplete fasting ( < 8 h ) , and 316 with missing information for hypertension and inflammation markers . a final sampling frame of 9,740 individuals ( 71% of those in the full cohort without diabetes at baseline ) was established after exclusions . of these , 1,105 participants ( 11.3% of the final sample ) were ascertained to have incident diabetes during follow - up . from the eligible members of this baseline cohort , we selected and measured analytes on ethnicity - stratified ( 50% white , 50% african american ) random samples of both cases of incident diabetes and eligible members of the full cohort ( 1,095 individuals in total ) . some participants within the cohort random sample developed diabetes during follow - up and were part of the case random sample as well . a score was computed to indicate low - grade systemic inflammation , ranging from 0 to 6 ; one point was attributed to a value greater than the median of the cohort random sample for each of the six markers of systemic inflammation : il-6 , crp , orosomucoid , sialic acid , white blood cell ( wbc ) count , and fibrinogen ( 4 ) . glucose was measured at baseline and at follow - up visits by standard hexokinase method . for education , we used a four - level categorical variable : 1 , no high school diploma or less ; 2 , high school or some college ; 3 , beyond bachelors ; and 4 , missing data . cases of incident diabetes were defined on the basis of : 1 ) a reported physician diagnosis , 2 ) reported use of antidiabetic medications during the 2 weeks prior to the visit , or 3 ) a fasting ( 8 h ) glucose value of at least 7.0 the date of diabetes incidence was estimated by linear interpolation using glucose values at the ascertaining visit and the previous visit ( 4,6 ) . circulating levels of il-18 were measured in a centralized laboratory in plasma samples that were continuously stored at 70c prior to analysis . weighted analyses were done based on the case - cohort sampling design to allow for inference to the entire cohort . quartile values were used in the models , and test of trend was generated across the quartiles of il-18 . cox proportional hazards models were fitted to investigate the relationship between il-18 and time to onset of diabetes . wald tests of interaction terms were used to test heterogeneity in the association between race and il-18 quartile . all statistical testing was performed using two - sided tests with p = 0.05 as the cutoff for statistical significance . aric is a population - based study involving both whites and african americans aged 4564 years in four u.s . a stratified , nested case - cohort design was used in an ancillary study investigating inflammatory precursors of diabetes . this design has been used to investigate the role of total and high - molecular - weight ( hmw ) adiponectin , leptin , ferritin , free fatty acids , and other plasma biomarkers in the development of diabetes in the aric study ( 4,6,1517 ) . from the baseline cohort , we excluded 2,018 participants with prevalent diabetes , 95 members of ethnic groups other than african americans or whites , 853 participants not returning for any follow - up visit , 26 with no valid diabetes determination at follow - up , 6 with restrictions on stored plasma use , 12 with missing baseline anthropometric data , 2,514 participants in previous aric case - control and case - cohort studies involving cardiovascular disease for whom stored plasma was either previously exhausted or held in reserve , 212 for incomplete fasting ( < 8 h ) , and 316 with missing information for hypertension and inflammation markers . a final sampling frame of 9,740 individuals ( 71% of those in the full cohort without diabetes at baseline ) was established after exclusions . of these , 1,105 participants ( 11.3% of the final sample ) were ascertained to have incident diabetes during follow - up . from the eligible members of this baseline cohort , we selected and measured analytes on ethnicity - stratified ( 50% white , 50% african american ) random samples of both cases of incident diabetes and eligible members of the full cohort ( 1,095 individuals in total ) . some participants within the cohort random sample developed diabetes during follow - up and were part of the case random sample as well . a score was computed to indicate low - grade systemic inflammation , ranging from 0 to 6 ; one point was attributed to a value greater than the median of the cohort random sample for each of the six markers of systemic inflammation : il-6 , crp , orosomucoid , sialic acid , white blood cell ( wbc ) count , and fibrinogen ( 4 ) . glucose was measured at baseline and at follow - up visits by standard hexokinase method . for education , we used a four - level categorical variable : 1 , no high school diploma or less ; 2 , high school or some college ; 3 , beyond bachelors ; and 4 , missing data . cases of incident diabetes were defined on the basis of : 1 ) a reported physician diagnosis , 2 ) reported use of antidiabetic medications during the 2 weeks prior to the visit , or 3 ) a fasting ( 8 h ) glucose value of at least 7.0 the date of diabetes incidence was estimated by linear interpolation using glucose values at the ascertaining visit and the previous visit ( 4,6 ) . circulating levels of il-18 were measured in a centralized laboratory in plasma samples that were continuously stored at 70c prior to analysis . quartile values were used in the models , and test of trend was generated across the quartiles of il-18 . cox proportional hazards models were fitted to investigate the relationship between il-18 and time to onset of diabetes . wald tests of interaction terms were used to test heterogeneity in the association between race and il-18 quartile . all statistical testing was performed using two - sided tests with p = 0.05 as the cutoff for statistical significance . our case - cohort sample of 1,084 aric study participants included 488 ( 45% ) african american and 596 ( 55% ) white individuals . the cohort random sample comprised 630 individuals , and the incident diabetes random sample included 548 individuals . of the participants selected in the cohort random sample , 94 developed diabetes during follow - up , and 71 of them were included in the incident diabetes random sample . thus , there were 23 incident diabetes cases in addition to those in the incident diabetes sample . among cases , 146 were white men , 142 were white women , 68 were african american men , and 192 were african american women . baseline il-18 levels were significantly higher in white compared with african american participants ( median [ interquartile range ] ; 235.6 [ 129.7 ] vs. 190.6 [ 116.4 ] pg / ml ; p < 0.001 ) . this difference was seen in both men ( p < 0.001 ) and women ( p < 0.001 ) ( table 2 ) . a statistically significant interaction between race and il-18 quartile for incident diabetes was found in the study population after adjusting for age , sex , and race ( p = 0.01 ) . however , after additional adjustment for parental history of diabetes , bmi , sbp , and waist - to - hip ratio ( whr ) , the interaction between race and il-18 quartile lost significance ( p = 0.15 ) . baseline characteristics in participants who developed diabetes and those who did not stratified by race / ethnicity distribution of baseline il-18 levels in the cohort random sample by race and sex hrs for developing diabetes by quartiles of il-18 are shown in supplementary table 2 . a statistically significant hr for developing diabetes was seen for study participants in the fourth ( versus first ) quartile of il-18 levels ( hr : 1.5 ; 95% ci : 1.12.1 ) in the crude analysis . the hrs for fourth versus first quartile remained significant even after adjusting for age , sex , study center , and race ( 1.7 ; 1.22.3 ) . the effects were attenuated , and the hr were no longer statistically significant after sequential additional adjustment for parental history of diabetes , bmi , sbp , and whr ( 1.2 ; 0.81.7 ) ; sport index , education level , and smoking status ( 0.9 ; 0.71.7 ) ; glucose , hdl - c , and tgs ( 0.9 , 95% ci : 0.61.4 ) ; inflammation score ( 1.0 ; 0.71.5 ) ; or total and hmw adiponectin ( 1.1 ; 0.81.6 ) ( supplementary table 2 ) . in a similar subanalysis by race ( table 3 ) , risk for developing diabetes in the fourth ( versus first ) quartile of il-18 levels was statistically significant among whites in the crude analysis ( hr : 2.5 ; 95% ci : 1.64.0 ) . this association remained significant after adjustment for age , sex , and study center ( 2.1 ; 1.33.4 ) . however , statistical significance was lost after additional adjustment for clinical variables ( 1.2 ; 0.72.0 ) and when adjustments were made for biochemical and inflammatory markers and adipocytokines . hrs for developing diabetes by quartiles of il-18 stratified by race correlations between il-18 levels and various baseline demographic , clinical , biochemical , inflammatory , and anti - inflammatory variables markedly varied among the whites and the african americans in the cohort random sample ( supplementary table 3 ) . unlike those in african americans , il-18 levels in whites had a significant correlation with age ( p < 0.01 ) and anthropometric characteristics such as waist circumference ( p < 0.001 ) , height ( p = 0.04 ) , whr ( p < 0.001 ) , and bmi ( p < 0.01 ) . similarly , il-18 levels among whites were significantly correlated with sbp ( p < 0.01 ) , fasting glucose ( p < 0.001 ) , and insulin ( p < 0.001 ) . il-18 levels among whites showed a highly significant correlation with hdl - c ( p < 0.001 ) , ldl cholesterol ( ldl - c ; p < 0.001 ) , and tgs ( p < 0.001 ) and moderate correlation with oxidized ldl ( p = 0.04 ) , but no correlation with nonesterified fatty acids ( p = 0.7 ) . in contrast , il-18 levels among african americans had the strongest correlation with nonesterified fatty acids ( p < 0.001 ) and a weaker correlation with hdl - c ( p = 0.05 ) , oxidized ldl ( p = 0.04 ) , and tgs ( p = 0.02 ) . for inflammatory markers , il-18 had a highly statistically significant correlation with il-6 ( p < 0.001 ) , crp ( p < 0.001 ) , intercellular adhesion molecule1 ( p 0.001 ) among whites and a highly significant correlation only with intercellular adhesion molecule1 ( p < 0.001 ) among african americans . although il-18 levels were significantly correlated with total ( p < 0.001 ) and hmw ( p < 0.001 ) adiponectin among whites , no correlation was seen in african americans . this is the first study to look at the association of il-18 levels with incident diabetes in a racially heterogeneous population . two previous studies have looked at this association in predominantly white populations ( 13,14 ) . in our study , higher il-18 levels showed a significant association with incident diabetes in the entire study cohort and in whites , but not in african americans . upon adjustment for clinical risk factors associated with diabetes , including age , parental history of diabetes , bmi , sbp , and whr , this association almost reached statistical significance . further addition of fasting glucose and inflammation score to the models weakened the independent association of il-18 levels with incident diabetes in our study . the findings of our study support the hypothesis that elevated fasting glucose may itself be a regulator of il-18 levels . it has been shown that the degree of lipid peroxidation is directly proportional to blood glucose concentrations ( 20 ) . nf-b is a transcription factor that plays a major role in mediating inflammatory responses and in the production of proinflammatory cytokines like il-6 , tnf- , and il-18 . the inflammatory cytokines in turn lead to insulin resistance and overt diabetes ( 21 ) , further increasing the plasma glucose levels . this leads to a vicious cascade phenomenon with a positive - feedback loop , causing difficulty in determining whether fasting glucose is upstream or downstream of il-18 in the pathway for onset of diabetes . this hypothesis is further strengthened by our observation that elevated il-18 levels were not associated with incident diabetes independent of inflammatory markers including il-6 , crp , sialic acid , and orosomucoid . in another model in our study , addition of hdl - c and tgs weakened the association of il-18 with incident diabetes . because il-18 is a proinflammatory molecule , further adjustments with lipid parameters associated with inflammation may have resulted in overadjustment and weakening of the independent association of il-18 with incident diabetes . il-18 levels showed a weak association with incident diabetes when adjusted for plasma adiponectin levels in our study . this is consistent with previous studies showing that future risk of diabetes was associated with lower levels of total and hmw adiponectin ( 13 ) . it may imply that though a greater amount of il-18 is produced in tissues outside the adipose tissue ( 23 ) , adiposity may drive the il-18 production in later stages . this is supported by a significant , but weak , correlation of il-18 levels with total and hmw adiponectin levels as well as obesity in the white subgroup of our study . ( 13 ) showed an association of il-18 levels with incident diabetes independent of hmw adiponectin and bmi . the authors , however , did not adjust the models for fasting glucose , inflammatory markers , and lipid parameters , all relevant factors in the occurrence of diabetes . the study also showed that the association of il-18 with diabetes tends to be stronger in women with lower bmi , suggesting that increasing adiposity masks the association of il-18 with diabetes ( 13 ) . our second aim was to evaluate racial differences in il-18 levels and their association with incident diabetes . in addition , il-18 levels were moderately associated with incident diabetes in whites but not at all in african americans . there was a significant interaction between il-18 and race in their associations with incident diabetes . however , this interaction was attenuated on adjustment for parental history of diabetes , bmi , sbp , and whr . ( 4 ) found an association of low - grade inflammation with diabetes in whites but not in african americans in the aric cohort . it is well known that white cell counts differ in african americans compared with whites ( 24 ) . these findings suggest racial differences in inflammatory burden , triggering factors for inflammatory pathways , and the association of inflammation with diabetes . interestingly , in our study , il-18 levels were correlated strongly with anthropometric measures related to obesity in whites but not in african americans . however , the absolute lack of correlation of il-18 with either bmi or whr in african americans in our study is both a novel and interesting finding . moreover , there was no correlation of adiponectin with il-18 levels in african americans , but there was a significant correlation in whites , again raising questions regarding the difference in the source of inflammatory markers and adipocytokines in the two races . similarly , fasting glucose and insulin levels were correlated with il-18 levels in whites but not in african americans , whereas free fatty acids were correlated with il-18 levels in african americans and not in whites . although african americans have long been recognized to have different characteristics of the metabolic syndrome compared with whites ( low tg , high hdl - c , and high glucose ) and increased frequency of incident diabetes , only more recently has there been an appreciation of the differences in inflammation markers associated with cardiometabolic risk . african americans have lower wbc count , il-18 , fetuin - a , cc chemokine ligand5 , and regulated on activation , normal t - cell expressed , and secreted ( rantes ) ( 25 ) than whites , but have higher levels of crp , sialic acid , fibrinogen , c3 , and il-6 than whites in the aric cohort . such differences are also seen in the association of these inflammatory markers with incident diabetes ( 4 ) . one possible explanation for the racial differences in levels of various chemokines and inflammatory markers may be the polymorphism in the duffy antigen receptor for chemokines ( darc ) ( 27 ) . the difference in the wbc count in african americans compared with whites is due to the common african - derived null variant of the darc gene ( 28 ) . evolutionary diversity along with associated social and environmental factors may contribute to the variable association of diabetes with insulin resistance , markers of inflammation , and anthropometric parameters in different races ( 29 ) . however , these novel markers of inflammation and their association with incident diabetes need further validation . communities , suitably designed to study racial differences and provide a long prospective follow - up of incident cases of diabetes ( 15 ) . in this study , we used a single baseline measurement of il-18 . whether obtaining multiple measurements over time would have strengthened the association seen in the study elevated il-18 levels are associated with incident diabetes mellitus ( dm ) , but there is a racial heterogeneity in this association . further studies are needed to elucidate the details of this mechanism of dm and the potential interplay of racial differences , anthropometric characteristics , and inflammation . in this study , we used a single baseline measurement of il-18 . whether obtaining multiple measurements over time would have strengthened the association seen in the study elevated il-18 levels are associated with incident diabetes mellitus ( dm ) , but there is a racial heterogeneity in this association . further studies are needed to elucidate the details of this mechanism of dm and the potential interplay of racial differences , anthropometric characteristics , and inflammation .

over the past few years , semiconductor nanowires ( nws ) have become a large research field due to their interesting electrical and optical properties due to quantum effects , large surface to volume ratio , and capability for bottom - up assembly . nw - based memory devices , lasers , single - molecule sensors , and solar cells have been demonstrated in recent years . additionally , one outstanding feature of the nw growth is that the crystal structure can be different from the stable bulk crystal structure , with possible striking effects on their optical properties . the stable bulk crystal structure for most iii v binary semiconductor materials is cubic zincblende ( zb ) , while synthesis of the hexagonal wurtzite ( wz ) structure in bulk gaas requires high pressure and temperature . however , nws can adopt both zb and wz crystal structures , and previous research has demonstrated the control of wz and zb crystal structure in gaas nws grown by metal organic chemical vapor deposition ( mocvd ) . v nws is [ 111 ] with a stacking sequence of abcabc , which could also be denoted as 3c using the ramsdell notation . the equivalent growth direction in the hexagonal case is [ 000.1 ] , with the stacking sequence ababab for wz , or 2h in the ramsdell notation . 3c ( zb ) and 2h ( wz ) thus differ only in the stacking sequence in the [ 111]/[000.1 ] direction and exhibit a small difference in cohesive energies , with 3c being the highest , hence the stable phase in bulk . however , with the large surface to volume ratio of nws , compared to bulk , the relative contribution of lateral surface energies to the total free energy of formed nws has to be considered and is a key parameter in growth models attempting to explain formation of polytypes in iii nws with a structure different from their bulk counterpart should be treated as metastable , and the growth is kinetically controlled . treating nws as metastable implies that their phase could change to a more stable one upon perturbation . in addition , if it is the large surface to volume ratio that enables growth of metastable nws , will the structure be maintained under large radial overgrowth , where the surface to volume ratio decreases ? phase transformation of grown gaas nws has been observed previously , where 2h structure epitaxially buried in planar 3c overgrowth gradually adopted the structure of the burying layer . phase transformation has also been observed in ingaas nws , using an electron beam to deliver a high energy dose to the structure , enough to transform 2h to a mixed 3c/2h structure . in the case of epitaxial burying , one layer at a time of the metastable 2h is gradually surrounded with a layer of the stable 3c structure . with a lateral mismatch of the top layer burying the nw , a linear defect is created at the interface between the burying layer and nw , which could be viewed as a shockley partial dislocation in the 3c matrix . once the dislocation has formed along at least two sides of the nw , it could propagate into the nw without changing its length . as the surrounding 3c layer has a higher cohesive energy , the total energy associated with the linear defect is decreased as the dislocation propagates through the nw and changes its structure to 3c . by completely burying the structure in a 3c matrix , the low energy surfaces of 2h can not stabilize the structure any longer , and the structure is forced into 3c . to further develop the model to apply to free - standing structures , contributions from the surface energies in addition , the dislocation line between a buried structure and its surrounding matrix compared to the dislocation line formed between free - standing structures has different geometries , which could have a big impact on the propagation of the dislocation line . in this work , we investigated what happens when adjacent wz nws radially merge with each other , for different amounts of radial overgrowth and merging . the approach resembles the epitaxial lateral overgrowth ( elo ) technique , which has been used since the 1960s to reduce threading dislocation densities in growth of lattice - mismatched thin films . elo is widely used to grow high quality gan films , but is not as common for non - nitride iii - vs . using a mocvd system , we first grew gaas 2h nws on 3c gaas substrate , and by radial overgrowth in a second step increased the nw diameter . during radial growth , the shell will adopt the same crystal structure as the core , which has been observed in several nanowire core shell material combinations . with increasing radial overgrowth time , more and more of the initial 2h nws radially merge with each other . for the longest growth times , the initial gaas 2h nws have merged to form a nearly continuous film . the crystal structure of samples with different amounts of radial overgrowth and merging was investigated . we used high resolution xrd and high resolution transmission electron microscopy ( hrtem ) for structural investigations . although one might expect to find a 2h film after the radial overgrowth , our observations show that this is not the case . once individual nws merge together , the crystal structure of the merged nws is transformed progressively from the initial pure 2h structure to a mixed 2h/3c structure . we use a rather simple combinatorial approach to explain the main features in the resulting crystal structure of radially merged nws . comparing energy contributions from differences in cohesive energy , surface energies and dislocation line energy , only a small energy difference for transforming 2h compared to 3c is found , which justifies the use of a combinatorial model . gaas 2h nws were grown on gaas ( 111)b substrates using mocvd and 70 nm size - selected au seed particles with a surface density of 1 m with 2h growth parameters as reported previously . a pregrowth annealing step was performed in a 100 mbar mocvd reactor under hydrogen ( h2 ) and arsine ( ash3 ) for 10 min at 650 c to remove the surface oxides and form catalyst droplets . after annealing , the temperature was reduced to the growth temperature of 550 c , and tmga was introduced to initiate the growth . the molar fractions of tmga and ash3 flow were 1.46 10 and 5.5 10 , respectively ( v / iii ratio of 4 ) , with a total gas flow of 6 slm . after 2h nw growth , the samples were cooled down under h2 only to prevent formation of a 3c neck region . after cooldown , the samples were removed from the mocvd system in order to remove the au - alloy seed particles by etching in a cyanide based etching solution ( tfac from transene ) . removing the au seed particles is essential to limit axial elongation of the nws during subsequent radial overgrowth . for this step , the samples were reintroduced into the mocvd system and annealed under a h2/ash3 environment at 650 c for 10 min to remove surface oxides formed as the nws were exposed to air . the temperature was then reduced to 630 c , and tmga was introduced to initiate radial overgrowth . the growth time ranged from 5 to 240 min with molar fractions of tmga and ash3 of 1.5 10 and 2.2 10 , respectively ( v / iii ratio 150 ) . ( a ) growth of wz gaas nws , ( b ) removal of au particles , ( c ) radial overgrowth of gaas nws , ( d ) merging of nws in order to form the designed structure . for structural investigations of large ensembles of nws and parasitic substrate growth , we performed high - resolution x - ray diffraction ( xrd ) measurements on as - grown samples . two laboratory diffractometers with cu anode and channel - cut monochromators were used together with one - dimensional detectors to record reciprocal space maps ( rsms ) . the measurements were performed in the scattering plane spanned by the [ 111 ] and [ 112 ] directions of the substrate . symmetric rsms around the 3c gaas ( 333 ) reciprocal lattice point and 2h gaas ( 000.6 ) as well as asymmetric rsms between 3c gaas substrate ( 224 ) bragg peak and ( 331 ) twinned gaas bragg peak were recorded , which also covers the 2h gaas ( 101.5 ) . the occurrence of the 3c ( 331 ) from twinned nws in the same azimuth as the 3c substrate ( 224 ) is due to the 180 rotation around [ 111 ] caused by twin defects . to characterize the local crystal structure and the quality at the interface between merged nws , we used focused ion beam ( fib ) to prepare cross sectional lamellae samples for transmission electron microscopy ( tem ) . using fib could potentially induce defects , but our method is gentle enough to preserve high crystalline quality of the prepared sample . high resolution tem ( hrtem ) , dark field tem ( dftem ) , scanning tem ( stem ) , and selected area electron diffraction ( saed ) were used for analysis . the grown nws have an average length of 2 m and average diameter of 100 nm prior to radial overgrowth , with 2h structure including low densities of stacking defects in the range of < 1020 m , as revealed by hrtem ; see figure 2a c . after the etching process , most of the au catalyst particles on the tops of the nws were removed , but the nws were insignificantly affected by the etchant ( see detail in supporting information , figure s1 ) . ( a c ) tem and hrtem images of a 2h structured nw before overgrowth imaged in the 112.0 zone axis . ( d i ) sem images of nws with radial overgrowth for different times . with 30 tilting angle : ( d ) 5 min ; ( e ) 20 min ; ( f ) 60 min ; top view sem images of nws radial overgrowth with different time : ( g ) 120 min ; ( h ) 180 min ; ( i ) 240 min . the insets shown in ( g ) and ( h ) are sem images taken at a 30 tilting angle , scale bars are 2000 nm . figure 2d i shows the nw overgrowth at different stages ranging from 5 to 240 min overgrowth time . after the first 5 min , we observed mainly an increase in the volume of the pyramidal base of the nws ; see figure 2d . the 2h nws are terminated by low energy { 101.0 } facets , which gives a low possibility of nucleation directly at the side facets . the pyramidal base consists of higher index facets , which have a higher surface energy due to the increased density of dangling bonds , resulting in an increased rate of nucleation of new layers . after 20 min ( see figure 2e ) , the base starts to form { 101.0 } facets , and the morphology evolution of the nws changes with radial overgrowth time of 60 min ( compare figure 2e , f ) . at the kink site between the base and the nw side facets , the higher coordination number is expected to have an even higher rate of nucleation . nucleation at the kink site followed by growth of layers along the nw [ 000.1 ] direction could explain the homogeneous shell at 60 min with steps at the top of the nw . as the growth time is increased further , the area between the nws becomes smaller , and the nws eventually merge . after 240 min of growth , most of the nws have merged with each other , and a nearly continuous film is formed . in parallel with the radial growth of the nws , we observe continuous growth on the substrate surface . as the parasitic substrate growth proceeds , some of the lower parts of the initial 2h nws are buried , which explains the apparently thinner base in figure 2f compared to 2d and 2e . however , most parts of the nws are radially merged before being buried in parasitic substrate growth , as seen in the insets in figure 2g , h . average nw length and diameter and parasitic substrate growth thickness are plotted in supporting information , figure s3 . the nws never become fully buried in the parasitic substrate growth , as the openings between the nws become too small to allow adatoms to reach the bottom of the pits between them . instead , growth on the nw top facets increases with decreased openings between the nws . using high resolution xrd to characterize the crystal structure of the samples , the measurements collect diffraction data from several mm area of the sample , and signals from the substrate and grown structures can not be separated directly . however , as the 2h structure only originates from the nws , the nw signal can be distinguished from the substrate . as 3c occurs in two different twin directions , two sets of 3c diffraction peaks occur ; one originates from the substrate and possible 3c formed during the growth in the same twin direction , and the other set solely from 3c formed during the growth with the other twin direction . figure 3 displays rsms from the sample with 120 min radial overgrowth time . in the symmetric rsm , the 3c gaas ( 333 ) peak at q[111 ] = 5.777 and 2h gaas ( 000.6 ) at q[111 ] both the substrate and twinned 3c contribute to the signal of the 3c ( 333 ) bragg peak and can not be distinguished . between the 2h and 3c peaks , a weaker signal is visible at q[111 ] = 5.758 , which originates from gaas 4h . x - ray diffraction reciprocal space maps ( rsms ) of the sample with 120 min radial overgrowth time . panel ( a ) shows a symmetric rsm around the 3c ( 333 ) and 2h ( 000.6 ) bragg peaks , ( b ) shows asymmetric rsm including zb substrate ( 224 ) , ( 331 ) twin 3c , 2h ( 101.5 ) , and 4h ( 101.9 ) , ( 101.10 ) , and ( 101.11 ) bragg reflections . in ( c ) and ( d ) , details of the twin 3c and 2h peaks are shown . with roman numerals , the positions of possible detector streak ( i , white line ) , broadening due to tilt distribution ( ii , red line ) and finite nw radius ( iii , black line ) are marked . linecuts from the rsms are shown in ( e ) and ( f ) for the symmetric and asymmetric case , respectively . linecuts from rsms recorded in both ( 331 ) and ( 224 ) azimuths are overlaid in ( e ) and ( f ) . in the asymmetric rsms in figure 3b , the peaks are attributed to the 3c ( 224 ) bragg peak , which originates from the substrate and grown 3c material with same twin orientation as the substrate as the peak with highest intensity and the 3c ( 331 ) bragg peak from 3c grown with opposite twin - orientation as the second strongest peak . in between the two 3c peaks , the 2h ( 101.5 ) is visible next to a weak 4h gaas ( 101.10 ) peak . on either side of the 3c peaks , two more 4h peaks are visible , 4h gaas ( 101.9 ) and 4h gaas ( 101.11 ) . for asymmetric rsms for all six growth shell times figure 3c , d shows details in the rsm around the 2h ( 101.5 ) and 3c ( 331 ) bragg peaks . the position of possible detector streak is marked with white ( i ) , broadening due to tilt distribution with red ( ii ) and broadening due to finite radius of the nanowires as black ( iii ) . in figure 3e , f , line cuts from the symmetric and asymmetric rsms for both ( 331 ) and ( 224 ) are shown . using the bragg peak integrated intensity , we estimate the change in scattering volume . first , line cuts along [ 111 ] are made in the asymmetric rsms ; see figure 4a . to each peak , a voigt shape curve was fitted after removal of background signal , and the curves integrated intensity was normalized with respect to the substrate peak intensity . as a consistency check , peaks from both azimuths on each sample were used . using different azimuths , the difference in structure factors for ( 224 ) and ( 331 ) as well as the difference in illuminated area the peak intensities for the 2h , 4h ( 101.10 ) and twinned 3c peaks are plotted . as seen , the 2h initially increases , and the largest 2h volume was found for the sample with 60 min radial overgrowth time . for the twinned 3c , the volume increases for the full radial overgrowth series . for the 4h ( 101.10 ) peak intensity , the scattering intensity follows a similar trend as for 2h , but with the strongest scattering signal at 120 min radial overgrowth time . ( a ) linecuts along q_[111 ] from asymmetric rsm with substrate peak at ( 224 ) plotted with offsets . the position for different bragg peaks decreasing intensities for the 4h and 2h peaks are visible for the longer radial overgrowth times . ( b ) integrated intensities for the 2h ( red , square ) , 4h ( triangles , green ) , and the 3c ( blue , circles ) peaks arising from the twinned zb . the trend lines are guides for the eyes only . by preparing a cross sectional lamella sample by fib , the crystal structure along the nws and the parasitic substrate growth were investigated after 120 min radial growth . figure 5a , b shows merged sections in bright - field ( bf ) and dark - field ( df ) tem images , respectively . the dark field is taken around ( 111 ) and ( 002 ) reflection for the two different 3c twin directions , and ( 000.1 ) for the 2h . the top part includes two free - standing nws , which have pure 2h crystal structure with no visible stacking faults . beneath this section , the merged region includes two crystal orientations with twin boundaries perpendicular to the nw growth direction , and the barcode - like pattern of twin regions span the majority of the formerly individual nanowires . the parasitic substrate growth results in 3c crystal structure which is heavily twinned including boundaries not perpendicular to the nw growth direction ( diffraction pattern and additional tem image in supporting information , figure s5 ) . the crystal structure of the merged region is confirmed by the saed pattern shown in the inset of figure 5b . the saed pattern indicates two dominant 3c crystals twinned in the nw growth direction only . ( a ) bftem images from an fib lamella with two partly merged nanowires . ( b ) dftem image of the same region as in ( a ) highlighting the crystal structure in different parts marked with different colors . the free - standing nanowires have 2h crystal structure with no visible stacking faults , while the merged regions have 3c crystal structure . the substrate growth has 3c crystal structure which is heavily twinned including boundaries not perpendicular to the growth direction . the inset in ( b ) shows saed from the merged region , which indicates two dominant 3c crystals twinned in the growth direction only . figure 6 shows the tem image of a fib lamella with two merged nws after 120 min radial overgrowth in which a large section of 4h was found . the corresponding saed patterns of the 2h and 4h phases are shown in figure 6b , c , respectively . the independent gaas nw segment before the merging exhibits a pure 2h crystal structure with almost no stacking faults , which is clear from the saed pattern in figure 6b . the appearance of a ( 000.1 ) reflection indicated by a small arrow in figure 6c and two times shorter distance between ( 000.0 ) and ( 000.1 ) reflection compared to saed pattern of 2h in figure 6b confirms the existence of 4h crystal structure . the hrtem image shown in figure 6d and the fft shown in the inset the length of the 4h segment is approximately 200 nm , which is consistent with the observation in xrd ( see below ) . ( b ) saed from the region in the blue circle in ( a ) showing 2h structure , and ( c ) saed from the region in the green circle in ( a ) showing the 4h crystal structure . ( d ) hrtem image from the region marked with rectangular in ( a ) showing a band of 4h crystal structure merging the two nanowires . the inset in ( d ) shows a fft from the left nanowire , which is a typical 4h diffractogram . xrd performed on nanostructures is affected by finite size effects , which in the case of nws are usually dominated by their small diameter , causing a broadening in the [ 112 ] direction . nws also tend to not grow exactly perpendicular to the substrate surface , but a small mosaicity due to a random nw tilt distribution causes a broadening in the same direction as the finite width in the symmetric rsms . with both finite size and tilt contributing to the same broadening in symmetric rsms , it is not possible to distinguish one from the other . since the effects scale differently for different reflections as well as show up in slightly different directions in asymmetric rsms , a distinction of the two effects is possible in the asymmetric rsms . the finite size contribution is always inversely proportional to the width of the nws , while the broadening due to tilt distribution increases linearly with q. in the asymmetric scans , the tilt distribution and finite width broadens the peak in different directions , which is indicated in figure 3c , d . for the 120 min radial overgrowth time shown in figure 3 , the broadening due to tilt distribution is larger than that due to finite nw width . the tilt distribution found is on the order of 0.1 deg , which is on the same order of magnitude as for other nw samples , such inas 2h and insb 2h on inas 3c and gap tilt distribution is a general feature in nw samples and is a limiting factor when performing high resolution xrd on such samples . another finite size effect present in the asymmetric rsms is the broadening of the diffraction peaks in the [ 111 ] direction , caused by short segments of the respective polytype in the nw growth direction . the inverse width of the diffraction peaks is proportional to the average segment length and for the 4h peaks segment lengths of 100200 nm were estimated . worth noting is that no 4h was found in the reference nws prior to radial overgrowth . for the 3c and 2h peaks , the widths are limited by the instrumental resolution and length estimations are therefore not possible . with a very high crystal quality as seen in both the hrtem analysis and xrd , we are able to accurately calculate the lattice parameters of both gaas 2h and 4h using the asymmetric rsms . the accuracy of the measurements is similar to the case of inp 2h , limited mainly by experimental setup and beam broadening due to measurement geometries . table 1 summarizes the calculated lattice parameters with relative differences with respect to ideal tetrahedral structures , using geometrically converted cubic 3c lattice parameters . both the 2h and 4h unit cells are elongated in the nw growth direction and contracted in the radial direction compared to 3c . from the sem images shown in figure 2 , a continuous increase in 2h volume would be expected for the full radial overgrowth time series , since the radii of the nws continuously increase with time . however , from the xrd data , a decrease in 2h is found for radial overgrowth times longer than 60 min . intuitively , one could argue that the decrease in 2h is due to epitaxial burying in a 3c substrate layer , similar to what has been observed in refs ( 18 and 19 ) , which would also be supported by the continuous increase in 3c diffraction signal with increasing growth time . however , comparing the volume of not - yet - buried nws for 5 and 180 min radial overgrowth , the volume is 2 orders of magnitude larger for the longer growth time , yet the 2h diffraction signal is weaker . hence , the burying of the nws is not sufficient to explain the drastic decrease in the 2h signal . in addition , epitaxial burying of the 2h nws in a 3c matrix would not explain the appearance of 4h segments , estimated to be 100200 nm long in the samples with the most 4h , as an epitaxial burying driven phase transformation would force the structure to adopt the burying matrix structure , in this case 3c . interestingly , the largest increase in 4h scattering intensity is found as the 2h scattering signal starts to decrease . the decreasing 2h signal coincides with the onset of the radial merging of adjacent nws , as seen in the inset in figure 2g , h . as reported by ng et al . , a high energy electron beam could potentially induce crystal phase transformation , which could affect the results obtained by tem . however , the agreement of the xrd and tem results suggests that the crystal phase transformation is independent of measurement technique . in addition , reference nws and the parts not yet merged showed only pure 2h structure under similar tem analysis ; therefore , the possibility of electron beam induced transformation is discarded . instead of explaining the phase transformation by epitaxial burying , we propose that the transformation could be explained by a nw nw interaction . to understand the process of the merging better we come back to the stacking sequences of the different crystallographic phases . the stacking along the hexagonal [ 000.1 ] direction of the 2h crystal structure can be described as ababab , while for 3c crystal structure it is described as abcabc along the equivalent [ 111 ] direction . each capital letter denotes a pair of layers consisting of one group v and group iii atom , a bilayer , at three distinct lateral positions . as the nws grow in [ 111]/[000.1 ] , the position of as in each new bilayer will determine whether the layer is in cubic or hexagonal position . the stacking sequences of 2h can also equivalently be described as bcbcbc , acacac , etc . ( nw 1 ) retains the stacking order as ababab and the second 2h structured nw ( nw 2 ) retains the stacking order as bababa. when the bottom bilayer of nw 1 reaches the periphery of nw 2 , the as atoms in the bottom bilayer of nw 1 is not in lateral registry with that of nw 2 . in principle now two scenarios compete with each other . either this mismatch is kept at the cost of having a line defect at the interface of the merging nws , or in order to release this mismatch , the line defect may propagate through one of the nws to accommodate the corresponding as plane . for such a gliding process , three different translation vectors ( the burger vectors , b ) are possible , 1/3[101.0 ] , 1/3[110.0 ] , and 1/3[011.0 ] ; see figure 7 . this edge dislocation could be seen as a shockley partial dislocation , and when propagating through the crystal changes the configuration of the as from hexagonal to cubic position ( or vice versa ) . ( a ) schematic of two miss - matched 2h nws viewed in the { 000.1}/{111 } direction . the mismatch causes a translation in the [ 110.0 ] direction of the as atoms in nw 1 closest to the interface . ( b d ) schematics displaying propagation of a defect through one of the nanowires . changing a 2h gaas to 3c gaas would to a first approximation result in a lower total energy for the crystal , as the cohesive energy is 24 mev / iii v pair higher for 3c . changing from 2h { 101.0}-type facets to 3c { 112}-type facets increases the surface energy due to the increase of dangling bonds , with a net energy difference of 3.06 mev / nm . in addition to the change in cohesive energy and surface energies , the dislocation line itself has an energy per unit length . the dislocation line energy originates from the strain field around it and the distorted bonds at the dislocation core . detailed calculations on defects in gaas 2h are lacking , but as an approximation the dislocation line energy could be calculated as edis = gb , where g is the shear modulus and b is the magnitude of the burgers vector . using the shear modulus of gaas 3c , as the shear modulus of 2h is unknown , the dislocation line energy is estimated to be 10.74 ev / nm . the 2h nws are hexagonal and have { 101.0 } type facets , which are perpendicular to the burgers vector , and the nws all have facets parallel to each other . for two nws merging along the full length of a side facet , as in figure 7 , the dislocation line has initially the same length as the side facet of the nws . for the dislocation line to propagate into one of the two nws by only changing the position of one as , it has to increase its length ; see figure 7b d . the minimum increase in length would then be b / cos(30 ) , if the propagation occurs at a nw facet . in total similarly , if the dislocation propagates into a 3c structure , the total energy increases by 2.88 ev for the first as moved . the difference between the two values is less than thermal energy at the present growth temperature . with the dislocation line energy as the dominant factor for the energies related to the phase transformation , the initial structures of the two merging nws are of minor importance the probability of either one to transform is roughly equal . comparing our phase transformation with the case of epitaxial burying described by patriarche et al . , the dislocation is of the same type , and both transformations are triggered by the interaction between two crystals with as atoms at different lateral positions . however , in the case of epitaxial burying where the burying matrix completely surrounds the nw , the nw has to adopt the structure of the burying matrix or keep the dislocation at the periphery of the nw . to let the dislocation propagate through the nw makes the dislocation shorter , and eventually disappear , which is the thermodynamically most favorable case , and the final structure will be easy to predict the one of the burying matrix . in our case , with a nw nw interaction , the dislocation could propagate into either crystal , and the final structure is much harder to predict . as the dislocation line energy depends on the shear modulus of the crystal and the burgers vector , changing growth conditions would not change the dynamics of the crystal phase transformation . changing the growth temperature could however change the propagation rate of the dislocation , as the peierls energy barrier sets an activation energy for the propagation . hypothetically , by reducing growth temperature enough , propagation of the defect could be kinetically hindered such that it stays at the interface between the nws , which then would retain their initial structure . however , since growth temperature in this study is high enough to ensure reasonable growth rates , we assume that the defect propagation is not kinetically hindered . assuming equal probability for the dislocation to propagate in either nw , a combinatorial approach gives the probabilities of different structures . assuming that a dislocation can propagate with the same probability into either nw as two nws merge , each merging bilayer gives rise to two equally likely outcomes . for merging of two nws with 10 bilayers each there will be 2 possible outcomes . if we involve a third 10 bilayer nw in the merging process , there will be ( 2 ) outcomes . to estimate the amount of 3c , 2h , and 4h in each outcome , we define a 3c segment to be of at least six bilayers ( two unit cells ) , 2h as six bilayers ( three unit cells ) and 4h as eight bilayers ( two unit cells ) . if the 10 bilayer long segment does not include any of the above segments , it is considered to be a mixed structure . using a simple matlab script , we simulate the merging of two , three , and four 2h nws with each 10 bilayers ( where the 10 bilayer limit is chosen to limit computation time ) . a schematic view on the merging process the simulations shows that the merging of just two nws gives a mixed fraction of around 80%that is , most of the possible outcomes have only segments consisting of less than six bilayers of any considered polytype . 2h is still the most common polytype , with 1516% , depending on the initial stacking sequences of the nws and how many nws merge . both 3c and 4h show up with lower amounts ( 36% and 0.30.5% , respectively ; see supporting information , figure s7 ) . this result fits well with our experimental observation : the most likely structure for radially merged 2h nws is a mixture between 2h and 3c . the increase in twinned 3c seen in the xrd measurements comes not only from the nws , but also from the parasitic substrate growth , and the 36% of 3c found in the nws in our combinatorial model is not the major contribution to the signal . for a structure with very short segments of 2h and 3c sandwiched , streaking between the xrd peaks is expected , and this is also what we observe in our rsms ; see figure 3b and supporting information figure s4 . using this simple combinatorial approach , we are able to describe the main features of the merging process . the lattice parameter for 2h gaas has been reported previously , both using xrd and electron diffraction on nws and on 2h in gaas powder obtained at high pressure . however , the 2h lattice parameters in the former report were measured from nws with a mixed crystal structure , consisting of 2h segments sandwiched between 3c , and most likely strained by the 3c segments . for our structures , the very few sfs in the pure 2h before any merging process are not likely to influence the overall 2h structure , and the 2h crystal is fully relaxed . using the sample with 60 min radial overgrowth , we measured the 2h and 4h unit cell parameters on the sample with the largest volume of pure 2h . the results are summarized in table 1 . with a larger lattice plane spacing along the c - axis in 2h as compared to the 3c counterpart and a smaller in - plane distance , the c / a ratio is measured to be 1.649 , which is 0.98% higher than the ideal 1.633 for hexagonal close - packed structures with perfect tetrahedral coordination . the value found is in good agreement to theoretical values of 1.6461.651 . similarly , we found a c / a ratio of 3.282 for 4h , which is 0.49% higher than ideal . the measured gaas 4h lattice parameters reported here are to the authors best knowledge the first experimental report on gaas 4h lattice parameters . the higher c / a ratio compared to the ideal is typical for metastable hexagonal structures and gives bonding slightly distorted from an ideal tetrahedron . for 4h , the deviation from the ideal close - packing is smaller than for 2h , suggesting 4h to be the more stable structure , but still with 3c as the most stable one . hence , the cohesive energy for 2h should be lower than for 3c , with 4h in between the two . the difference in cohesive energy gives a higher nucleation barrier for 2h compared to 4h or 3c in gaas , which should result in a higher probability of 4h than 2h during growth . however , the surface energies of the nucleus and its surroundings play a crucial role in determining the structure formed during nw growth . v nws is the assumption that the surface energies of 2h are considerably lower than those of 3c , which overcomes the difference in cohesive energy . assuming surface energies of 2h to be considerably lower than those of 4h as well explains why observations of 4h grown in nws are very few , and mostly only occur in a transition region between 2h and 3c . in this work , we studied the crystal structure of radially overgrown gaas wurtzite ( 2h ) nws , especially the resulting crystal structure as nws progressively grow into each other and merge . we characterized the grown structure by high resolution xrd and tem with diffraction analysis . we observed that the free - standing gaas nws retain the pure 2h structure until they merge with each other , at which point the merged segments transform progressively from pure 2h structure to a mixture between zinc blende ( 3c ) structure with twins and 2h . we also found sections with the 4h crystal structure in the merged nws and determined lattice parameters of both 2h and 4h gaas . the phase transformation continues as long as the merging process proceeds , driven by a dislocation formed between adjacent nws due to lateral mismatch of as in the ( 111)/(000.1 ) plane . using a combinatorial approach , we estimated the probability of the resulting structure after radial merging of 2h nws , which shows that a heavily mixed structure is the most probably outcome , in agreement with our xrd and tem measurements .

iii v nanowires ( nws ) grown with metal organic chemical vapor deposition commonly show a polytypic crystal structure , allowing growth of structures not found in the bulk counterpart . in this paper we studied the radial overgrowth of pure wurtzite ( wz ) gaas nanowires and characterized the samples with high resolution x - ray diffraction ( xrd ) to reveal the crystal structure of the grown material . in particular , we investigated what happens when adjacent wz nws radially merge with each other by analyzing the evolution of xrd peaks for different amounts of radial overgrowth and merging . by preparing cross - sectional lamella samples we also analyzed the local crystal structure of partly merged nws by transmission electron microscopy . once individual nws start to merge , the crystal structure of the merged segments is transformed progressively from initial pure wz to a mixed wz / zb structure . the merging process is then modeled using a simple combinatorial approach , which predicts that merging of two or more wz nws will result in a mixed crystal structure containing wz , zb , and 4h . the existence large and relaxed segments of 4h structure within the merged nws was confirmed by xrd , allowing us to accurately determine the lattice parameters of gaas 4h . we compare the measured wz and 4h unit cells with an ideal tetrahedron and find that both the polytypes are elongated in the c - axis and compressed in the a - axis compared to the geometrically converted cubic zb unit cell .

Introduction Experimental Details Results Discussion Conclusion

over the past few years , semiconductor nanowires ( nws ) have become a large research field due to their interesting electrical and optical properties due to quantum effects , large surface to volume ratio , and capability for bottom - up assembly . additionally , one outstanding feature of the nw growth is that the crystal structure can be different from the stable bulk crystal structure , with possible striking effects on their optical properties . the stable bulk crystal structure for most iii v binary semiconductor materials is cubic zincblende ( zb ) , while synthesis of the hexagonal wurtzite ( wz ) structure in bulk gaas requires high pressure and temperature . however , nws can adopt both zb and wz crystal structures , and previous research has demonstrated the control of wz and zb crystal structure in gaas nws grown by metal organic chemical vapor deposition ( mocvd ) . 3c ( zb ) and 2h ( wz ) thus differ only in the stacking sequence in the [ 111]/[000.1 ] direction and exhibit a small difference in cohesive energies , with 3c being the highest , hence the stable phase in bulk . however , with the large surface to volume ratio of nws , compared to bulk , the relative contribution of lateral surface energies to the total free energy of formed nws has to be considered and is a key parameter in growth models attempting to explain formation of polytypes in iii nws with a structure different from their bulk counterpart should be treated as metastable , and the growth is kinetically controlled . phase transformation has also been observed in ingaas nws , using an electron beam to deliver a high energy dose to the structure , enough to transform 2h to a mixed 3c/2h structure . with a lateral mismatch of the top layer burying the nw , a linear defect is created at the interface between the burying layer and nw , which could be viewed as a shockley partial dislocation in the 3c matrix . by completely burying the structure in a 3c matrix , the low energy surfaces of 2h can not stabilize the structure any longer , and the structure is forced into 3c . to further develop the model to apply to free - standing structures , contributions from the surface energies in addition , the dislocation line between a buried structure and its surrounding matrix compared to the dislocation line formed between free - standing structures has different geometries , which could have a big impact on the propagation of the dislocation line . in this work , we investigated what happens when adjacent wz nws radially merge with each other , for different amounts of radial overgrowth and merging . using a mocvd system , we first grew gaas 2h nws on 3c gaas substrate , and by radial overgrowth in a second step increased the nw diameter . during radial growth , the shell will adopt the same crystal structure as the core , which has been observed in several nanowire core shell material combinations . with increasing radial overgrowth time , more and more of the initial 2h nws radially merge with each other . the crystal structure of samples with different amounts of radial overgrowth and merging was investigated . we used high resolution xrd and high resolution transmission electron microscopy ( hrtem ) for structural investigations . although one might expect to find a 2h film after the radial overgrowth , our observations show that this is not the case . once individual nws merge together , the crystal structure of the merged nws is transformed progressively from the initial pure 2h structure to a mixed 2h/3c structure . we use a rather simple combinatorial approach to explain the main features in the resulting crystal structure of radially merged nws . after annealing , the temperature was reduced to the growth temperature of 550 c , and tmga was introduced to initiate the growth . after cooldown , the samples were removed from the mocvd system in order to remove the au - alloy seed particles by etching in a cyanide based etching solution ( tfac from transene ) . the temperature was then reduced to 630 c , and tmga was introduced to initiate radial overgrowth . ( a ) growth of wz gaas nws , ( b ) removal of au particles , ( c ) radial overgrowth of gaas nws , ( d ) merging of nws in order to form the designed structure . for structural investigations of large ensembles of nws and parasitic substrate growth , we performed high - resolution x - ray diffraction ( xrd ) measurements on as - grown samples . the occurrence of the 3c ( 331 ) from twinned nws in the same azimuth as the 3c substrate ( 224 ) is due to the 180 rotation around [ 111 ] caused by twin defects . to characterize the local crystal structure and the quality at the interface between merged nws , we used focused ion beam ( fib ) to prepare cross sectional lamellae samples for transmission electron microscopy ( tem ) . high resolution tem ( hrtem ) , dark field tem ( dftem ) , scanning tem ( stem ) , and selected area electron diffraction ( saed ) were used for analysis . the grown nws have an average length of 2 m and average diameter of 100 nm prior to radial overgrowth , with 2h structure including low densities of stacking defects in the range of < 1020 m , as revealed by hrtem ; see figure 2a c . after the first 5 min , we observed mainly an increase in the volume of the pyramidal base of the nws ; see figure 2d . after 20 min ( see figure 2e ) , the base starts to form { 101.0 } facets , and the morphology evolution of the nws changes with radial overgrowth time of 60 min ( compare figure 2e , f ) . after 240 min of growth , most of the nws have merged with each other , and a nearly continuous film is formed . in parallel with the radial growth of the nws , we observe continuous growth on the substrate surface . as the parasitic substrate growth proceeds , some of the lower parts of the initial 2h nws are buried , which explains the apparently thinner base in figure 2f compared to 2d and 2e . using high resolution xrd to characterize the crystal structure of the samples , the measurements collect diffraction data from several mm area of the sample , and signals from the substrate and grown structures can not be separated directly . in the symmetric rsm , the 3c gaas ( 333 ) peak at q[111 ] = 5.777 and 2h gaas ( 000.6 ) at q[111 ] both the substrate and twinned 3c contribute to the signal of the 3c ( 333 ) bragg peak and can not be distinguished . x - ray diffraction reciprocal space maps ( rsms ) of the sample with 120 min radial overgrowth time . in the asymmetric rsms in figure 3b , the peaks are attributed to the 3c ( 224 ) bragg peak , which originates from the substrate and grown 3c material with same twin orientation as the substrate as the peak with highest intensity and the 3c ( 331 ) bragg peak from 3c grown with opposite twin - orientation as the second strongest peak . as seen , the 2h initially increases , and the largest 2h volume was found for the sample with 60 min radial overgrowth time . by preparing a cross sectional lamella sample by fib , the crystal structure along the nws and the parasitic substrate growth were investigated after 120 min radial growth . beneath this section , the merged region includes two crystal orientations with twin boundaries perpendicular to the nw growth direction , and the barcode - like pattern of twin regions span the majority of the formerly individual nanowires . the crystal structure of the merged region is confirmed by the saed pattern shown in the inset of figure 5b . ( b ) dftem image of the same region as in ( a ) highlighting the crystal structure in different parts marked with different colors . the free - standing nanowires have 2h crystal structure with no visible stacking faults , while the merged regions have 3c crystal structure . the inset in ( b ) shows saed from the merged region , which indicates two dominant 3c crystals twinned in the growth direction only . figure 6 shows the tem image of a fib lamella with two merged nws after 120 min radial overgrowth in which a large section of 4h was found . the independent gaas nw segment before the merging exhibits a pure 2h crystal structure with almost no stacking faults , which is clear from the saed pattern in figure 6b . the appearance of a ( 000.1 ) reflection indicated by a small arrow in figure 6c and two times shorter distance between ( 000.0 ) and ( 000.1 ) reflection compared to saed pattern of 2h in figure 6b confirms the existence of 4h crystal structure . the hrtem image shown in figure 6d and the fft shown in the inset the length of the 4h segment is approximately 200 nm , which is consistent with the observation in xrd ( see below ) . ( b ) saed from the region in the blue circle in ( a ) showing 2h structure , and ( c ) saed from the region in the green circle in ( a ) showing the 4h crystal structure . nws also tend to not grow exactly perpendicular to the substrate surface , but a small mosaicity due to a random nw tilt distribution causes a broadening in the same direction as the finite width in the symmetric rsms . since the effects scale differently for different reflections as well as show up in slightly different directions in asymmetric rsms , a distinction of the two effects is possible in the asymmetric rsms . the finite size contribution is always inversely proportional to the width of the nws , while the broadening due to tilt distribution increases linearly with q. in the asymmetric scans , the tilt distribution and finite width broadens the peak in different directions , which is indicated in figure 3c , d . another finite size effect present in the asymmetric rsms is the broadening of the diffraction peaks in the [ 111 ] direction , caused by short segments of the respective polytype in the nw growth direction . worth noting is that no 4h was found in the reference nws prior to radial overgrowth . with a very high crystal quality as seen in both the hrtem analysis and xrd , we are able to accurately calculate the lattice parameters of both gaas 2h and 4h using the asymmetric rsms . table 1 summarizes the calculated lattice parameters with relative differences with respect to ideal tetrahedral structures , using geometrically converted cubic 3c lattice parameters . both the 2h and 4h unit cells are elongated in the nw growth direction and contracted in the radial direction compared to 3c . hence , the burying of the nws is not sufficient to explain the drastic decrease in the 2h signal . in addition , epitaxial burying of the 2h nws in a 3c matrix would not explain the appearance of 4h segments , estimated to be 100200 nm long in the samples with the most 4h , as an epitaxial burying driven phase transformation would force the structure to adopt the burying matrix structure , in this case 3c . the decreasing 2h signal coincides with the onset of the radial merging of adjacent nws , as seen in the inset in figure 2g , h . however , the agreement of the xrd and tem results suggests that the crystal phase transformation is independent of measurement technique . to understand the process of the merging better we come back to the stacking sequences of the different crystallographic phases . either this mismatch is kept at the cost of having a line defect at the interface of the merging nws , or in order to release this mismatch , the line defect may propagate through one of the nws to accommodate the corresponding as plane . this edge dislocation could be seen as a shockley partial dislocation , and when propagating through the crystal changes the configuration of the as from hexagonal to cubic position ( or vice versa ) . the mismatch causes a translation in the [ 110.0 ] direction of the as atoms in nw 1 closest to the interface . changing a 2h gaas to 3c gaas would to a first approximation result in a lower total energy for the crystal , as the cohesive energy is 24 mev / iii v pair higher for 3c . the 2h nws are hexagonal and have { 101.0 } type facets , which are perpendicular to the burgers vector , and the nws all have facets parallel to each other . with the dislocation line energy as the dominant factor for the energies related to the phase transformation , the initial structures of the two merging nws are of minor importance the probability of either one to transform is roughly equal . , the dislocation is of the same type , and both transformations are triggered by the interaction between two crystals with as atoms at different lateral positions . however , in the case of epitaxial burying where the burying matrix completely surrounds the nw , the nw has to adopt the structure of the burying matrix or keep the dislocation at the periphery of the nw . to let the dislocation propagate through the nw makes the dislocation shorter , and eventually disappear , which is the thermodynamically most favorable case , and the final structure will be easy to predict the one of the burying matrix . if we involve a third 10 bilayer nw in the merging process , there will be ( 2 ) outcomes . to estimate the amount of 3c , 2h , and 4h in each outcome , we define a 3c segment to be of at least six bilayers ( two unit cells ) , 2h as six bilayers ( three unit cells ) and 4h as eight bilayers ( two unit cells ) . using a simple matlab script , we simulate the merging of two , three , and four 2h nws with each 10 bilayers ( where the 10 bilayer limit is chosen to limit computation time ) . a schematic view on the merging process the simulations shows that the merging of just two nws gives a mixed fraction of around 80%that is , most of the possible outcomes have only segments consisting of less than six bilayers of any considered polytype . the increase in twinned 3c seen in the xrd measurements comes not only from the nws , but also from the parasitic substrate growth , and the 36% of 3c found in the nws in our combinatorial model is not the major contribution to the signal . for a structure with very short segments of 2h and 3c sandwiched , streaking between the xrd peaks is expected , and this is also what we observe in our rsms ; see figure 3b and supporting information figure s4 . using this simple combinatorial approach , we are able to describe the main features of the merging process . however , the 2h lattice parameters in the former report were measured from nws with a mixed crystal structure , consisting of 2h segments sandwiched between 3c , and most likely strained by the 3c segments . for our structures , the very few sfs in the pure 2h before any merging process are not likely to influence the overall 2h structure , and the 2h crystal is fully relaxed . using the sample with 60 min radial overgrowth , we measured the 2h and 4h unit cell parameters on the sample with the largest volume of pure 2h . with a larger lattice plane spacing along the c - axis in 2h as compared to the 3c counterpart and a smaller in - plane distance , the c / a ratio is measured to be 1.649 , which is 0.98% higher than the ideal 1.633 for hexagonal close - packed structures with perfect tetrahedral coordination . the measured gaas 4h lattice parameters reported here are to the authors best knowledge the first experimental report on gaas 4h lattice parameters . the higher c / a ratio compared to the ideal is typical for metastable hexagonal structures and gives bonding slightly distorted from an ideal tetrahedron . the difference in cohesive energy gives a higher nucleation barrier for 2h compared to 4h or 3c in gaas , which should result in a higher probability of 4h than 2h during growth . assuming surface energies of 2h to be considerably lower than those of 4h as well explains why observations of 4h grown in nws are very few , and mostly only occur in a transition region between 2h and 3c . in this work , we studied the crystal structure of radially overgrown gaas wurtzite ( 2h ) nws , especially the resulting crystal structure as nws progressively grow into each other and merge . we characterized the grown structure by high resolution xrd and tem with diffraction analysis . we observed that the free - standing gaas nws retain the pure 2h structure until they merge with each other , at which point the merged segments transform progressively from pure 2h structure to a mixture between zinc blende ( 3c ) structure with twins and 2h . we also found sections with the 4h crystal structure in the merged nws and determined lattice parameters of both 2h and 4h gaas . the phase transformation continues as long as the merging process proceeds , driven by a dislocation formed between adjacent nws due to lateral mismatch of as in the ( 111)/(000.1 ) plane . using a combinatorial approach , we estimated the probability of the resulting structure after radial merging of 2h nws , which shows that a heavily mixed structure is the most probably outcome , in agreement with our xrd and tem measurements .

the psychological complications are becoming more significant and common in postmenopausal women with the increase of postmenopausal periods . for example , depression is widespread and associated with functional disability and decreased quality of life in postmenopausal women . several studies , including animal models and clinical observations in women , indicate that the low - estrogen state may be related to depression . therefore , clinical trials have been performed in postmenopausal women using estrogen or estrogen - associated therapies ; however , observed effects have been inconsistent . moreover , investigations have indicated that the use of hormone therapy was associated with endometrial hyperplasia and cancer and may increase the risk of dementia and coronary heart disease . as mentioned above , thus , there is an increased interest in biochemical factors , such as neurotransmitter systems . gamma - aminobutyric acid ( gaba ) is the main inhibitory neurotransmitter in the central nervous system ( cns ) . it is increasingly being recognized that alterations of the gabaergic system are implicated in the pathophysiology of depression . moreover , studies have suggested that the normalization of cerebral gaba deficits is associated with positive treatment effects in depression . many studies have detected changes in gaba - inhibitory function during the menstrual cycle , pregnancy , and postpartum , using methods including proton magnetic resonance spectroscopy ( h - mrs ) , transcranial magnetic stimulation , and cerebrospinal fluid ( csf ) analysis . however , there is little information on cerebral gaba levels in postmenopausal women with depression . h - mrs methods permit researchers to noninvasively quantify brain metabolites , such as n - acetyl - aspartate ( naa ) , myo - inositol ( mi ) , choline ( cho ) , glutamate - glutamine ( glx ) , total creatine ( tcr ) , and lactate ( lac ) . however , detection of gaba using the conventional h - mrs is limited due to its relatively low concentration and the spectral overlap of signals from other major metabolites . an advanced mrs method , mescher - garwood point resolved spectroscopy ( mega - press ) has been used to detect gaba levels in the healthy brain and various psychiatric diseases . herein , we explore the cerebral gaba levels in postmenopausal women using the edited mrs technique mega - press . the present study hypothesized that the gaba levels would be lower in postmenopausal women with depression . nineteen postmenopausal women suffering from depression and 13 healthy postmenopausal women who were age- , body index- and educationally matched controls participated in this study . this study was approved by the local ethical committee , and all participants provided written informed consent before the initiation of the study . the 13 healthy controls ( hcs ) were self - referred in response to fliers or paid advertising . the patients were recruited through the outpatient departments of mental psychology and gynecology of the no . 2 affiliated hospital of shandong traditional chinese medicine and shandong university medical center . both patients and hcs had no current or past personal or family history of any axis i disorder . the 19 postmenopausal women had to meet the icd-10 criteria for depression ( code : f32.0 , 32.1 ) . the symptomatic severity of depression was assessed by 17-item hamilton depression scale ( hamd ) , a standard measure for current symptoms of depression . two trained neuropsychologists , blinded to the magnetic resonance imaging ( mri ) data , performed the tests on all the subjects . all the participants were required to be at least 2 years past the cessation of menses to ensure stabilization of low estrogen values . other inclusion criteria for the 2 groups were intact uterus ; natural menopause at least 2 to 8 years and no subjective complaints of hot flushes ; absence of previous history of psychiatric disorders , endocrine and metabolic illnesses ; at least 6 months free of drugs ( i.e. , psychotropic medication , antidepressants , hormones ) and alcohol abuse ; and no major ischemic vascular disease ( e.g. , cerebral ischemic stroke , subcortical arteriosclerotic encephalopathy ) diagnosed by conventional brain mri . all subjects underwent thorough medical examination and laboratory test ( including estrogen levels ) , and no positive results were observed that would necessitate medical interventions . all mri / mrs experiments were performed on a 3.0 tesla ( t ) scanner ( philips achieva tx , best , the netherlands ) equipped with an 8-channel phased - array head coil . prior to the mrs examination , t1-weighted 3-dimensional ( 3d ) turbo field echo images were used for localization , and the scan parameters were as follows : tr ( repetition time ) = 8.1 ms ; te ( echo time ) = 3.7 ms ; slice thickness = 1 mm ; matrix : 256 256 ; field of view = 24 24 cm ; and flip angle = 8. the mrs voxels were set on anterior cingulate cortex / medial prefrontal cortex ( acc / mpfc ; 3 3 3 cm ) and posterior cingulate cortex ( pcc ; 3 2 3 cm ) ( fig . the median sagittal plane was selected as a reference slice for voxel localization : the voxel in the acc / mpfc was prescribed superior to the genu of the corpus callosum , aligned with the long axis of the corpus callosum knee , and positioned on the medial aspect of the axial plane ; the voxel in the pcc was prescribed posterior and superior to the splenium of the corpus callosum , aligned with its tangent , and positioned again on the medial aspect of the axial plane . both voxels were positioned in a manner such as to avoid the lateral ventricle and skull . magnetic resonance spectroscopy voxel placement and resulting spectra . t1-weighted turbo field echo images show single - voxel placements centered on the acc / mpfc in the sagittal ( a ) , axial ( b ) projections and on the pcc in the sagittal ( d ) , axial ( e ) projections . the corresponding results of brain segmentation are shown in the acc / mpfc ( c ) and pcc ( f ) . ( g , h ) representative mega - press spectra processed using the gannet 2.0 toolkit in acc / mpfc and pcc of postmenopausal women with depression . ( g ) the curve - fitting of the gaba peak using gannet , the red lines in the panels are the results of the gannetfit curve - fitting , the blue lines show the postphase and frequency aligned gaba data , and the black line is the residual difference between the experimental data and the curve - fit . acc / mpfc = anterior cingulate cortex / medial prefrontal cortex , gaba = gamma - aminobutyric acid , mega - press = mescher - garwood point resolved spectroscopy , pcc = posterior cingulate cortex . the mega - press sequence was used for gaba editing , with the following parameters : tr = 2000 ms ; te = 68 ms ; 320 signal averages ; acquisition bandwidth = 1000 hz ; and scan duration 8 min 48 s. j - evolution for gaba was refocused during the odd - numbered acquisitions ( on ) but not during the even numbered acquisitions ( off ) by applying gaussian inversion pulse to the ch2 resonance of gaba at 1.9 ppm ( on ) and at 7.5 ppm , symmetrically about the water peak ( off ) , respectively . fastmap shimming of the volume of interest ( voi ) was conducted automatically prior to each acquisition . the difference between on and off spectra provided an edited spectrum of gaba . the signal detected at 3 ppm with these parameters and the mega - press technique is known to contain contributions from both the macromolecules ( mm ) and homocarnosine , as well as gaba , and hence , the detected signal is referred to as gaba+ . ( gaba - mrs analysis tool ) in matlab 2010b ( mathworks ) with gaussian curve - fitting to the gaba+ peaks . gannet provides the standard deviation ( sd ) of the fitting residues divided by the amplitude of the fitted peaks , generating the overall fitting error which reflects the quality of the data . only spectra with a relative fitting error of gaba+ below 10% were included in the statistical analysis . different voxel proportions of gray matter ( gm ) , white matter ( wm ) , and csf in h - mrs may confound group differences in metabolite measurement . to determine if the differences in the tissue composition of the subjects from both the groups could account for differences in gaba+ levels , each mrs voxel was segmented as gm , wm , or csf using the 3d t1-weighted brain images and the automatic brain segmentation program ( fig . 1 ) , fast ( fmrib 's automated segmentation tool ) in the fsl package ( oxford university , oxford , uk ) . the vois were co - registered to the anatomical images using the re - creation of voi matlab tool . tissue gm fractions were obtained by calculating the ratio of gm volume to the gm + wm volumes in the vois . statistical analyses were conducted using the statistical package for social sciences software ( spss 17.0 ) . mrs data , normally distributed , were presented as mean sd values . the differences of gaba+ levels from patients and controls were tested using 1-way analysis of variance . spearman correlation coefficients were used to evaluate the linear associations between gaba+ levels and hamd / hama scores , as well as estrogen levels . all statistical tests were 2-sided and the level of significance was set at p < 0.05 . nineteen postmenopausal women suffering from depression and 13 healthy postmenopausal women who were age- , body index- and educationally matched controls participated in this study . this study was approved by the local ethical committee , and all participants provided written informed consent before the initiation of the study . the 13 healthy controls ( hcs ) were self - referred in response to fliers or paid advertising . the patients were recruited through the outpatient departments of mental psychology and gynecology of the no . 2 affiliated hospital of shandong traditional chinese medicine and shandong university medical center . both patients and hcs had no current or past personal or family history of any axis i disorder . the 19 postmenopausal women had to meet the icd-10 criteria for depression ( code : f32.0 , 32.1 ) . the symptomatic severity of depression was assessed by 17-item hamilton depression scale ( hamd ) , a standard measure for current symptoms of depression . two trained neuropsychologists , blinded to the magnetic resonance imaging ( mri ) data , performed the tests on all the subjects . all the participants were required to be at least 2 years past the cessation of menses to ensure stabilization of low estrogen values . other inclusion criteria for the 2 groups were intact uterus ; natural menopause at least 2 to 8 years and no subjective complaints of hot flushes ; absence of previous history of psychiatric disorders , endocrine and metabolic illnesses ; at least 6 months free of drugs ( i.e. , psychotropic medication , antidepressants , hormones ) and alcohol abuse ; and no major ischemic vascular disease ( e.g. , cerebral ischemic stroke , subcortical arteriosclerotic encephalopathy ) diagnosed by conventional brain mri . all subjects underwent thorough medical examination and laboratory test ( including estrogen levels ) , and no positive results were observed that would necessitate medical interventions . all mri / mrs experiments were performed on a 3.0 tesla ( t ) scanner ( philips achieva tx , best , the netherlands ) equipped with an 8-channel phased - array head coil . prior to the mrs examination , t1-weighted 3-dimensional ( 3d ) turbo field echo images were used for localization , and the scan parameters were as follows : tr ( repetition time ) = 8.1 ms ; te ( echo time ) = 3.7 ms ; slice thickness = 1 mm ; matrix : 256 256 ; field of view = 24 24 cm ; and flip angle = 8. the mrs voxels were set on anterior cingulate cortex / medial prefrontal cortex ( acc / mpfc ; 3 3 3 cm ) and posterior cingulate cortex ( pcc ; 3 2 3 cm ) ( fig . the median sagittal plane was selected as a reference slice for voxel localization : the voxel in the acc / mpfc was prescribed superior to the genu of the corpus callosum , aligned with the long axis of the corpus callosum knee , and positioned on the medial aspect of the axial plane ; the voxel in the pcc was prescribed posterior and superior to the splenium of the corpus callosum , aligned with its tangent , and positioned again on the medial aspect of the axial plane . both voxels were positioned in a manner such as to avoid the lateral ventricle and skull . magnetic resonance spectroscopy voxel placement and resulting spectra . t1-weighted turbo field echo images show single - voxel placements centered on the acc / mpfc in the sagittal ( a ) , axial ( b ) projections and on the pcc in the sagittal ( d ) , axial ( e ) projections . the corresponding results of brain segmentation are shown in the acc / mpfc ( c ) and pcc ( f ) . ( g , h ) representative mega - press spectra processed using the gannet 2.0 toolkit in acc / mpfc and pcc of postmenopausal women with depression . ( g ) the curve - fitting of the gaba peak using gannet , the red lines in the panels are the results of the gannetfit curve - fitting , the blue lines show the postphase and frequency aligned gaba data , and the black line is the residual difference between the experimental data and the curve - fit . acc / mpfc = anterior cingulate cortex / medial prefrontal cortex , gaba = gamma - aminobutyric acid , mega - press = mescher - garwood point resolved spectroscopy , pcc = posterior cingulate cortex . the mega - press sequence was used for gaba editing , with the following parameters : tr = 2000 ms ; te = 68 ms ; 320 signal averages ; acquisition bandwidth = 1000 hz ; and scan duration 8 min 48 s. j - evolution for gaba was refocused during the odd - numbered acquisitions ( on ) but not during the even numbered acquisitions ( off ) by applying gaussian inversion pulse to the ch2 resonance of gaba at 1.9 ppm ( on ) and at 7.5 ppm , symmetrically about the water peak ( off ) , respectively . fastmap shimming of the volume of interest ( voi ) was conducted automatically prior to each acquisition . the difference between on and off spectra provided an edited spectrum of gaba . the signal detected at 3 ppm with these parameters and the mega - press technique is known to contain contributions from both the macromolecules ( mm ) and homocarnosine , as well as gaba , and hence , the detected signal is referred to as gaba+ . ( gaba - mrs analysis tool ) in matlab 2010b ( mathworks ) with gaussian curve - fitting to the gaba+ peaks . gannet provides the standard deviation ( sd ) of the fitting residues divided by the amplitude of the fitted peaks , generating the overall fitting error which reflects the quality of the data . only spectra with a relative fitting error of gaba+ below 10% were included in the statistical analysis . different voxel proportions of gray matter ( gm ) , white matter ( wm ) , and csf in h - mrs may confound group differences in metabolite measurement . to determine if the differences in the tissue composition of the subjects from both the groups could account for differences in gaba+ levels , each mrs voxel was segmented as gm , wm , or csf using the 3d t1-weighted brain images and the automatic brain segmentation program ( fig . 1 ) , fast ( fmrib 's automated segmentation tool ) in the fsl package ( oxford university , oxford , uk ) . the vois were co - registered to the anatomical images using the re - creation of voi matlab tool . tissue gm fractions were obtained by calculating the ratio of gm volume to the gm + wm volumes in the vois . statistical analyses were conducted using the statistical package for social sciences software ( spss 17.0 ) . mrs data , normally distributed , were presented as mean sd values . the differences of gaba+ levels from patients and controls were tested using 1-way analysis of variance . spearman correlation coefficients were used to evaluate the linear associations between gaba+ levels and hamd / hama scores , as well as estrogen levels . all statistical tests were 2-sided and the level of significance was set at p < 0.05 . the demographic information and results of the 17-hamd/14-hama of all subjects are presented in table 1 . there was no difference in age ( p = 0.15 ) , cessation of menstruation time ( p = 0.34 ) , hormone estrogen levels ( p = 0.17 ) , body index ( p = 0.21 ) , and educational background ( p = 0.37 ) between the 2 groups . the scores of the patient group on the 17-hamd and 14-hama ( range 1521 , mean = 17.32 1.73 and range 1419 , mean = 15.42 1.30 , respectively ) are significantly higher than the hc group ( range 47 , mean = 5.23 1.01 and range 36 , mean = 5.62 0.96 , respectively ) ( p < 0.001 ) . according to the scores , the patients fulfilled criteria for mild - to - moderate depression . demographic , mrs , and segmentation data of postmenopausal women with patients group and healthy controls group . the mean gm tissue fraction gm/(gm + wm ) in the patient group was 54.21 1.50% and 53.84 1.96% in the acc / mpfc region and the pcc , respectively , and 53.28 0.48% and 54.77 2.05% , respectively , in hcs ( table 1 ) . there were no significant difference in gm fraction of the vois between the 2 groups ( acc / mpfc : f(1 , 30 ) = 1.740 , p = 0.224 ; p = 0.224 ; pcc : f(1 , 30 ) = 0.529 , p = 0.488 ) . edited spectra were successfully obtained from the acc / mpfc region and the pcc region in all the 32 subjects ( 19 patients and 13 hcs ) , and the fitting error of gaba+ in each spectrum was below 10% . significantly lower gaba+ levels were found in acc / mpfc region of the patients , relative to hcs ( f(1 , 30 ) = 11.901 , p = 0.002 ) . slightly lower mean gaba+ levels were also found in the pcc region ( 1.00 0.14 ) of the patients group as compared to the hcs ( 1.05 0.13 ) , but did not reach statistical significance ( f(1 , 30 ) = 0.792 , p = 0.380 ) ( table 1 ; fig . the mean gaba+ fitting error was 5.31% and 6.64% for acc / mpfc and pcc voxels , respectively , in the patients group , and 5.24% and 6.57% for hcs group ( table 1 ) , with no significant differences between the groups ( acc / mpfc : f(1 , 30 ) = 0.025 , p = 0.875 ; pcc : f(1 , 30 ) = 0.048 , p = 0.828 ) . the gaba+ levels of depression patients and healthy control in acc / mpfc ( a ) and pcc ( b ) regions . the mean and standard deviation of gaba+ levels ( mm ) are displayed . ( a ) gaba+ levels of patients group are significantly decreased in the acc / mpfc ( p = 0.002 ) . ( b ) mean gaba+ levels are lower in patients group than in healthy controls groups in pcc ( p = 0.380 ) , but no statistically significant difference is detected . acc / mpfc = anterior cingulate cortex / medial prefrontal cortex , gaba = gamma - aminobutyric acid , pcc = posterior cingulate cortex . no significant correlations were found between 17-hamd/14-hama and gaba+ levels , either in acc / mpfc ( p = 0.486 ; r = 0.170/p = 0.814 ; r = 0.058 ) or pcc ( p = 0.887 ; r = 0.035/p = 0.987 ; r = 0.004 ) in the patients group . also , with regard to the correlation of 17-hamd/14-hama scores and gaba+ levels in hcs group , no statistical correlation was detected ( acc / mpfc : p = 0.337 ; r = 0.290/p = 0.584 ; r = 0.168 ; pcc : p = 0.667 ; r = 0.132/p = 0.716 ; r = 0.112 ) . significant correlations could not be established between gaba+ levels and estrogen levels , either in the patients group ( acc / mpfc : p = 0.629 , r = 0.118 ; pcc : p = 0.861 , r = 0.043 ) or in the hcs group ( acc / mpfc : p = 0.471 , r = 0.220 ; pcc : p = 0.415 , r = 0.247 ) . the demographic information and results of the 17-hamd/14-hama of all subjects are presented in table 1 . there was no difference in age ( p = 0.15 ) , cessation of menstruation time ( p = 0.34 ) , hormone estrogen levels ( p = 0.17 ) , body index ( p = 0.21 ) , and educational background ( p = 0.37 ) between the 2 groups . the scores of the patient group on the 17-hamd and 14-hama ( range 1521 , mean = 17.32 1.73 and range 1419 , mean = 15.42 1.30 , respectively ) are significantly higher than the hc group ( range 47 , mean = 5.23 1.01 and range 36 , mean = 5.62 0.96 , respectively ) ( p < 0.001 ) . according to the scores , the patients fulfilled criteria for mild - to - moderate depression . demographic , mrs , and segmentation data of postmenopausal women with patients group and healthy controls group . the mean gm tissue fraction gm/(gm + wm ) in the patient group was 54.21 1.50% and 53.84 1.96% in the acc / mpfc region and the pcc , respectively , and 53.28 0.48% and 54.77 2.05% , respectively , in hcs ( table 1 ) . there were no significant difference in gm fraction of the vois between the 2 groups ( acc / mpfc : f(1 , 30 ) = 1.740 , p = 0.224 ; p = 0.224 ; pcc : f(1 , 30 ) = 0.529 , p = 0.488 ) . edited spectra were successfully obtained from the acc / mpfc region and the pcc region in all the 32 subjects ( 19 patients and 13 hcs ) , and the fitting error of gaba+ in each spectrum was below 10% . significantly lower gaba+ levels were found in acc / mpfc region of the patients , relative to hcs ( f(1 , 30 ) = 11.901 , p = 0.002 ) . slightly lower mean gaba+ levels were also found in the pcc region ( 1.00 0.14 ) of the patients group as compared to the hcs ( 1.05 0.13 ) , but did not reach statistical significance ( f(1 , 30 ) = 0.792 , p = 0.380 ) ( table 1 ; fig . the mean gaba+ fitting error was 5.31% and 6.64% for acc / mpfc and pcc voxels , respectively , in the patients group , and 5.24% and 6.57% for hcs group ( table 1 ) , with no significant differences between the groups ( acc / mpfc : f(1 , 30 ) = 0.025 , p = 0.875 ; pcc : f(1 , 30 ) = 0.048 , p = 0.828 ) . the gaba+ levels of depression patients and healthy control in acc / mpfc ( a ) and pcc ( b ) regions . the mean and standard deviation of gaba+ levels ( mm ) are displayed . ( a ) gaba+ levels of patients group are significantly decreased in the acc / mpfc ( p = 0.002 ) . ( b ) mean gaba+ levels are lower in patients group than in healthy controls groups in pcc ( p = 0.380 ) , but no statistically significant difference is detected . acc / mpfc = anterior cingulate cortex / medial prefrontal cortex , gaba = gamma - aminobutyric acid , pcc = posterior cingulate cortex . no significant correlations were found between 17-hamd/14-hama and gaba+ levels , either in acc / mpfc ( p = 0.486 ; r = 0.170/p = 0.814 ; r = 0.058 ) or pcc ( p = 0.887 ; r = 0.035/p = 0.987 ; r = 0.004 ) in the patients group . also , with regard to the correlation of 17-hamd/14-hama scores and gaba+ levels in hcs group , no statistical correlation was detected ( acc / mpfc : p = 0.337 ; r = 0.290/p = 0.584 ; r = 0.168 ; pcc : p = 0.667 ; r = 0.132/p = 0.716 ; r = 0.112 ) . significant correlations could not be established between gaba+ levels and estrogen levels , either in the patients group ( acc / mpfc : p = 0.629 , r = 0.118 ; pcc : p = 0.861 , r = 0.043 ) or in the hcs group ( acc / mpfc : p = 0.471 , r = 0.220 ; pcc : p = 0.415 , r = 0.247 ) . the key outcome of this study is that significantly decreased gaba+ levels are present in the acc / mpfc region of postmenopausal women suffering from depression , as compared to hc subjects . as the main inhibitory neurotransmitter in the cns , gaba is mainly localized to inhibitory neurons , critically influencing the prefrontal cortical and acc function . therefore , the reduced gaba+ level could indicate either loss or dysfunction of gabaergic neurons in postmenopausal women with depression . our findings are consistent with the previous studies , which detected reduced gaba levels in the anterior cingulate , the dorsomedial prefrontal cortex , and the dorsal anterolateral prefrontal cortex in depressed patients . moreover , normalization of gaba levels in the brain by treatment with selective serotonin reuptake inhibitors or electroconvulsive therapy was associated with the improvement of depression , which further implied that our findings of reduced gaba+ levels may be involved in the pathogenesis of depression in postmenopausal women . as mentioned above , the deficiency of gaba+ levels may be either due to gabaergic neuronal loss or dysfunction . if due to dysfunction , either gaba synthesis itself or enzymatic deficiency in glutamate / glutamine cycling may be the plausible cause . postmortem studies exhibited reduced glutamate decarboxylase ( gad ) in the prefrontal cortex of patients with depression . furthermore , animal models of depression indicated that glutamate / gaba - glutamine cycling was decreased . on the other hand , the alterations in density or size of neuron and glial - cell may be involved in the decreased gaba levels in a postmenopausal woman with depression . previous studies suggested that the size or density of neurons in depressed patients was reduced compared to the hcs in the dorsolateral prefrontal cortex , occipital cortex , and acc . this further indicated that the decreased levels of gad-67 may be consistent with the reduced densities and size of calbindin - positive gabaergic interneurons in major depressive disorders . however , the underlying mechanisms need to be elucidated in future studies . around the time of final menstrual period , the levels of estrogen gradually decline with the aging of the ovarian function and then is stable in the early stage of postmenopause . studies suggested that the basal number of gad65-immunoreactive cells decreased in rats after long - term ovariectomy . moreover , several reports indicated that estrogen and progesterone differentially regulated the gabaergic system ; for example , weiland established that mrna expression of gad in the hippocampus ( hpc ) was stimulated by estradiol administration in ovariectomized rats and suppressed by the addition of progesterone ; while another study indicated that estradiol decreased the expression of gad in hippocampal neurons within 24 h , thus overall increasing the excitatory drive . in addition , estrogen plus progesterone attenuated gad gene expression in the hpc and amygdala ( amd ) , but not in the medial basal hypothalamus ( mbh ) ; while the gaba receptor subunit gene expression was generally higher in the amd and hpc than in the mbh . taken together , the deficient gaba+ levels may be an appropriate adapted response for the postmenopausal stage , which may cause a change in behavior and emotion in certain postmenopausal women . furthermore , the lack of relationship between gaba+ levels and the estrogen levels in patients group may be partially explained by the differential hormonal regulation of the gad and gaba receptor expression . there is also no significant correlation between the scores assessed by 17-hamd/14-hama and cerebral gaba+ levels , which is consistent with previous studies . it is well known that depression is a multifactor disease , influence socially and psychologically influences . therefore , a deeper insight is essential for elucidating the underlying mechanism of depression , gabaergic system , and hormones in postmenopausal women . first , the sample size of 19 in postmenopausal women with mild - to - moderate depression and 13 healthy postmenopausal women is rather small for a clinical study of this kind , and so , the present results should be viewed as preliminary . further studies with a larger sample size will be indispensable to confirm the current results . it is possible that the lack of significant difference in estrogen levels between the 2 groups and gaba+ levels in the pcc is due to the small sample size and limited statistical power . secondly , the severity of depression in postmenopausal women in our study is mild - to - moderate , not including those with severe depression . it also might be one of the factors for the lack of correlations between gaba+ levels and the scores of 17-hamd/14-hama or estrogen levels . therefore , further study including patients with severe major depression will be performed to evaluate this correlation . thirdly , similar to our previous study , the voxel volume was relatively large ( 3 3 3 cm ; 3 3 2 cm ) due to the low intensity of the gaba+ signal . examination of other brain regions will be obligatory to determine whether gaba abnormalities are specific to acc / mpfc or more broadly distributed throughout the brain . gaba decrease previously reported in the plasma and csf of the depressed patients might suggest a broad distribution of reductions . in the future , a smaller - voi mrs method could prove more efficient for region - specific analyses of brain gaba levels . lastly , the mrs measure of gaba+ includes non - gaba contributions from co - edited mm and homocarnosine . in summary , the decreased gaba+ levels were found in the acc / mpfc of postmenopausal women with mild - to - moderate depression . our findings suggest that the aberrant gabaergic system may also be involved in the pathogenesis of depression in postmenopausal women .

abstractbackground : it is increasingly being recognized that alterations of the gabaergic system are implicated in the pathophysiology of depression . this study aimed to explore in vivo gamma - aminobutyric acid ( gaba ) levels in the anterior cingulate cortex / medial prefrontal cortex ( acc / mpfc ) and posterior - cingulate cortex ( pcc ) of postmenopausal women with depression using magnetic resonance spectroscopy ( 1h - mrs).methods : nineteen postmenopausal women with depression and thirteen healthy controls were enrolled in the study . all subjects underwent 1h - mrs of the acc / mpfc and pcc using the mega point resolved spectroscopy sequence ( mega - press ) technique . the severity of depression was assessed by 17-item hamilton depression scale ( hamd ) . quantification of mrs data was performed using gannet program . differences of gaba+ levels from patients and controls were tested using one - way analysis of variance . spearman correlation coefficients were used to evaluate the linear associations between gaba+ levels and hamd scores , as well as estrogen levels.results:significantly lower gaba+ levels were detected in the acc / mpfc of postmenopausal women with depression compared to healthy controls ( p = 0.002 ) . no significant correlations were found between 17-hamd/14-hama and gaba+ levels , either in acc / mpfc ( p = 0.486 ; r = 0.170/p = 0.814 ; r = 0.058 ) or pcc ( p = 0.887 ; r = 0.035/ p = 0.987 ; r = 0.004 ) in the patients ; there is also no significant correlation between gaba+ levels and estrogen levels in patients group ( acc / mpfc : p = 0.629 , r = 0.018 ; pcc : p = 0.861 , r = 0.043).conclusion : significantly lower gaba+ levels were found in the acc / mpfc of postmenopausal women with depression , suggesting that the dysfunction of the gabaergic system may also be involved in the pathogenesis of depression in postmenopausal women .

Introduction Materials and methods Participants MRI/MRS study protocol VOI segmentation Statistical analysis Results Demographic characteristics of study subjects VOI segmentation results Comparisons of GABA+ levels between the 2 groups Correlation between GABA+ levels and the results of 17-HAMD/14-HAMA Correlation between GABA+ levels and estrogen levels Discussion Conclusion

the psychological complications are becoming more significant and common in postmenopausal women with the increase of postmenopausal periods . gamma - aminobutyric acid ( gaba ) is the main inhibitory neurotransmitter in the central nervous system ( cns ) . it is increasingly being recognized that alterations of the gabaergic system are implicated in the pathophysiology of depression . many studies have detected changes in gaba - inhibitory function during the menstrual cycle , pregnancy , and postpartum , using methods including proton magnetic resonance spectroscopy ( h - mrs ) , transcranial magnetic stimulation , and cerebrospinal fluid ( csf ) analysis . however , there is little information on cerebral gaba levels in postmenopausal women with depression . an advanced mrs method , mescher - garwood point resolved spectroscopy ( mega - press ) has been used to detect gaba levels in the healthy brain and various psychiatric diseases . herein , we explore the cerebral gaba levels in postmenopausal women using the edited mrs technique mega - press . the present study hypothesized that the gaba levels would be lower in postmenopausal women with depression . nineteen postmenopausal women suffering from depression and 13 healthy postmenopausal women who were age- , body index- and educationally matched controls participated in this study . the symptomatic severity of depression was assessed by 17-item hamilton depression scale ( hamd ) , a standard measure for current symptoms of depression . prior to the mrs examination , t1-weighted 3-dimensional ( 3d ) turbo field echo images were used for localization , and the scan parameters were as follows : tr ( repetition time ) = 8.1 ms ; te ( echo time ) = 3.7 ms ; slice thickness = 1 mm ; matrix : 256 256 ; field of view = 24 24 cm ; and flip angle = 8. the mrs voxels were set on anterior cingulate cortex / medial prefrontal cortex ( acc / mpfc ; 3 3 3 cm ) and posterior cingulate cortex ( pcc ; 3 2 3 cm ) ( fig . the median sagittal plane was selected as a reference slice for voxel localization : the voxel in the acc / mpfc was prescribed superior to the genu of the corpus callosum , aligned with the long axis of the corpus callosum knee , and positioned on the medial aspect of the axial plane ; the voxel in the pcc was prescribed posterior and superior to the splenium of the corpus callosum , aligned with its tangent , and positioned again on the medial aspect of the axial plane . t1-weighted turbo field echo images show single - voxel placements centered on the acc / mpfc in the sagittal ( a ) , axial ( b ) projections and on the pcc in the sagittal ( d ) , axial ( e ) projections . the corresponding results of brain segmentation are shown in the acc / mpfc ( c ) and pcc ( f ) . ( g , h ) representative mega - press spectra processed using the gannet 2.0 toolkit in acc / mpfc and pcc of postmenopausal women with depression . acc / mpfc = anterior cingulate cortex / medial prefrontal cortex , gaba = gamma - aminobutyric acid , mega - press = mescher - garwood point resolved spectroscopy , pcc = posterior cingulate cortex . the mega - press sequence was used for gaba editing , with the following parameters : tr = 2000 ms ; te = 68 ms ; 320 signal averages ; acquisition bandwidth = 1000 hz ; and scan duration 8 min 48 s. j - evolution for gaba was refocused during the odd - numbered acquisitions ( on ) but not during the even numbered acquisitions ( off ) by applying gaussian inversion pulse to the ch2 resonance of gaba at 1.9 ppm ( on ) and at 7.5 ppm , symmetrically about the water peak ( off ) , respectively . the signal detected at 3 ppm with these parameters and the mega - press technique is known to contain contributions from both the macromolecules ( mm ) and homocarnosine , as well as gaba , and hence , the detected signal is referred to as gaba+ . to determine if the differences in the tissue composition of the subjects from both the groups could account for differences in gaba+ levels , each mrs voxel was segmented as gm , wm , or csf using the 3d t1-weighted brain images and the automatic brain segmentation program ( fig . the differences of gaba+ levels from patients and controls were tested using 1-way analysis of variance . spearman correlation coefficients were used to evaluate the linear associations between gaba+ levels and hamd / hama scores , as well as estrogen levels . nineteen postmenopausal women suffering from depression and 13 healthy postmenopausal women who were age- , body index- and educationally matched controls participated in this study . the symptomatic severity of depression was assessed by 17-item hamilton depression scale ( hamd ) , a standard measure for current symptoms of depression . prior to the mrs examination , t1-weighted 3-dimensional ( 3d ) turbo field echo images were used for localization , and the scan parameters were as follows : tr ( repetition time ) = 8.1 ms ; te ( echo time ) = 3.7 ms ; slice thickness = 1 mm ; matrix : 256 256 ; field of view = 24 24 cm ; and flip angle = 8. the mrs voxels were set on anterior cingulate cortex / medial prefrontal cortex ( acc / mpfc ; 3 3 3 cm ) and posterior cingulate cortex ( pcc ; 3 2 3 cm ) ( fig . the median sagittal plane was selected as a reference slice for voxel localization : the voxel in the acc / mpfc was prescribed superior to the genu of the corpus callosum , aligned with the long axis of the corpus callosum knee , and positioned on the medial aspect of the axial plane ; the voxel in the pcc was prescribed posterior and superior to the splenium of the corpus callosum , aligned with its tangent , and positioned again on the medial aspect of the axial plane . t1-weighted turbo field echo images show single - voxel placements centered on the acc / mpfc in the sagittal ( a ) , axial ( b ) projections and on the pcc in the sagittal ( d ) , axial ( e ) projections . the corresponding results of brain segmentation are shown in the acc / mpfc ( c ) and pcc ( f ) . ( g , h ) representative mega - press spectra processed using the gannet 2.0 toolkit in acc / mpfc and pcc of postmenopausal women with depression . acc / mpfc = anterior cingulate cortex / medial prefrontal cortex , gaba = gamma - aminobutyric acid , mega - press = mescher - garwood point resolved spectroscopy , pcc = posterior cingulate cortex . the mega - press sequence was used for gaba editing , with the following parameters : tr = 2000 ms ; te = 68 ms ; 320 signal averages ; acquisition bandwidth = 1000 hz ; and scan duration 8 min 48 s. j - evolution for gaba was refocused during the odd - numbered acquisitions ( on ) but not during the even numbered acquisitions ( off ) by applying gaussian inversion pulse to the ch2 resonance of gaba at 1.9 ppm ( on ) and at 7.5 ppm , symmetrically about the water peak ( off ) , respectively . the signal detected at 3 ppm with these parameters and the mega - press technique is known to contain contributions from both the macromolecules ( mm ) and homocarnosine , as well as gaba , and hence , the detected signal is referred to as gaba+ . to determine if the differences in the tissue composition of the subjects from both the groups could account for differences in gaba+ levels , each mrs voxel was segmented as gm , wm , or csf using the 3d t1-weighted brain images and the automatic brain segmentation program ( fig . the differences of gaba+ levels from patients and controls were tested using 1-way analysis of variance . spearman correlation coefficients were used to evaluate the linear associations between gaba+ levels and hamd / hama scores , as well as estrogen levels . there was no difference in age ( p = 0.15 ) , cessation of menstruation time ( p = 0.34 ) , hormone estrogen levels ( p = 0.17 ) , body index ( p = 0.21 ) , and educational background ( p = 0.37 ) between the 2 groups . demographic , mrs , and segmentation data of postmenopausal women with patients group and healthy controls group . the mean gm tissue fraction gm/(gm + wm ) in the patient group was 54.21 1.50% and 53.84 1.96% in the acc / mpfc region and the pcc , respectively , and 53.28 0.48% and 54.77 2.05% , respectively , in hcs ( table 1 ) . there were no significant difference in gm fraction of the vois between the 2 groups ( acc / mpfc : f(1 , 30 ) = 1.740 , p = 0.224 ; p = 0.224 ; pcc : f(1 , 30 ) = 0.529 , p = 0.488 ) . edited spectra were successfully obtained from the acc / mpfc region and the pcc region in all the 32 subjects ( 19 patients and 13 hcs ) , and the fitting error of gaba+ in each spectrum was below 10% . significantly lower gaba+ levels were found in acc / mpfc region of the patients , relative to hcs ( f(1 , 30 ) = 11.901 , p = 0.002 ) . slightly lower mean gaba+ levels were also found in the pcc region ( 1.00 0.14 ) of the patients group as compared to the hcs ( 1.05 0.13 ) , but did not reach statistical significance ( f(1 , 30 ) = 0.792 , p = 0.380 ) ( table 1 ; fig . the mean gaba+ fitting error was 5.31% and 6.64% for acc / mpfc and pcc voxels , respectively , in the patients group , and 5.24% and 6.57% for hcs group ( table 1 ) , with no significant differences between the groups ( acc / mpfc : f(1 , 30 ) = 0.025 , p = 0.875 ; pcc : f(1 , 30 ) = 0.048 , p = 0.828 ) . the gaba+ levels of depression patients and healthy control in acc / mpfc ( a ) and pcc ( b ) regions . ( a ) gaba+ levels of patients group are significantly decreased in the acc / mpfc ( p = 0.002 ) . ( b ) mean gaba+ levels are lower in patients group than in healthy controls groups in pcc ( p = 0.380 ) , but no statistically significant difference is detected . acc / mpfc = anterior cingulate cortex / medial prefrontal cortex , gaba = gamma - aminobutyric acid , pcc = posterior cingulate cortex . no significant correlations were found between 17-hamd/14-hama and gaba+ levels , either in acc / mpfc ( p = 0.486 ; r = 0.170/p = 0.814 ; r = 0.058 ) or pcc ( p = 0.887 ; r = 0.035/p = 0.987 ; r = 0.004 ) in the patients group . also , with regard to the correlation of 17-hamd/14-hama scores and gaba+ levels in hcs group , no statistical correlation was detected ( acc / mpfc : p = 0.337 ; r = 0.290/p = 0.584 ; r = 0.168 ; pcc : p = 0.667 ; r = 0.132/p = 0.716 ; r = 0.112 ) . significant correlations could not be established between gaba+ levels and estrogen levels , either in the patients group ( acc / mpfc : p = 0.629 , r = 0.118 ; pcc : p = 0.861 , r = 0.043 ) or in the hcs group ( acc / mpfc : p = 0.471 , r = 0.220 ; pcc : p = 0.415 , r = 0.247 ) . there was no difference in age ( p = 0.15 ) , cessation of menstruation time ( p = 0.34 ) , hormone estrogen levels ( p = 0.17 ) , body index ( p = 0.21 ) , and educational background ( p = 0.37 ) between the 2 groups . demographic , mrs , and segmentation data of postmenopausal women with patients group and healthy controls group . the mean gm tissue fraction gm/(gm + wm ) in the patient group was 54.21 1.50% and 53.84 1.96% in the acc / mpfc region and the pcc , respectively , and 53.28 0.48% and 54.77 2.05% , respectively , in hcs ( table 1 ) . there were no significant difference in gm fraction of the vois between the 2 groups ( acc / mpfc : f(1 , 30 ) = 1.740 , p = 0.224 ; p = 0.224 ; pcc : f(1 , 30 ) = 0.529 , p = 0.488 ) . edited spectra were successfully obtained from the acc / mpfc region and the pcc region in all the 32 subjects ( 19 patients and 13 hcs ) , and the fitting error of gaba+ in each spectrum was below 10% . significantly lower gaba+ levels were found in acc / mpfc region of the patients , relative to hcs ( f(1 , 30 ) = 11.901 , p = 0.002 ) . slightly lower mean gaba+ levels were also found in the pcc region ( 1.00 0.14 ) of the patients group as compared to the hcs ( 1.05 0.13 ) , but did not reach statistical significance ( f(1 , 30 ) = 0.792 , p = 0.380 ) ( table 1 ; fig . the mean gaba+ fitting error was 5.31% and 6.64% for acc / mpfc and pcc voxels , respectively , in the patients group , and 5.24% and 6.57% for hcs group ( table 1 ) , with no significant differences between the groups ( acc / mpfc : f(1 , 30 ) = 0.025 , p = 0.875 ; pcc : f(1 , 30 ) = 0.048 , p = 0.828 ) . the gaba+ levels of depression patients and healthy control in acc / mpfc ( a ) and pcc ( b ) regions . ( a ) gaba+ levels of patients group are significantly decreased in the acc / mpfc ( p = 0.002 ) . ( b ) mean gaba+ levels are lower in patients group than in healthy controls groups in pcc ( p = 0.380 ) , but no statistically significant difference is detected . acc / mpfc = anterior cingulate cortex / medial prefrontal cortex , gaba = gamma - aminobutyric acid , pcc = posterior cingulate cortex . no significant correlations were found between 17-hamd/14-hama and gaba+ levels , either in acc / mpfc ( p = 0.486 ; r = 0.170/p = 0.814 ; r = 0.058 ) or pcc ( p = 0.887 ; r = 0.035/p = 0.987 ; r = 0.004 ) in the patients group . also , with regard to the correlation of 17-hamd/14-hama scores and gaba+ levels in hcs group , no statistical correlation was detected ( acc / mpfc : p = 0.337 ; r = 0.290/p = 0.584 ; r = 0.168 ; pcc : p = 0.667 ; r = 0.132/p = 0.716 ; r = 0.112 ) . significant correlations could not be established between gaba+ levels and estrogen levels , either in the patients group ( acc / mpfc : p = 0.629 , r = 0.118 ; pcc : p = 0.861 , r = 0.043 ) or in the hcs group ( acc / mpfc : p = 0.471 , r = 0.220 ; pcc : p = 0.415 , r = 0.247 ) . the key outcome of this study is that significantly decreased gaba+ levels are present in the acc / mpfc region of postmenopausal women suffering from depression , as compared to hc subjects . therefore , the reduced gaba+ level could indicate either loss or dysfunction of gabaergic neurons in postmenopausal women with depression . our findings are consistent with the previous studies , which detected reduced gaba levels in the anterior cingulate , the dorsomedial prefrontal cortex , and the dorsal anterolateral prefrontal cortex in depressed patients . moreover , normalization of gaba levels in the brain by treatment with selective serotonin reuptake inhibitors or electroconvulsive therapy was associated with the improvement of depression , which further implied that our findings of reduced gaba+ levels may be involved in the pathogenesis of depression in postmenopausal women . on the other hand , the alterations in density or size of neuron and glial - cell may be involved in the decreased gaba levels in a postmenopausal woman with depression . furthermore , the lack of relationship between gaba+ levels and the estrogen levels in patients group may be partially explained by the differential hormonal regulation of the gad and gaba receptor expression . there is also no significant correlation between the scores assessed by 17-hamd/14-hama and cerebral gaba+ levels , which is consistent with previous studies . therefore , a deeper insight is essential for elucidating the underlying mechanism of depression , gabaergic system , and hormones in postmenopausal women . first , the sample size of 19 in postmenopausal women with mild - to - moderate depression and 13 healthy postmenopausal women is rather small for a clinical study of this kind , and so , the present results should be viewed as preliminary . it is possible that the lack of significant difference in estrogen levels between the 2 groups and gaba+ levels in the pcc is due to the small sample size and limited statistical power . secondly , the severity of depression in postmenopausal women in our study is mild - to - moderate , not including those with severe depression . it also might be one of the factors for the lack of correlations between gaba+ levels and the scores of 17-hamd/14-hama or estrogen levels . in summary , the decreased gaba+ levels were found in the acc / mpfc of postmenopausal women with mild - to - moderate depression . our findings suggest that the aberrant gabaergic system may also be involved in the pathogenesis of depression in postmenopausal women .

microvascular decompression ( mvd ) was introduced in the late 1960s and is a widely accepted treatment for neurovascular compression , including hemifacial spasm ( hfs ) , trigeminal neuralgia ( tn ) , and glossopharyngeal neuralgia . technological advances , combined with microsurgical innovations , make this procedure effective and safe9 ) . additionally , mvd offers significant advantages for both hfs and tn in terms of success rates without further treatment when compared with alternative treatments , such as botulinum toxin injections for hfs and radiofrequency lesioning of the gasserian ganglion for tn5 ) . however , to obtain these benefits , patients are subjected to a slightly higher treatment risk , including the risk of cerebellar injury , hearing loss , cerebrospinal fluid ( csf ) leakage , facial palsy , etc9 ) . therefore , many efforts have been made to reduce the rates of complications following mvd via neurophysiologic monitoring and/or modified surgical techniques2,8 - 10,12 ) . currently , the rate of facial palsy following mvd is very low , and the rates of cerebellar injury and hearing loss have been down by half , approximately less than 1%9 ) . however , the rate of csf leakage , often causing fatal meningitis , remains high and has been reported to range from approximately 1% to 5%3,9,13,14 ) . one of the most important aspects of mvd with respect to the avoidance of csf leakage is a watertight dural closure9 ) . moreover , the appropriate choice of dural substitutes can also lower the risk of csf leakage11,12 ) . closed - suction drainage has traditionally been used to prevent postoperative hematoma or in cases of uncertain hemostasis6 ) . however , the quoted incidence of intracranial hematoma following mvd is only 0.4 - 0.5%5,7 ) . moreover , epidural hematoma at the surgical site , for which only closed - suction drainage can work , appears to be even rarer . furthermore , closed - suction drainage following mvd can cause csf leakage ( jacques magnan ; personal communication , september 9 , 2010 ) . we compared a traditional retrosigmoid suboccipital craniotomy using closed - suction drainage to a minimally invasive small craniectomy without drainage . we performed this study to investigate whether closed - suction drainage following mvd causes csf leakage by comparing the incidences of csf leakage following mvd for both of these surgical procedures . between 2004 and 2011 , a total of 157 patients with neurovascular compression were treated with mvd . of these , mvd was performed for hfs in 150 ( 95.5% ) cases and for tn in 7 ( 4.5% ) cases . our surgical procedures for mvd included a traditional retromastoid craniotomy and a minimally invasive approach in which a small 2 - 2.5 cm retromastoid craniectomy using a bony landmark of the incisura mastoidea or the mastoid notch , as described elsewhere4,9 ) . a three - point head - fixation was applied while the patient is in the supine lateral position . after shaving the area 4 to 5 cm behind the ear , the mastoid tip and notch were identified and marked . a lazy - s skin incision of 8 to 10 cm in length and a half above the mastoid notch was made along the hair line . a keyhole was drawn on the asterion , and the junction of the transverse sinus and sigmoid sinus was identified . a bone flap of approximately 4 cm in size was removed using a high - speed drill , and bony exposure was laterally extended to identify the posterior margin of the sigmoid sinus . the incision of the dura mater was made along the posterior margin of the sigmoid sinus . following decompression of the offending vessel(s ) from the cranial nerves , the bone edges of the mastoid air cells were thoroughly sealed with bone wax . a watertight dural closure was subsequently performed , and dural substitutes were used as necessary . fibrin glue was applied on the epidural space , and the bone flap was repositioned usually with a titanium mesh . before approximation of the deep and superficial muscles , a closed - suction drainage system ( hemovac ; generally 400 cc ) was inserted into the subgaleal space between the bone flap and the muscle layer . early mobilization of the patients was encouraged on postoperative day 1 , and the patients were discharged following resolution of postoperative dizziness and headache . with the patient in the supine position , the head was rotated approximately 20 to 30 away from the affected side without the use of head - fixation . the mastoid tip and notch were identified and marked , and a 1 cm - wide area was shaved behind the hairline . a 4 to 5 cm curvilinear skin incision is made along the hairline , in which three quarters were below the mastoid notch in cases of hfs and a half above the mastoid notch in cases of tn . following the identification of the digastric groove , a 2 to 2.5 cm craniectomy was performed below the digastric groove for hfs and above the digastric groove for tn4,9 ) . an incision of the dura mater was made along the inferoposterior margin of the sigmoid sinus . the offending vessel(s ) were decompressed from the cranial nerves , and the bone edges of the mastoid air cells were waxed , as described above . if required , pieces of muscle from the adjacent neck muscles were used to perform the plugging muscle method , as described elsewhere12 ) . following application of fibrinogen / thrombin - based collagen fleece ( tachocomb ; nycomed , linz , austria ) for additional sealing of the dural incision and to achieve hemostasis of the outer surface of the exposed dura mater , a cranioplasty was performed usually using polymethylmethacrylate bone cement . finally , the deep and superficial muscles were approximated without using closed - suction drainage . csf leakage following mvd was defined as follows : 1 ) rhinorrhea , otorrhea , pseudomeningocele , and/or an incisional leak at any time following mvd irrespective of the development of central nervous system infections1 ) ; 2 ) middle ear effusions , generally accompanied by ear fullness , identified by thorough otological evaluations7 ) ; 3 ) csf leakage developing on postoperative day 2 or later , considering the time required for drainage of the fluid that potentially flowed into the mastoid air cells before waxing of the inlet during the procedure ; and 4 ) persistence of csf leakage for 2 or more days ; in cases of middle ear effusion , effusion was identified at the follow - up otological evaluation 2 or 3 days following the initial exam . the duration of closed - suction drainage was defined as the interval between skin closure to the removal or clamping of the drainage system . a single rater , who was blind to the clinical outcome data and the development of csf leakage , reviewed all of the immediate postoperative computed tomography ( ct ) scans . this was performed to determine whether the mastoid air cells were opened during mvd or not , which may influence the development of middle ear effusion due to csf leakage . the ct scan was obtained at a 2.5 - 5-mm thickness ( brilliance ct ; philips medical systems , wa , usa ) . the clinical outcome of the neurovascular compression with respect to the surgical method was not a principal concern of this study and was not evaluated . all of the patient data were based on information that was contained in hospital charts . the radiological studies were collected in accordance with the case record form , which was approved by the institutional review board . the incidence of csf leakage was compared between the two groups of patients using fisher 's exact test . student 's t - test was performed to compare the means of the continuous variables between the groups . to reduce the probability of type ii errors considering the modest sample size , variables were considered for the multivariate analysis only if they were associated with a dependent variable in each analysis at the p<0.10 level . when the estimate of the coefficient was zero or extreme , exact logistic regression was used to obtain a median unbiased estimate . these statistical analyses were performed using pasw statistics software version 17.0.2 ( spss inc . , chicago , il , usa ) and the stata software package version 12.0 ( stata corp . , college station , tx , usa ) . a three - point head - fixation was applied while the patient is in the supine lateral position . after shaving the area 4 to 5 cm behind the ear , a lazy - s skin incision of 8 to 10 cm in length and a half above the mastoid notch was made along the hair line . a keyhole was drawn on the asterion , and the junction of the transverse sinus and sigmoid sinus was identified . a bone flap of approximately 4 cm in size was removed using a high - speed drill , and bony exposure was laterally extended to identify the posterior margin of the sigmoid sinus . the incision of the dura mater was made along the posterior margin of the sigmoid sinus . following decompression of the offending vessel(s ) from the cranial nerves , the bone edges of the mastoid air cells were thoroughly sealed with bone wax . a watertight dural closure was subsequently performed , and dural substitutes were used as necessary . fibrin glue was applied on the epidural space , and the bone flap was repositioned usually with a titanium mesh . before approximation of the deep and superficial muscles , a closed - suction drainage system ( hemovac ; generally 400 cc ) was inserted into the subgaleal space between the bone flap and the muscle layer . early mobilization of the patients was encouraged on postoperative day 1 , and the patients were discharged following resolution of postoperative dizziness and headache . with the patient in the supine position , the head was rotated approximately 20 to 30 away from the affected side without the use of head - fixation . the mastoid tip and notch were identified and marked , and a 1 cm - wide area was shaved behind the hairline . a 4 to 5 cm curvilinear skin incision is made along the hairline , in which three quarters were below the mastoid notch in cases of hfs and a half above the mastoid notch in cases of tn . following the identification of the digastric groove , a 2 to 2.5 cm craniectomy was performed below the digastric groove for hfs and above the digastric groove for tn4,9 ) . an incision of the dura mater was made along the inferoposterior margin of the sigmoid sinus . the offending vessel(s ) were decompressed from the cranial nerves , and the bone edges of the mastoid air cells were waxed , as described above . if required , pieces of muscle from the adjacent neck muscles were used to perform the plugging muscle method , as described elsewhere12 ) . following application of fibrinogen / thrombin - based collagen fleece ( tachocomb ; nycomed , linz , austria ) for additional sealing of the dural incision and to achieve hemostasis of the outer surface of the exposed dura mater , a cranioplasty was performed usually using polymethylmethacrylate bone cement . finally , the deep and superficial muscles were approximated without using closed - suction drainage . csf leakage following mvd was defined as follows : 1 ) rhinorrhea , otorrhea , pseudomeningocele , and/or an incisional leak at any time following mvd irrespective of the development of central nervous system infections1 ) ; 2 ) middle ear effusions , generally accompanied by ear fullness , identified by thorough otological evaluations7 ) ; 3 ) csf leakage developing on postoperative day 2 or later , considering the time required for drainage of the fluid that potentially flowed into the mastoid air cells before waxing of the inlet during the procedure ; and 4 ) persistence of csf leakage for 2 or more days ; in cases of middle ear effusion , effusion was identified at the follow - up otological evaluation 2 or 3 days following the initial exam . the duration of closed - suction drainage was defined as the interval between skin closure to the removal or clamping of the drainage system . a single rater , who was blind to the clinical outcome data and the development of csf leakage , reviewed all of the immediate postoperative computed tomography ( ct ) scans . this was performed to determine whether the mastoid air cells were opened during mvd or not , which may influence the development of middle ear effusion due to csf leakage . the ct scan was obtained at a 2.5 - 5-mm thickness ( brilliance ct ; philips medical systems , wa , usa ) . the clinical outcome of the neurovascular compression with respect to the surgical method was not a principal concern of this study and was not evaluated . all of the patient data were based on information that was contained in hospital charts . the radiological studies were collected in accordance with the case record form , which was approved by the institutional review board . the incidence of csf leakage was compared between the two groups of patients using fisher 's exact test . student 's t - test was performed to compare the means of the continuous variables between the groups . to reduce the probability of type ii errors considering the modest sample size , variables were considered for the multivariate analysis only if they were associated with a dependent variable in each analysis at the p<0.10 level . when the estimate of the coefficient was zero or extreme , exact logistic regression was used to obtain a median unbiased estimate . these statistical analyses were performed using pasw statistics software version 17.0.2 ( spss inc . , chicago , il , usa ) and the stata software package version 12.0 ( stata corp . , college station , tx , usa ) . ninety - two ( 58.6% ) patients underwent mvd using closed - suction drainage and were entered into the ' drainage group ' . mvd without closed - suction drainage was performed in 65 ( 41.4% ) patients , designated the ' no - drainage group ' . in 44 ( 28.0% ) patients , dural substitutes , such as neuro - patch ( b. braun ) or muscle pieces were used ; a primary watertight dural closure without the use of dural substitutes was performed in the remaining patients . the mastoid air cells were opened during the surgery in 78 ( 49.7% ) patients based on the routine postoperative ct scan . among 92 patients in the drainage group , the closed - suction drainage was removed or clamped due to csf drainage via the drainage system in 57 patients ( 62.0% ) , due to air drainage in 10 patients ( 10.9% ) , and due to no or very little drainage in 23 patients ( 25.0% ) . in two ( 2.2% ) patients , the closed - suction drainage was removed due to an additional surgery for postoperative supratentorial epidural hematoma . the mean duration of the closed - suction drainage was 20.911.6 hours ( range , 0 - 48.3 ) . in two ( 2.2% ) patients , drainage was not performed following the placement of the closed - suction drainage because the csf was drained immediately after the surgery ; the drainage was therefore subsequently clamped and removed . a total of 11 ( 7.0% ) patients experienced csf leakage following mvd based on the criteria of this study . interestingly , all of these patients were in the drainage group ( 12.0% of the drainage group ) . eight of these patients were conservatively treated with bed rest and local management , such as a mastoid compression dressing . in two of these patients , continuous lumbar drainage was put in place for 5 days , and csf leakage was successfully controlled . one 50-year - old male , whose drainage system drained air immediately following the surgery , underwent an additional surgery on the 13th postoperative day for a cerebellar abscess combined with meningitis and middle ear effusion ( fig . one patient exhibited a trace of slightly yellow fluid on her pillow on postoperative day 4 . however , no evidence of middle ear effusion or wound leakage was identified , even following a thorough otological evaluation . no further event suggesting csf leakage developed thereafter . thus , based on our criteria of csf leakage , no patient in the no - drainage group experienced csf leakage following mvd . in the unadjusted analyses , the incidence of csf leakage was significantly related with the use of closed - suction drainage following mvd ( 12.0% in the drainage group vs. 0% in the no - drainage group , respectively ; p=0.003 ; fisher 's exact test ) . six ( 13.6% ) of the 44 patients for whom dural substitutes were used experienced csf leakage , as did 5 ( 4.4% ) of the 113 patients who did not receive dural substitutes ; however , this effect did not reach statistical significance ( p=0.075 ; fisher 's exact test ) . lastly , the elderly patients ( cut - off value=60 years ) also exhibited a trend toward an increased risk of csf leakage ( p=0.090 ; fisher 's exact test ) ; however , this effect also did not reach statistical significance . the opening of the mastoid air cells during surgery was unrelated to the development of csf leakage following mvd ( p=0.369 ; fisher 's exact test ) . in the multivariate analysis , only the use of closed - suction drainage was significantly and independently related with the development of csf leakage following mvd ( odds ratio=9.900 ; 95% confidence interval , 1.418 to infinity ; p=0.017 ) . the results of the statistical analyses are summarized in table 3 . a postoperative infection developed in 5 ( 3.2% ) patients . one patient in the drainage group experienced a cerebellar abscess following a middle ear effusion , as mentioned above . another patient in the drainage group developed pseudomeningocele combined with meningitis and was treated conservatively . in the no - drainage group , 3 patients experienced postoperative infection ; one with aseptic meningitis and two successive cases of superficial wound infection caused by serratia marcescens . an asymptomatic small amount of supratentorial subdural hematoma was observed on the routine immediate postoperative ct scan in 3 ( 1.9% ) patients ( 2 in the drainage group and 1 in the no - drainage group ) . two ( 1.3% ) patients in the no - drainage group developed ecchymosis in the neck near the surgical wound ; however , they did not exhibit related symptoms or require further management . interestingly , 4 ( 2.5% ) patients experienced an immediate postoperative seizure without any evidence of intracranial hemorrhage . a total of 11 ( 7.0% ) patients experienced csf leakage following mvd based on the criteria of this study . interestingly , all of these patients were in the drainage group ( 12.0% of the drainage group ) . eight of these patients were conservatively treated with bed rest and local management , such as a mastoid compression dressing . in two of these patients , continuous lumbar drainage was put in place for 5 days , and csf leakage was successfully controlled . one 50-year - old male , whose drainage system drained air immediately following the surgery , underwent an additional surgery on the 13th postoperative day for a cerebellar abscess combined with meningitis and middle ear effusion ( fig . one patient exhibited a trace of slightly yellow fluid on her pillow on postoperative day 4 . however , no evidence of middle ear effusion or wound leakage was identified , even following a thorough otological evaluation . no further event suggesting csf leakage developed thereafter thus , based on our criteria of csf leakage , no patient in the no - drainage group experienced csf leakage following mvd . in the unadjusted analyses , the incidence of csf leakage was significantly related with the use of closed - suction drainage following mvd ( 12.0% in the drainage group vs. 0% in the no - drainage group , respectively ; p=0.003 ; fisher 's exact test ) . six ( 13.6% ) of the 44 patients for whom dural substitutes were used experienced csf leakage , as did 5 ( 4.4% ) of the 113 patients who did not receive dural substitutes ; however , this effect did not reach statistical significance ( p=0.075 ; fisher 's exact test ) . lastly , the elderly patients ( cut - off value=60 years ) also exhibited a trend toward an increased risk of csf leakage ( p=0.090 ; fisher 's exact test ) ; however , this effect also did not reach statistical significance . the opening of the mastoid air cells during surgery was unrelated to the development of csf leakage following mvd ( p=0.369 ; fisher 's exact test ) . in the multivariate analysis , only the use of closed - suction drainage was significantly and independently related with the development of csf leakage following mvd ( odds ratio=9.900 ; 95% confidence interval , 1.418 to infinity ; p=0.017 ) . one patient in the drainage group experienced a cerebellar abscess following a middle ear effusion , as mentioned above . another patient in the drainage group developed pseudomeningocele combined with meningitis and was treated conservatively . in the no - drainage group , 3 patients experienced postoperative infection ; one with aseptic meningitis and two successive cases of superficial wound infection caused by serratia marcescens . an asymptomatic small amount of supratentorial subdural hematoma was observed on the routine immediate postoperative ct scan in 3 ( 1.9% ) patients ( 2 in the drainage group and 1 in the no - drainage group ) . two ( 1.3% ) patients in the no - drainage group developed ecchymosis in the neck near the surgical wound ; however , they did not exhibit related symptoms or require further management . interestingly , 4 ( 2.5% ) patients experienced an immediate postoperative seizure without any evidence of intracranial hemorrhage . the role of closed - suction drainage following surgery has been thoroughly investigated for various procedures to prevent postoperative hematoma and infection - related hematoma in the dead space6 ) . however , the usefulness of closed - suction drainage following mvd has not been thoroughly investigated despite the quoted low incidence of intracranial hematoma following mvd and the potential harm of closed - suction drainage . fortunately , we were able to address this issue by virtue of our modification of the mvd procedure , which improves the surgical outcome and reduces the rates of complications . based on our data , closed - suction drainage appears to be significantly associated with csf leakage and postoperative infection following mvd . and the majority of the placed drainage systems were removed or clamped within one day or immediately after the surgery . furthermore , considering the rate of 10% ( one patient ) of cerebellar abscess and meningitis among the 10 patients who exhibited air drainage via their closed - suction drainage systems , air drainage may cause a fatal central nervous system infection , such as a cerebellar abscess . although the pathogenesis of csf leakage following mvd is still debated , one of the most important aspects of mvd with respect to the avoidance of csf leakage is a primary watertight dural closure9,12 ) . many dural substitutes have been developed and are used to make a watertight dural closure when a primary watertight dural closure is impossible due to shrinkage of the dural margins . the appropriate choice of dural substitute also appears to affect the risk of csf leakage11 ) . nonetheless , the use of dural substitutes tended to encourage csf leakage in our study , although this effect did not reach statistical significance and was dropped from the multivariate analysis . one of the most notable aspects of this study was the definition of csf leakage . many studies have examined csf leakage following lateral skull base surgeries for neurovascular compression , vestibular schwannoma , etc1,7,15 ) . , middle ear effusion was regarded as csf leakage when it was identified by otological evaluations and persisted for 2 or more days following mvd . this definition appears to be a possible reason for the relatively high incidence of csf leakage ( 12.0% ) observed in the drainage group . however , considering that not even middle ear effusion developed in the no - drainage group , for which there was a higher incidence of opening of the mastoid aircells , closed - suction drainage appears to be one of major causes of csf leakage following mvd . another interesting finding was that remote supratentorial epidural hematoma developed only in the drainage group . although the pathogenesis of remote supratentorial epidural hematoma following mvd is unclear , excessive csf draining via the closed - suction drainage system may be a principal factor . the same explanation may be applied for supratentorial subdural hematoma , although this condition did not cause any neurological symptoms in our cohort . this study has several limitations , including a retrospective design , a relatively small sample size , and possible biases , such as the use of dural substitutes and different surgical methods . however , the effects of these limitations on the primary conclusions of this manuscript are likely minimal . the use of closed - suction drainage following mvd appears to be related to the development of csf leakage .

objectivewe performed this study to investigate whether the use of closed - suction drainage following microvascular decompression ( mvd ) causes cerebrospinal fluid ( csf ) leakage.methodsbetween 2004 and 2011 , a total of 157 patients with neurovascular compression were treated with mvd . mvd was performed for hemifacial spasm in 150 ( 95.5% ) cases and for trigeminal neuralgia in 7 ( 4.5% ) cases . the mean age of the patients was 49.89.6 years ( range , 20 - 69 ) . dural substitutes were used in 44 ( 28.0% ) patients . ninety - two patients ( 58.6% ) were underwent a 4 - 5 cm craniotomy using drainage ( drainage group ) , and 65 ( 41.4% ) did a small 2 - 2.5 cm retromastoid craniectomy without closed - suction drainage ( no - drainage group).resultseleven ( 7.0% ) patients experienced csf leakage following mvd based on the criteria of this study ; all of these patients were in the drainage group . in the unadjusted analyses , the incidence of csf leakage was significantly related with the use of closed - suction drainage following mvd ( 12.0% in the drainage group vs. 0% in the no - drainage group , respectively ; p=0.003 ; fisher 's exact test ) . those who received dural substitutes and the elderly ( cut - off value=60 years ) exhibited a tendency to develop csf leakage ( p=0.075 and p=0.090 , respectively ; fisher 's exact test ) . in the multivariate analysis , only the use of closed - suction drainage was significantly and independently associated with the development of csf leakage following mvd ( odds ratio=9.900 ; 95% confidence interval , 1.418 to infinity ; p=0.017).conclusionthe use of closed - suction drainage following mvd appears to be related to the development of csf leakage .

INTRODUCTION MATERIALS AND METHODS A traditional retromastoid craniotomy with closed-suction drainage A mastoid notch keyhole craniectomy without closed-suction drainage Definition of CSF leakage following MVD and statistical analysis RESULTS CSF leakage and closed-suction drainage Other complications DISCUSSION CONCLUSION

microvascular decompression ( mvd ) was introduced in the late 1960s and is a widely accepted treatment for neurovascular compression , including hemifacial spasm ( hfs ) , trigeminal neuralgia ( tn ) , and glossopharyngeal neuralgia . however , to obtain these benefits , patients are subjected to a slightly higher treatment risk , including the risk of cerebellar injury , hearing loss , cerebrospinal fluid ( csf ) leakage , facial palsy , etc9 ) . currently , the rate of facial palsy following mvd is very low , and the rates of cerebellar injury and hearing loss have been down by half , approximately less than 1%9 ) . however , the rate of csf leakage , often causing fatal meningitis , remains high and has been reported to range from approximately 1% to 5%3,9,13,14 ) . one of the most important aspects of mvd with respect to the avoidance of csf leakage is a watertight dural closure9 ) . moreover , the appropriate choice of dural substitutes can also lower the risk of csf leakage11,12 ) . closed - suction drainage has traditionally been used to prevent postoperative hematoma or in cases of uncertain hemostasis6 ) . however , the quoted incidence of intracranial hematoma following mvd is only 0.4 - 0.5%5,7 ) . moreover , epidural hematoma at the surgical site , for which only closed - suction drainage can work , appears to be even rarer . furthermore , closed - suction drainage following mvd can cause csf leakage ( jacques magnan ; personal communication , september 9 , 2010 ) . we compared a traditional retrosigmoid suboccipital craniotomy using closed - suction drainage to a minimally invasive small craniectomy without drainage . we performed this study to investigate whether closed - suction drainage following mvd causes csf leakage by comparing the incidences of csf leakage following mvd for both of these surgical procedures . between 2004 and 2011 , a total of 157 patients with neurovascular compression were treated with mvd . of these , mvd was performed for hfs in 150 ( 95.5% ) cases and for tn in 7 ( 4.5% ) cases . our surgical procedures for mvd included a traditional retromastoid craniotomy and a minimally invasive approach in which a small 2 - 2.5 cm retromastoid craniectomy using a bony landmark of the incisura mastoidea or the mastoid notch , as described elsewhere4,9 ) . a keyhole was drawn on the asterion , and the junction of the transverse sinus and sigmoid sinus was identified . a watertight dural closure was subsequently performed , and dural substitutes were used as necessary . fibrin glue was applied on the epidural space , and the bone flap was repositioned usually with a titanium mesh . before approximation of the deep and superficial muscles , a closed - suction drainage system ( hemovac ; generally 400 cc ) was inserted into the subgaleal space between the bone flap and the muscle layer . early mobilization of the patients was encouraged on postoperative day 1 , and the patients were discharged following resolution of postoperative dizziness and headache . with the patient in the supine position , the head was rotated approximately 20 to 30 away from the affected side without the use of head - fixation . following the identification of the digastric groove , a 2 to 2.5 cm craniectomy was performed below the digastric groove for hfs and above the digastric groove for tn4,9 ) . the offending vessel(s ) were decompressed from the cranial nerves , and the bone edges of the mastoid air cells were waxed , as described above . following application of fibrinogen / thrombin - based collagen fleece ( tachocomb ; nycomed , linz , austria ) for additional sealing of the dural incision and to achieve hemostasis of the outer surface of the exposed dura mater , a cranioplasty was performed usually using polymethylmethacrylate bone cement . finally , the deep and superficial muscles were approximated without using closed - suction drainage . csf leakage following mvd was defined as follows : 1 ) rhinorrhea , otorrhea , pseudomeningocele , and/or an incisional leak at any time following mvd irrespective of the development of central nervous system infections1 ) ; 2 ) middle ear effusions , generally accompanied by ear fullness , identified by thorough otological evaluations7 ) ; 3 ) csf leakage developing on postoperative day 2 or later , considering the time required for drainage of the fluid that potentially flowed into the mastoid air cells before waxing of the inlet during the procedure ; and 4 ) persistence of csf leakage for 2 or more days ; in cases of middle ear effusion , effusion was identified at the follow - up otological evaluation 2 or 3 days following the initial exam . the duration of closed - suction drainage was defined as the interval between skin closure to the removal or clamping of the drainage system . a single rater , who was blind to the clinical outcome data and the development of csf leakage , reviewed all of the immediate postoperative computed tomography ( ct ) scans . this was performed to determine whether the mastoid air cells were opened during mvd or not , which may influence the development of middle ear effusion due to csf leakage . the clinical outcome of the neurovascular compression with respect to the surgical method was not a principal concern of this study and was not evaluated . all of the patient data were based on information that was contained in hospital charts . the incidence of csf leakage was compared between the two groups of patients using fisher 's exact test . student 's t - test was performed to compare the means of the continuous variables between the groups . to reduce the probability of type ii errors considering the modest sample size , variables were considered for the multivariate analysis only if they were associated with a dependent variable in each analysis at the p<0.10 level . a keyhole was drawn on the asterion , and the junction of the transverse sinus and sigmoid sinus was identified . a watertight dural closure was subsequently performed , and dural substitutes were used as necessary . fibrin glue was applied on the epidural space , and the bone flap was repositioned usually with a titanium mesh . before approximation of the deep and superficial muscles , a closed - suction drainage system ( hemovac ; generally 400 cc ) was inserted into the subgaleal space between the bone flap and the muscle layer . early mobilization of the patients was encouraged on postoperative day 1 , and the patients were discharged following resolution of postoperative dizziness and headache . with the patient in the supine position , the head was rotated approximately 20 to 30 away from the affected side without the use of head - fixation . following the identification of the digastric groove , a 2 to 2.5 cm craniectomy was performed below the digastric groove for hfs and above the digastric groove for tn4,9 ) . the offending vessel(s ) were decompressed from the cranial nerves , and the bone edges of the mastoid air cells were waxed , as described above . following application of fibrinogen / thrombin - based collagen fleece ( tachocomb ; nycomed , linz , austria ) for additional sealing of the dural incision and to achieve hemostasis of the outer surface of the exposed dura mater , a cranioplasty was performed usually using polymethylmethacrylate bone cement . finally , the deep and superficial muscles were approximated without using closed - suction drainage . csf leakage following mvd was defined as follows : 1 ) rhinorrhea , otorrhea , pseudomeningocele , and/or an incisional leak at any time following mvd irrespective of the development of central nervous system infections1 ) ; 2 ) middle ear effusions , generally accompanied by ear fullness , identified by thorough otological evaluations7 ) ; 3 ) csf leakage developing on postoperative day 2 or later , considering the time required for drainage of the fluid that potentially flowed into the mastoid air cells before waxing of the inlet during the procedure ; and 4 ) persistence of csf leakage for 2 or more days ; in cases of middle ear effusion , effusion was identified at the follow - up otological evaluation 2 or 3 days following the initial exam . the duration of closed - suction drainage was defined as the interval between skin closure to the removal or clamping of the drainage system . a single rater , who was blind to the clinical outcome data and the development of csf leakage , reviewed all of the immediate postoperative computed tomography ( ct ) scans . this was performed to determine whether the mastoid air cells were opened during mvd or not , which may influence the development of middle ear effusion due to csf leakage . the clinical outcome of the neurovascular compression with respect to the surgical method was not a principal concern of this study and was not evaluated . all of the patient data were based on information that was contained in hospital charts . the incidence of csf leakage was compared between the two groups of patients using fisher 's exact test . to reduce the probability of type ii errors considering the modest sample size , variables were considered for the multivariate analysis only if they were associated with a dependent variable in each analysis at the p<0.10 level . ninety - two ( 58.6% ) patients underwent mvd using closed - suction drainage and were entered into the ' drainage group ' . mvd without closed - suction drainage was performed in 65 ( 41.4% ) patients , designated the ' no - drainage group ' . in 44 ( 28.0% ) patients , dural substitutes , such as neuro - patch ( b. braun ) or muscle pieces were used ; a primary watertight dural closure without the use of dural substitutes was performed in the remaining patients . the mastoid air cells were opened during the surgery in 78 ( 49.7% ) patients based on the routine postoperative ct scan . among 92 patients in the drainage group , the closed - suction drainage was removed or clamped due to csf drainage via the drainage system in 57 patients ( 62.0% ) , due to air drainage in 10 patients ( 10.9% ) , and due to no or very little drainage in 23 patients ( 25.0% ) . in two ( 2.2% ) patients , the closed - suction drainage was removed due to an additional surgery for postoperative supratentorial epidural hematoma . the mean duration of the closed - suction drainage was 20.911.6 hours ( range , 0 - 48.3 ) . in two ( 2.2% ) patients , drainage was not performed following the placement of the closed - suction drainage because the csf was drained immediately after the surgery ; the drainage was therefore subsequently clamped and removed . a total of 11 ( 7.0% ) patients experienced csf leakage following mvd based on the criteria of this study . interestingly , all of these patients were in the drainage group ( 12.0% of the drainage group ) . eight of these patients were conservatively treated with bed rest and local management , such as a mastoid compression dressing . in two of these patients , continuous lumbar drainage was put in place for 5 days , and csf leakage was successfully controlled . thus , based on our criteria of csf leakage , no patient in the no - drainage group experienced csf leakage following mvd . in the unadjusted analyses , the incidence of csf leakage was significantly related with the use of closed - suction drainage following mvd ( 12.0% in the drainage group vs. 0% in the no - drainage group , respectively ; p=0.003 ; fisher 's exact test ) . six ( 13.6% ) of the 44 patients for whom dural substitutes were used experienced csf leakage , as did 5 ( 4.4% ) of the 113 patients who did not receive dural substitutes ; however , this effect did not reach statistical significance ( p=0.075 ; fisher 's exact test ) . lastly , the elderly patients ( cut - off value=60 years ) also exhibited a trend toward an increased risk of csf leakage ( p=0.090 ; fisher 's exact test ) ; however , this effect also did not reach statistical significance . the opening of the mastoid air cells during surgery was unrelated to the development of csf leakage following mvd ( p=0.369 ; fisher 's exact test ) . in the multivariate analysis , only the use of closed - suction drainage was significantly and independently related with the development of csf leakage following mvd ( odds ratio=9.900 ; 95% confidence interval , 1.418 to infinity ; p=0.017 ) . one patient in the drainage group experienced a cerebellar abscess following a middle ear effusion , as mentioned above . another patient in the drainage group developed pseudomeningocele combined with meningitis and was treated conservatively . in the no - drainage group , 3 patients experienced postoperative infection ; one with aseptic meningitis and two successive cases of superficial wound infection caused by serratia marcescens . an asymptomatic small amount of supratentorial subdural hematoma was observed on the routine immediate postoperative ct scan in 3 ( 1.9% ) patients ( 2 in the drainage group and 1 in the no - drainage group ) . two ( 1.3% ) patients in the no - drainage group developed ecchymosis in the neck near the surgical wound ; however , they did not exhibit related symptoms or require further management . interestingly , 4 ( 2.5% ) patients experienced an immediate postoperative seizure without any evidence of intracranial hemorrhage . a total of 11 ( 7.0% ) patients experienced csf leakage following mvd based on the criteria of this study . interestingly , all of these patients were in the drainage group ( 12.0% of the drainage group ) . eight of these patients were conservatively treated with bed rest and local management , such as a mastoid compression dressing . in two of these patients , continuous lumbar drainage was put in place for 5 days , and csf leakage was successfully controlled . no further event suggesting csf leakage developed thereafter thus , based on our criteria of csf leakage , no patient in the no - drainage group experienced csf leakage following mvd . in the unadjusted analyses , the incidence of csf leakage was significantly related with the use of closed - suction drainage following mvd ( 12.0% in the drainage group vs. 0% in the no - drainage group , respectively ; p=0.003 ; fisher 's exact test ) . six ( 13.6% ) of the 44 patients for whom dural substitutes were used experienced csf leakage , as did 5 ( 4.4% ) of the 113 patients who did not receive dural substitutes ; however , this effect did not reach statistical significance ( p=0.075 ; fisher 's exact test ) . lastly , the elderly patients ( cut - off value=60 years ) also exhibited a trend toward an increased risk of csf leakage ( p=0.090 ; fisher 's exact test ) ; however , this effect also did not reach statistical significance . the opening of the mastoid air cells during surgery was unrelated to the development of csf leakage following mvd ( p=0.369 ; fisher 's exact test ) . in the multivariate analysis , only the use of closed - suction drainage was significantly and independently related with the development of csf leakage following mvd ( odds ratio=9.900 ; 95% confidence interval , 1.418 to infinity ; p=0.017 ) . one patient in the drainage group experienced a cerebellar abscess following a middle ear effusion , as mentioned above . another patient in the drainage group developed pseudomeningocele combined with meningitis and was treated conservatively . in the no - drainage group , 3 patients experienced postoperative infection ; one with aseptic meningitis and two successive cases of superficial wound infection caused by serratia marcescens . an asymptomatic small amount of supratentorial subdural hematoma was observed on the routine immediate postoperative ct scan in 3 ( 1.9% ) patients ( 2 in the drainage group and 1 in the no - drainage group ) . two ( 1.3% ) patients in the no - drainage group developed ecchymosis in the neck near the surgical wound ; however , they did not exhibit related symptoms or require further management . interestingly , 4 ( 2.5% ) patients experienced an immediate postoperative seizure without any evidence of intracranial hemorrhage . the role of closed - suction drainage following surgery has been thoroughly investigated for various procedures to prevent postoperative hematoma and infection - related hematoma in the dead space6 ) . however , the usefulness of closed - suction drainage following mvd has not been thoroughly investigated despite the quoted low incidence of intracranial hematoma following mvd and the potential harm of closed - suction drainage . based on our data , closed - suction drainage appears to be significantly associated with csf leakage and postoperative infection following mvd . furthermore , considering the rate of 10% ( one patient ) of cerebellar abscess and meningitis among the 10 patients who exhibited air drainage via their closed - suction drainage systems , air drainage may cause a fatal central nervous system infection , such as a cerebellar abscess . although the pathogenesis of csf leakage following mvd is still debated , one of the most important aspects of mvd with respect to the avoidance of csf leakage is a primary watertight dural closure9,12 ) . nonetheless , the use of dural substitutes tended to encourage csf leakage in our study , although this effect did not reach statistical significance and was dropped from the multivariate analysis . one of the most notable aspects of this study was the definition of csf leakage . many studies have examined csf leakage following lateral skull base surgeries for neurovascular compression , vestibular schwannoma , etc1,7,15 ) . this definition appears to be a possible reason for the relatively high incidence of csf leakage ( 12.0% ) observed in the drainage group . however , considering that not even middle ear effusion developed in the no - drainage group , for which there was a higher incidence of opening of the mastoid aircells , closed - suction drainage appears to be one of major causes of csf leakage following mvd . another interesting finding was that remote supratentorial epidural hematoma developed only in the drainage group . although the pathogenesis of remote supratentorial epidural hematoma following mvd is unclear , excessive csf draining via the closed - suction drainage system may be a principal factor . this study has several limitations , including a retrospective design , a relatively small sample size , and possible biases , such as the use of dural substitutes and different surgical methods . however , the effects of these limitations on the primary conclusions of this manuscript are likely minimal . the use of closed - suction drainage following mvd appears to be related to the development of csf leakage .

the issue of pharmaceuticals in the environment has come to the fore in recent years with the realization that these drugs , designed to act at specific mammalian targets , may have effects in non - target organisms provided that the molecular target ( usually a receptor or enzyme ) is conserved . was first articulated by huggett et al . , but the concept of target conservation between vertebrate groups had been recognized previously . the hypothesis also presumes that a specific interaction between a drug and a target will result in a pharmacological response before a toxicological one , and will require a plasma concentration similar to that necessary to see a pharmacological effect in humans . if this hypothesis is correct , it should be possible to use information derived from the drug development process , such as molecular target , mode of action , effective dose and physicochemical properties , together with genomic and physiological knowledge of the non - target organism , to predict the likelihood of an effect occurring , assuming relevant exposure . this concept is not new ; the well - established use of rodents in the drug development process relies on the similarity of drug targets and physiology with humans . the use of model organisms such as danio rerio ( zebrafish ) in drug screening indicates that it is possible to read up from a lower vertebrate such as a fish to humans , and our increased knowledge of the genomes and physiology of fish has confirmed that they are in many respects very similar to mammals , including the presence of drug targets and similar responses . it should be noted that this hypothesis relates to biological read - across , as opposed to the chemical read - across approach used to predict toxicity from untested chemicals of the same class , or indeed addressing general toxicity from a battery of tests . the theoretical fish plasma model presented by huggett et al . aims to assess whether a pharmaceutical present in the environment is likely to have an effect on fish ( figure 1 ) . the model compares the human therapeutic plasma concentration ( cmax ) of a drug to a predicted steady state concentration in fish , calculated using log kow ( octanol : water partition coefficient , a measure of lipophilicity ) and environmental concentration ( measured or predicted ) . the closer the predicted fish plasma concentration is to cmax , the greater the likelihood that a pharmacological effect will be seen , although it should be noted that log kow is affected by the pka and may therefore not give an accurate estimate for ionogenic compounds . there are over 3000 human pharmaceuticals in general use , and many of these have been detected in surface waters and sewage treatment work effluents . thus , aquatic organisms may be continually exposed to a cocktail of drugs , in contrast to the transient nature of human drug exposure . it is unrealistic to assess each drug experimentally , and although environmental concentrations are low ( in the ng / l to low g / l range ) , it can not be presumed that no effects will occur ( figure 1 ) . experimental validation of the fish plasma model will provide a framework for identifying the drugs that are of most concern . the past decade has seen a proliferation of studies relating to effects of pharmaceuticals on fish and other organisms , although few of these have explicitly tested the read - across hypothesis . application of the fish plasma model to two pharmaceuticals , ethinylestradiol ( ee2 ) and atenolol . the model compares the human therapeutic plasma concentration ( htpc ) and the predicted fish steady - state plasma concentration ( fsspc ) . if htpc fsspc , the risk of a pharmaceutical having pharmacological effects in fish is high . ee2 at environmentally relevant concentrations ( i.e. , pec ) will produce fsspc htpc , indicating a high risk of pharmacological effects occurring . atenolol , which is highly hydrophilic , does not bioaccumulate to a significant extent , resulting in fsspc htpc and therefore no effect is expected . this review will provide a critical commentary of the extent to which these studies , mostly concerned with human pharmaceuticals , have validated the read - across hypothesis in general , and the fish plasma model in particular . it will also highlight many of the factors that influence the applicability of the hypothesis . we will focus mainly on fish , as these are recognized as obvious indicators of aquatic contamination , but the increasing evidence for extending the read - across concept to invertebrates , and even plants , will also be considered . it is beyond the scope of this review to discuss test substance concentrations in tissues other than plasma , as this would not add to the evidence - base for the read - across hypothesis , given that tissue concentrations are not reported in relation to pharmacological effects in humans . the evolutionary conservation of the primary drug target is a prerequisite for the read - across hypothesis , and the key paper by gunnarsson et al they looked at 1318 human drug targets across 16 species , and found 86% to be conserved in zebrafish , 61% in daphnia pulex ( water flea ) and 35% in chlamydomonas reinhardtii ( green algae ) , indicating the potential for lower vertebrates , and even invertebrates and plants , to respond to pharmaceuticals present in the environment . interestingly , enzymes were especially well conserved across all species , whereas receptors were not . this suggests that drugs targeting enzymes may potentially affect a greater number of species than a drug targeting a receptor , although there are insufficient studies to date to provide convincing evidence for this in practice . the increasing knowledge of gene sequences from a range of species should facilitate our predictive capacity for assessing target conservation . for example , 5-reductase , the enzyme converting testosterone to its more potent metabolite dihydrotestosterone , has only recently been identified and characterized in fish ( pimephales promelas , fathead minnow ) , and phylogenetic analysis suggests homologues to be present in molluscs and nematodes , as well as plants ( e.g. , arabidopsis ) ( figure 2 ) . the arabidopsis homologue , det2 , is particularly interesting , as it is known to play a role in light - regulated development , and is inhibited by 4-ma , one of the 4-azasteroids which are potent inhibitors of mammalian 5-reductase . thus , 5-reductase inhibitors used to treat benign prostatic hyperplasia and male - pattern baldness , if present in the aquatic environment , could potentially affect not just fish , but a range of aquatic organisms , including plants . phylogenetic tree of the enzymes 5-alpha reductase type 1 ( srd5a1 ) and type 2 ( srd5a2 ) from 25 species ranging from human to plants . the evolutionary distances were computed using the jones thornton taylor ( jtt ) matrix - based method . amino acid sequences were aligned using muscle and the evolutionary analyses were conducted in mega5 . the sequences and their accession numbers are detailed in supporting information table s1 . however , the potential complexity in crossing species boundaries should not be underestimated ; for example , many receptors exist in multiple subtypes . our understanding of the differential regulation and functional roles of receptor subtypes is often incomplete , and therefore the possibility that a drug may interact with any subtype present , irrespective of the specificity displayed in humans , can not be discounted . thus , the apparent absence of a particular subtype in a species does not mean that a specifically targeted drug will have no mode of action effect . it is also possible that a single receptor in a lower vertebrate fulfils the roles of several receptor subtypes in humans . little attention is usually paid to the conservation of secondary targets , such as proteins in the pathway downstream of the primary target , which is an implicit requirement for the read - across hypothesis to be fully valid . however , the importance of establishing this may be questionable , as generally an absence of the primary therapeutic effect would indicate that the particular pathway has not been conserved . a greater issue , therefore , is a lack of knowledge regarding the physiology of organisms such as fish at the level of , for example , receptor signaling . if we do not understand the particular role of a receptor in the normal physiology of an organism , that is , the consequences of its activation or inhibition , it becomes unclear whether effects seen following exposure to a particular pharmaceutical are due to specific effects caused at a particular target , a side - effect , or indeed a toxic effect . incidentally , the biochemical , metabolic and cell signaling pathways are often better understood in invertebrates such as drosophila and caenorhabditis elegans , but the sequence conservation of drug targets is generally lower ( 3842% ) . an understanding of what is a side effect and what is a secondary consequence of the primary drug action is also required ; thus , the cardiovascular risk associated with cyclo - oxygenase ( cox ) enzyme inhibition is a direct pharmacological consequence of inhibiting cox 2 , and not due to a side effect of nonsteroidal anti - inflammatory drugs ( nsaids ) . given our lack of knowledge of how physiological responses are linked to specific targets in many nonmammalian organisms , our ( in)ability to recognize effects , let alone side - effects , in non - target organisms will also compromise our ability to validate the read - across hypothesis . we currently do not have the validated techniques to assess if many drugs are affecting fish , let alone lower vertebrates , invertebrates or plants . further , high variability in specific physiological endpoints often makes realistic study design impossible , or analysis prohibitively expensive . conservation of the drug target alone does not guarantee a functional interaction , and therefore experimental studies are required . most studies have been carried out in fish , and in terms of assessing the impact of pharmaceuticals present in the environment , fish are one of the most likely ( but not always the most sensitive ) classes of organisms to be affected . despite the large number of publications where effects of drugs have been studied , there are relatively few ( see supporting information table s2 ) in which effects have been correlated to the internal exposure concentration ( i.e. , amount of substance in the body ) , usually indicated by plasma concentration as opposed to exposure concentration ( amount of substance in the water ) ) . table 1 proposes a classification of experimental studies according to how well they address the read - across hypothesis . a level 1 study provides little or no relevant information , whereas a level 4 study , incorporating measurements of drug exposure and internal concentrations , as well as a mode of action endpoint , will directly address all aspects of the hypothesis . currently , no level 4 study has been published , although one study by valenti et al . gets reasonably close , as discussed below , to this highest level of support for the hypothesis . only studies at level 4 address all aspects of the read - across hypothesis , and can relate effects to the human therapeutic plasma concentration . level 3 studies may be able to confirm that similar effects to those seen in humans occur in aquatic organisms , but without a measure of the internal concentration it is not possible to relate these effects to the human therapeutic plasma concentration , and therefore determine if an aquatic organism is more , or less , sensitive to a particular drug . the importance of having a measure of internal exposure is that some pharmaceuticals can bioconcentrate , perhaps resulting in an internal concentration several hundred times higher than the external concentration . for example , fick et al . demonstrated > 50-fold bioconcentration for 10 out of 16 pharmaceuticals present in sewage effluents . a measure of the internal drug concentration is also required in order to demonstrate that the pharmaceutical is taken up ; in the study by fick et al . it was reported that benzafibrate and telmisartan were not detected in fish plasma despite being present in the sewage effluent , and predicted to bioconcentrate . without knowledge of plasma concentrations , which reflect the pharmacokinetics of the drug , a comparison with human therapeutic concentrations can not be made and incorrect conclusions could be drawn regarding the exposure concentration at which a pharmaceutical elicits a response , and thereby incorrectly estimating the threat to non - target organisms if the drug is present in the environment . an additional requirement for validating the read - across hypothesis is that internal exposure is linked to a biological effect reflecting the primary therapeutic response , by measuring relevant endpoints in physiologically relevant tissues . given the general acceptance of the hypothesis , it is therefore somewhat surprising that only one study to date has confirmed the administered pharmaceutical dose by measuring water concentrations , obtained the internal exposure level by measuring plasma concentrations , linked exposure to a biological effect by measuring relevant endpoints , compared the results to those expected at cmax , and demonstrated that effects were seen only at concentrations similar to those in a human taking the drug . thus , in a poster presented at setac north america , moen et al . demonstrated that the beta - blocker propranolol had the expected cardiac effects in rainbow trout ( onchorynchuss mykiss ) only when plasma concentrations were similar to human therapeutic plasma concentrations . as this study remains , to our knowledge , the only one that fully addresses all aspects of the read - across hypothesis and can therefore be considered a level 4 study ( table 1 ) , it is unfortunate that further details are not available in the peer - reviewed literature . a recent study observed behavioral effects of sertraline ( a selective serotonin reuptake inhibitor used to modify behavior in humans ) in fathead minnows exposed to water concentrations of 2.828.1 g / l . the resulting plasma concentrations ( 305 ng / ml at the lowest exposure concentration ) were close to the normal human therapeutic range ( 50250 ng / ml ) , thus supporting the read - across hypothesis . however , it is unfortunate that the lowest exposure concentration tested resulted in plasma levels similar to human cmax , so we do not know if any effects would have been observed below cmax , a requirement to fully validate the read - across hypothesis . the highest exposure concentration of sertraline used ( 28.1 g / l ) gave a measured plasma concentration of 2000 ng / ml , and is therefore likely to have resulted in plasma concentrations in the toxic range for humans ; however , no ( further ) adverse effects were reported . a previous study , which did not measure plasma concentrations , reported that an exposure concentration of 72 g / l sertraline was lethal to fathead minnows , which would suggest that the plasma concentrations reached in the most recent study were well below the fatal range . the studies reported by zeilinger et al ( 33 ) and fick et al , when considered together , provide strong support for the read - across hypothesis . levonorgestrel , a synthetic progestagen present in oral contraceptives , has been shown to inhibit reproduction of adult fathead minnows at exposure concentrations of 0.8 ng / l and higher . subsequently , it was demonstrated that levonorgestrel was strongly bioconcentrated in rainbow trout exposed to sewage effluent , where a measured concentration of 1 ng / l resulted in a plasma concentration of 8.512 g / l . this exceeds the human therapeutic concentration of 2.4 g / l , and suggests that the mode of action effects observed by zeilinger et al . a number of studies have measured water and plasma concentrations of the pharmaceutical(s ) of interest , but have used endpoints that do not relate to primary therapeutic effects ( see supporting information table s2 for a list of publications ) . these may provide partial support for the read - across hypothesis , in that non - target effects are not seen at human therapeutic plasma concentrations : for example , in fathead minnows , propranolol produced no toxic effects until the plasma concentration exceeded cmax by 100-fold , which occurred at a water concentration of 1 mg / l . similarly , owen et al . observed an effect of propranolol on rainbow trout growth at plasma concentrations above cmax and sufficiently high to be toxic to humans . these studies did not measure cardiac effects , and so it is not clear whether a modulation of heart rate would have occurred at plasma concentrations equal to human therapeutic plasma concentrations . incidentally , owen et al . also reported changes in heart size , and remodelling of the heart is a reported secondary effect of long - term beta - blocker use in humans . there are now a reasonable number of high quality studies that , although they do not include measurement of plasma concentrations of the pharmaceuticals being studied , involve assessing one or more endpoints relevant to the mode of action . one of the first of these studies was a demonstration that ethinylestradiol , a synthetic estrogen used in oral contraceptives , stops fish reproducing , besides having other effects in fish indicative of its estrogenic nature . a number of other classes of steroidal pharmaceuticals have also been shown to have effects on fish appropriate to their mode of action in humans : this includes synthetic glucocorticoids and synthetic progestagens . pharmaceuticals that inhibit the actions of natural steroidal hormones , either because they act as receptor antagonists or inhibit the synthesis of hormones , also have the same effects in fish as they do in humans . for example , the human estrogen receptor antagonist tamoxifen inhibits fish reproduction , as do aromatase inhibitors . fibrates , a lipid - regulating class of drugs used to treat cardiovascular problems in humans , also lower plasma concentrations of lipoproteins in fish . furthermore , antidepressants such as fluoxetine and oxazapam , but not citalopram , affect various aspects of behavior in fish . in all these studies , and others , the effects of the pharmaceuticals on fish were those that would be anticipated if the drugs had the same mode of action in fish as they do in humans . however , because plasma concentrations of the pharmaceuticals were not determined , it is premature to conclude that these level 3 studies ( table 1 ) provide unequivocal support for the read - across hypothesis . they are undoubtedly not in conflict with the hypothesis ( they do not refute it ) , a conclusion supported by using the fish plasma model to predict internal concentrations ( see supporting information table s3 for details ) . however , because effective plasma concentrations of pharmaceuticals can not be compared between humans and fish , the studies provide at best only partial support for the hypothesis . it is more difficult to interpret studies where gene expression has been used as the endpoint of exposure . in particular , if the genes studied are not directly related to the mode of action , it needs to be remembered that gene expression can be responsive to a range of influences that may not be related to the pharmaceutical being tested . thus , the significance of changes in global fathead minnow brain gene expression in response to 4 g / l propranolol ( measured ) for 21 days , and increases in vitellogenin , cyp1a and p53 gene expression in medaka ( oryzia latipes ) liver , gill , and intestine following 4 day exposure to 1 g / l diclofenac , is unclear . indeed , they may not be reproducible , as in the study with propranolol , the exposure concentration was much lower than that required to obtain therapeutic levels . demonstrated that a 14 day exposure of juvenile rainbow trout to 1.681.5 g diclofenac / l , resulting in plasma concentrations 1.588% of cmax , affected hepatic gene expression . these findings could be interpreted to suggest that environmentally relevant concentrations of diclofenac could alter gene expression in fish , but it is unclear whether these genes would also be induced upon exposure to any pharmaceutical , or even as a consequence of something unrelated to drug exposure . however , the authors demonstrated that at plasma diclofenac concentrations close to cmax the majority of differentially regulated genes were functionally associated with inflammation and the immune response , and therefore consistent with the mode of action of diclofenac . a recent study has provided a cautionary note regarding gene expression studies : the lack of validation and knowledge of the complexity of gene expression , small sample size and differential biological impact on individual fish , make it difficult , perhaps even impossible presently , to relate changes in gene expression to drug exposure . the majority of studies concerned with pharmaceuticals in the environment have involved fish . however , huggett et al . recognized the need for studies to be done in invertebrates , algae , and plants , as conceptually these organisms could also respond to pharmaceuticals if the target is conserved . there is increasing evidence that this is the case ; for example , the rotifer brachionus manjavacas contains a proposed progesterone receptor in its reproductive organs , and daphnia magna an eiconsanoid biosynthesis pathway , the target for nsaids . the schistosoma flatworm contains many potential drug targets , such as receptors and enzymes , and appears to have cellular signaling , metabolic and neuroendocrine pathways similar to those present in higher organisms . however , presence of a target is not sufficient on its own , and a major challenge is interpretation of responses ; the response resulting from activation or inhibition of a particular target in an invertebrate or a plant may be very different , and perhaps unrecognizable , from the response seen in a human . it is likely that targets related to fundamental systems such as reproduction , metabolic pathways or detoxification are more conserved in their functions , and therefore easier to recognize as being affected by pharmaceuticals . an example of this would be the enzyme hmgr ( 3-hydroxy-3-methylglutaryl coenzyme a reductase ) , the rate - limiting enzyme in cholesterol biosynthesis and the target for statins . orthologues were predicted in almost all eukaryotes , and statins have been shown to inhibit hmgr activity in plants , fungi and invertebrates . in particular , the work of brain et al . supports the read - across hypothesis : the effects in the plant lemna gibba was seen at low g / l doses , and the molecular endpoint used ( mevalonic acid pathway biosynthesis , which is regulated by hmgr)directly related to the mode of action of the drugs tested was some 23 times more sensitive than a gross morphological endpoint ( wet weight ) . many of the studies carried out in lower eukaryotes are subject to the same criticisms as fish studies . for example , high doses of drugs have been administered , no measurement of the internal exposure concentration was made , or the endpoints used are not mode of action related . it therefore becomes unclear whether the effects observed are related to the equivalent human therapeutic dose ; represent the effects of an overdose ; or indeed occur at a lower exposure concentration than the nominal dose . however , it is probably unrealistic to expect that a full validation of the read - across hypothesis is possible in plants , fungi , and invertebrates , in a large part due to the technical difficulties in measuring internal concentrations in very small organisms . the mode of action principle underpinning the read - across hypothesis does not preclude other effects being seen , but it is presumed that these will occur when the internal concentration exceeds the cmax . therefore , any studies where significant , and perhaps toxic , effects are seen at or below the cmax , would suggest that the read - across hypothesis is not universally applicable , perhaps as a consequence of differences in detoxification mechanisms between humans and non - target organisms . the most well - known example of the effect of a pharmaceutical that does not appear to support the read - across hypothesis is the death of old world vultures ( gyps genus ) , due to kidney failure , following ingestion of diclofenac present in the cadavers of cattle on which they fed . diclofenac , an nsaid , inhibits the cox enzyme , reducing prostaglandin synthesis . the human therapeutic dose is approximately 1.59 mg / kg ( 5003,000 ng / ml plasma ) , with toxic effects , such as irreversible kidney and liver failure , occurring at 150 mg / kg ( 50 000 ng / ml plasma ) . side effects , including renal damage , occur in some 110% of treated patients , indicating that the relatively low safety margin of diclofenac is a problem in sensitive individuals . so were the gyps vultures highly sensitive to diclofenac , resulting in a toxic response at concentrations well below cmax ? diclofenac plasma concentrations may have been as high as 40 000 ng / ml in these vultures ( assuming no metabolism , based on an estimated body weight of 10 kg , an ingestion of 10 mg diclofenac , a plasma volume of 250 ml and a hematocrit of 0.5 ) . this is very close to the human toxic dose of a drug that few veterinary surgeons would prescribe directly for birds . additionally , polymorphic variations in cyp enzymes that influence the metabolism of diclofenac could be important in the sensitivity of gyps vultures to diclofenac . new world vultures , such as turkey vulture ( cathartes aura ) , are remarkably resistant to diclofenac , tolerating doses up to 25 mg / kg for 7 days . in contrast , old world vultures ingesting approximately 1 mg diclofenac / kg died of renal failure . no diclofenac was detected in cathares tissues and the estimated half - life was 6 h whereas , in the gyps vultures , diclofenac residues were found in the kidney and the estimated half - life was 1618 h. thus , the diclofenac and vultures story , while representing the only example where impact at a population level can be attributed to the presence of a pharmaceutical in the environment , does not invalidate the read - across hypothesis . in fact , our view would be that it validates it by demonstrating the expected toxic effects seen in humans . the literature on pharmaceuticals in the environment contains many studies reporting effects at low exposure concentrations . there is probably a need to recognize that drugs act over a range of doses , and in assessing whether a non - target organism displays greater sensitivity than a human , the no observable therapeutic effect level should be considered . probably more of an issue is the fact that these studies have not generally measured the internal exposure concentration or the administered dose ( for example ) , or calculated the theoretical bioconcentration factor , and therefore any observed effects can not be related to what would be expected based on mammalian drug development data . it is therefore not possible to determine whether the responses seen are due to bioconcentration of the drug , and therefore higher dose at target , or whether fish are more sensitive than humans , which could be the implication if no bioconcentration has occurred . in terms of assessing the potential impact of environmental exposures , the conclusions drawn would differ depending on which scenario was correct , and there is , therefore , a need for these studies to be replicated before too much emphasis can be placed on them . it is also difficult to fully interpret studies where tissue , as opposed to plasma , concentrations of a drug have been measured ( for example : diclofenac , antidepressants , gemfibrozil , oxazapam , ) . therefore , the suggestion that bluntnose minnow ( pimephales notatus ) is up to an order of magnitude more sensitive to ibuprofen than humans , based on gill ibuprofen concentrations and the effect on prostaglandin e2 synthesis , must be viewed with caution . while species differences can not be ruled out , the metabolism of ibuprofen in rainbow trout gill microsomes and liver cells appears to be similar to mammals . there are a large number of studies where extremely high doses ( mg / l ) of pharmaceuticals have been administered , or are required , to elicit an effect . for example , de felice et al . demonstrated differences in hepatic gene expression in zebrafish embryos exposed to a nominal concentration of 1.25 mg / l diclofenac for 48 h. similarly , saravanan et al . found changes in a range of blood parameters following exposure of indian major carp ( cirrhinus mrigala ) fingerlings to 14.2 mg / l ( nominal ) ibuprofen for 35 days . the observed changes appear to be related to toxicity rather than mode of action , and therefore may not be drug - specific . additionally , no plasma or body concentrations of the drugs were determined in these studies . the influence of polymorphisms on drug responses is increasingly being recognized in humans , forming the basis of personalized medicine . if polymorphic variation is also relevant for other organisms , we may need to be more aware of the potential side effects of drugs could these become a major effect in some species , or perhaps more likely , some individuals within each species ? it also highlights the fact that we must be aware of potential species differences ( which has implications for the test species used in regulatory testing ) , and allow a sufficient safety margin between environmental and effect concentrations . a recent paper reported variation among amphibian species in their sensitivity to the insecticide endosulfan , which was linked to their phylogenetic groupings . clearly , the aim of the research being carried out under the pharmaceuticals in the environment banner is to aid decision making with regards to the risks drugs may pose to non - target organisms exposed via the environment . several recent publications provide support for the fish plasma model as a tool to prioritise drugs , although it is recognized that the input parameters , in particular the predictor of bioconcentration , should be refined . it is unrealistic for experimental data to be obtained for all pharmaceuticals in use , or on all possible species receiving exposure , but the read - across hypothesis could be tested with a number of drugs , representing different modes of action , and the data obtained used to derive an improved model for predicting likely environmental impact of different drugs , or classes of drugs . the read - across hypothesis relies on a conserved mode of action , and it would therefore be expected that all drugs of a certain class , having the same mode of action , could contribute to an effect . therefore , in considering whether drugs are present in the environment at concentrations likely to cause effects , the total load of a particular class of drugs must be considered . while a mixture situation is not simple similar drugs may have different degrees of uptake , as illustrated by atenolol and propranolol a sufficiently large margin between the therapeutic concentrations , which is presumed to be below toxic concentrations , and environmental concentrations should allow for the worst - case scenario of totally additive effects to be predicted . few studies have attempted to assess the internal concentrations as a consequence of environmental exposure to pharmaceuticals . the studies by brown et al . , fick et al . , and lahti et al . are the only ones that , to our knowledge , have taken a field - work approach and exposed juvenile rainbow trout to sewage treatment work effluents for 23 weeks . the presence of five or 25 pharmaceuticals was measured in plasma and compared with the modeled uptake , and for most drugs the measured bioconcentration was in reasonably good agreement with the predicted values . exceptions included levonorgestrel and cilazapril , where measured concentrations were 260 and 100 times higher than modeled , respectively , and telmisartan and benzafibrate , which were not detected despite being predicted to bioconcentrate . the exact reasons for these apparent discrepancies are not known , but may be related to the log kow values used in the modeling . in the case of benzafibrate , there are variations in the log kow values generated by different algorithms ; the american chemical development ( acd ) prediction gives values lower than those generated by other algorithms ( 2.5 compared to 4.25 ) , and is the only one to provide a log d7.4 value , which at 1.09 would suggest that no bioaccumulation of this drug would occur . fick et al . used a log kow of 4.25 , resulting in a predicted bioconcentration factor of 168 which may well have been an overestimation . in the case of telmisartan , the modeling suggested a bioconcentration factor of 178649 ; whereas using the log d7.4 would give a predicted value of 380 . this is still higher than the observed bioconcentration , and consideration needs to be given to the highly lipophilic nature of this drug which may affect its bioavailability . lahti et al . did not compare measured with modeled uptake , but found that only three pharmaceuticals ( diclofenac , naproxen and ibuprofen ) , of the 15 drugs measured , were detectable in plasma . thus , it is clear that factors other than the log kow ( upon which the prediction of plasma concentrations was originally based in the model proposed by huggett et al . ) influences bioconcentration of drugs , and gaining this understanding will be crucial if we are to produce a robust , predictive model ( see ) . the counterintuitive observation of differences in measured plasma concentrations in fish placed in three different sewage treatment work effluent sites in sweden and finland , may perhaps be explained by the apparent considerable variation between individual fish . difficulties in reliably measuring plasma concentrations of pharmaceuticals may perhaps have contributed to this variation , but the nature of the exposure matrix , such as the presence of colloids or surfactants and diurnal variation in the exposure concentrations , may also affect uptake parameters . indeed , a study of three nsaids ( ketoprofen , ibuprofen and naproxen ) present in undiluted sewage effluent or clean water showed more efficient bioconcentration from the effluent . variation in pharmaceutical uptake depending on the characteristics of the exposure medium , in particular ph , has been recognized by others , and it has been suggested that any modeling must take account of this by using the ph - corrected octanol water partition coefficient ( dow ) or ph - corrected liposome - water partition coefficients ( dlipw ) . however , there remains considerable variation between individual fish exposed in the same clean laboratory tanks ( e.g. , owen et al . ) , suggesting that differences in uptake mechanisms , such as gill membrane based drug transporters , or metabolic clearance rates , may also play a significant role . understanding factors that affect uptake is clearly crucial ; poor uptake of citalopram , a selective serotonin reuptake inhibitor , is believed to explain the lack of effects in rainbow trout and guppies ( poecilia reticulata ) , even though modeling suggests the drug could present an environmental hazard . in fish , the presence of sex - steroid - binding globulin ( ssbg ) in the gills is believed to explain the very effective uptake of steroids , resulting in plasma concentrations 1000-fold higher than the surrounding environment and exceeding the cmax . however , this generalized assumption may not hold true for all steroids , as illustrated by the apparent contradictory results for two glucocorticoids , beclomethasone and dexamethasone . while exposure of fathead minnows to 1 g / l beclomethasone dipropionate resulted in mode of action - relevant biological effects , an exposure concentration of 500 g / l dexamethasone the explanation for these results is most likely related to the differing uptake of the two glucocorticoids ; although the studies did not measure plasma concentrations , modeling of the bioconcentration of beclomethasone diproprionate ( the formulation used by kugathas and sumpter ) , and dexamethasone indicates that only beclomethasone diproprionate would bioconcentrate to any significant extent ( table 2 ) . in this regard , it should be noted that due to its lower lipophilicity , dexamethasone is often administered by injection to patients . this highlights the importance of clearly stating the specific formulation of the pharmaceutical used in all exposure studies , as the absence / presence and identity of specific adjunctive chemical groups ( e.g. , acetate , valerate , propionate ) on the parent compound may significantly affect the bioaccumulation process , and hence the potential effects on fish . the read - across hypothesis can be viewed simply as what we see in one species is likely to be reflected in others , that conserve the same target , at the level of a pharmacological response ( mode of action ) ; that is , we see the same end result . although conservation of the molecular target is fundamental to the hypothesis , we do not understand the mechanism of action for most pharmaceuticals at a sufficiently detailed level for the molecular target to be a robust endpoint . most drugs have multiple mechanisms , multiple modes of action , and ultimately a range of effects . some are clearly the principle mechanism and the net effect is that a disease state or symptoms are alleviated in most people . however , the secondary pharmacology contributes to the side effects that are associated with many pharmaceuticals . any of these responses may be expected to read across to other species with similar systems . in terms of evidencing the read - across hypothesis , we should be able to test the intended therapeutic end point , or a major secondary endpoint . in principle , read - across would be expected to hold true for most , but not all , classes of drugs ( and indeed other chemicals ) . a notable exception would be some of the biopharmaceuticals , such as antibodies . these are exquisitely specific , and only work in one species , man , and as we have already alluded to , increased evolutionary distance from man will reduce the likelihood of read - across . further , antibodies will not be present in the environment , as they are unlikely to be excreted by patients , and even if they were , their poor stability outside the human body and large molecular size would prevent uptake into fish or other organisms . for example , in writing this paper , we have made the assumption that the binding kinetics of a drug to plasma proteins and tissue are the same in a fish as in a human . however , if a drug is less tightly bound in fish plasma than in a human , there will be a greater amount of drug available to flood the tissue compartment at the same plasma concentration , and in terms of effect it is the unbound concentration of a drug that matters . thus , understanding the pharmacokinetics in fish may be important , especially if the expected therapeutic end points are seen at plasma concentrations below human therapeutic concentrations . additionally , differential pharmacokinetics may contribute to apparent species differences in no observed effect concentration ( noec ) values . although the read - across hypothesis is generally accepted , this review has shown that there is currently scant evidence for it . this is not through a lack of research effort , as attested to by the large number of publications , but rather that most studies have been found wanting in some respects , mostly in relation to measurements of the pharmaceuticals in water and the exposed organism . such analyses require highly specialist and inherently expensive facilities , and are beyond the reach of many research groups . in our view , fewer and better designed studies , perhaps informed by the adverse outcome pathway conceptual framework , are required in order to provide the basis for a robust risk assessment of pharmaceuticals present in the environment . the importance of this is exemplified by a recently published study which makes the claim that 1 ng / l fluoxetine has significant effects on cuttlefish ( sepia officinalis ) . if these results , which are intuitively surprising , were repeatable ( for example , we have concerns because there was no analytical chemistry confirmation of doses in the static exposures ; there was high mortality ; the inverse dose effect is difficult to interpret ) , they would have profound implications it would suggest that current environmental concentrations of fluoxetine are sufficiently high enough to affect aquatic organisms . in order to ensure that environmental policy and regulations are not informed by studies that are unlikely to be repeatable , a consortium - based approach , as well as work within and between experienced , well - resourced laboratories , may be a way forward , ensuring that complete studies ( table 1 ) that will generate robust data are undertaken . it would be beneficial to agree standard operating protocols and blind analysis of samples presently , interpretation and comparisons of results are difficult and often impossible because of varying experimental conditions ( exposure length , age of fish , different species ) ; endpoints that are related or unrelated to mode of action ; lack of information about exposure and internal concentrations ; and even the particular formulation of the drug used is not always provided . a concerted , collaborative effort by the scientific community and industry can provide the resources required to fully understand the factors influencing bioconcentration ( perhaps the most important in assessing risks for aquatic organisms ) , and allow studies to be done on a sufficiently large scale to reduce the uncertainty created by the biological variability often observed . the publication process can be used to encourage more complete studies , as it is only on the basis of robust data that we can develop predictive models , which in our view , is the only feasible approach to protecting the environment .

pharmaceuticals in the environment have received increased attention over the past decade , as they are ubiquitous in rivers and waterways . concentrations are in sub - ng to low g / l , well below acute toxic levels , but there are uncertainties regarding the effects of chronic exposures and there is a need to prioritise which pharmaceuticals may be of concern . the read - across hypothesis stipulates that a drug will have an effect in non - target organisms only if the molecular targets such as receptors and enzymes have been conserved , resulting in a ( specific ) pharmacological effect only if plasma concentrations are similar to human therapeutic concentrations . if this holds true for different classes of pharmaceuticals , it should be possible to predict the potential environmental impact from information obtained during the drug development process . this paper critically reviews the evidence for read - across , and finds that few studies include plasma concentrations and mode of action based effects . thus , despite a large number of apparently relevant papers and a general acceptance of the hypothesis , there is an absence of documented evidence . there is a need for large - scale studies to generate robust data for testing the read - across hypothesis and developing predictive models , the only feasible approach to protecting the environment .

The Read-Across Hypothesis Target Conservation Studies That Support the Read-Across Hypothesis How Far Does the Concept of Read-Across Extend? Apparent Limitations of the ApproachStudies That Appear not to Support the Read-Across Hypothesis Environmental Relevance and a Predictive Model Concluding Remarks Looking Forward

the issue of pharmaceuticals in the environment has come to the fore in recent years with the realization that these drugs , designed to act at specific mammalian targets , may have effects in non - target organisms provided that the molecular target ( usually a receptor or enzyme ) is conserved . the hypothesis also presumes that a specific interaction between a drug and a target will result in a pharmacological response before a toxicological one , and will require a plasma concentration similar to that necessary to see a pharmacological effect in humans . if this hypothesis is correct , it should be possible to use information derived from the drug development process , such as molecular target , mode of action , effective dose and physicochemical properties , together with genomic and physiological knowledge of the non - target organism , to predict the likelihood of an effect occurring , assuming relevant exposure . the use of model organisms such as danio rerio ( zebrafish ) in drug screening indicates that it is possible to read up from a lower vertebrate such as a fish to humans , and our increased knowledge of the genomes and physiology of fish has confirmed that they are in many respects very similar to mammals , including the presence of drug targets and similar responses . it should be noted that this hypothesis relates to biological read - across , as opposed to the chemical read - across approach used to predict toxicity from untested chemicals of the same class , or indeed addressing general toxicity from a battery of tests . the closer the predicted fish plasma concentration is to cmax , the greater the likelihood that a pharmacological effect will be seen , although it should be noted that log kow is affected by the pka and may therefore not give an accurate estimate for ionogenic compounds . there are over 3000 human pharmaceuticals in general use , and many of these have been detected in surface waters and sewage treatment work effluents . it is unrealistic to assess each drug experimentally , and although environmental concentrations are low ( in the ng / l to low g / l range ) , it can not be presumed that no effects will occur ( figure 1 ) . the past decade has seen a proliferation of studies relating to effects of pharmaceuticals on fish and other organisms , although few of these have explicitly tested the read - across hypothesis . this review will provide a critical commentary of the extent to which these studies , mostly concerned with human pharmaceuticals , have validated the read - across hypothesis in general , and the fish plasma model in particular . we will focus mainly on fish , as these are recognized as obvious indicators of aquatic contamination , but the increasing evidence for extending the read - across concept to invertebrates , and even plants , will also be considered . it is beyond the scope of this review to discuss test substance concentrations in tissues other than plasma , as this would not add to the evidence - base for the read - across hypothesis , given that tissue concentrations are not reported in relation to pharmacological effects in humans . the evolutionary conservation of the primary drug target is a prerequisite for the read - across hypothesis , and the key paper by gunnarsson et al they looked at 1318 human drug targets across 16 species , and found 86% to be conserved in zebrafish , 61% in daphnia pulex ( water flea ) and 35% in chlamydomonas reinhardtii ( green algae ) , indicating the potential for lower vertebrates , and even invertebrates and plants , to respond to pharmaceuticals present in the environment . this suggests that drugs targeting enzymes may potentially affect a greater number of species than a drug targeting a receptor , although there are insufficient studies to date to provide convincing evidence for this in practice . thus , the apparent absence of a particular subtype in a species does not mean that a specifically targeted drug will have no mode of action effect . little attention is usually paid to the conservation of secondary targets , such as proteins in the pathway downstream of the primary target , which is an implicit requirement for the read - across hypothesis to be fully valid . however , the importance of establishing this may be questionable , as generally an absence of the primary therapeutic effect would indicate that the particular pathway has not been conserved . an understanding of what is a side effect and what is a secondary consequence of the primary drug action is also required ; thus , the cardiovascular risk associated with cyclo - oxygenase ( cox ) enzyme inhibition is a direct pharmacological consequence of inhibiting cox 2 , and not due to a side effect of nonsteroidal anti - inflammatory drugs ( nsaids ) . given our lack of knowledge of how physiological responses are linked to specific targets in many nonmammalian organisms , our ( in)ability to recognize effects , let alone side - effects , in non - target organisms will also compromise our ability to validate the read - across hypothesis . most studies have been carried out in fish , and in terms of assessing the impact of pharmaceuticals present in the environment , fish are one of the most likely ( but not always the most sensitive ) classes of organisms to be affected . despite the large number of publications where effects of drugs have been studied , there are relatively few ( see supporting information table s2 ) in which effects have been correlated to the internal exposure concentration ( i.e. a level 1 study provides little or no relevant information , whereas a level 4 study , incorporating measurements of drug exposure and internal concentrations , as well as a mode of action endpoint , will directly address all aspects of the hypothesis . only studies at level 4 address all aspects of the read - across hypothesis , and can relate effects to the human therapeutic plasma concentration . level 3 studies may be able to confirm that similar effects to those seen in humans occur in aquatic organisms , but without a measure of the internal concentration it is not possible to relate these effects to the human therapeutic plasma concentration , and therefore determine if an aquatic organism is more , or less , sensitive to a particular drug . without knowledge of plasma concentrations , which reflect the pharmacokinetics of the drug , a comparison with human therapeutic concentrations can not be made and incorrect conclusions could be drawn regarding the exposure concentration at which a pharmaceutical elicits a response , and thereby incorrectly estimating the threat to non - target organisms if the drug is present in the environment . given the general acceptance of the hypothesis , it is therefore somewhat surprising that only one study to date has confirmed the administered pharmaceutical dose by measuring water concentrations , obtained the internal exposure level by measuring plasma concentrations , linked exposure to a biological effect by measuring relevant endpoints , compared the results to those expected at cmax , and demonstrated that effects were seen only at concentrations similar to those in a human taking the drug . as this study remains , to our knowledge , the only one that fully addresses all aspects of the read - across hypothesis and can therefore be considered a level 4 study ( table 1 ) , it is unfortunate that further details are not available in the peer - reviewed literature . the resulting plasma concentrations ( 305 ng / ml at the lowest exposure concentration ) were close to the normal human therapeutic range ( 50250 ng / ml ) , thus supporting the read - across hypothesis . however , it is unfortunate that the lowest exposure concentration tested resulted in plasma levels similar to human cmax , so we do not know if any effects would have been observed below cmax , a requirement to fully validate the read - across hypothesis . the highest exposure concentration of sertraline used ( 28.1 g / l ) gave a measured plasma concentration of 2000 ng / ml , and is therefore likely to have resulted in plasma concentrations in the toxic range for humans ; however , no ( further ) adverse effects were reported . a previous study , which did not measure plasma concentrations , reported that an exposure concentration of 72 g / l sertraline was lethal to fathead minnows , which would suggest that the plasma concentrations reached in the most recent study were well below the fatal range . this exceeds the human therapeutic concentration of 2.4 g / l , and suggests that the mode of action effects observed by zeilinger et al . these may provide partial support for the read - across hypothesis , in that non - target effects are not seen at human therapeutic plasma concentrations : for example , in fathead minnows , propranolol produced no toxic effects until the plasma concentration exceeded cmax by 100-fold , which occurred at a water concentration of 1 mg / l . there are now a reasonable number of high quality studies that , although they do not include measurement of plasma concentrations of the pharmaceuticals being studied , involve assessing one or more endpoints relevant to the mode of action . in all these studies , and others , the effects of the pharmaceuticals on fish were those that would be anticipated if the drugs had the same mode of action in fish as they do in humans . however , because plasma concentrations of the pharmaceuticals were not determined , it is premature to conclude that these level 3 studies ( table 1 ) provide unequivocal support for the read - across hypothesis . however , because effective plasma concentrations of pharmaceuticals can not be compared between humans and fish , the studies provide at best only partial support for the hypothesis . thus , the significance of changes in global fathead minnow brain gene expression in response to 4 g / l propranolol ( measured ) for 21 days , and increases in vitellogenin , cyp1a and p53 gene expression in medaka ( oryzia latipes ) liver , gill , and intestine following 4 day exposure to 1 g / l diclofenac , is unclear . demonstrated that a 14 day exposure of juvenile rainbow trout to 1.681.5 g diclofenac / l , resulting in plasma concentrations 1.588% of cmax , affected hepatic gene expression . recognized the need for studies to be done in invertebrates , algae , and plants , as conceptually these organisms could also respond to pharmaceuticals if the target is conserved . the schistosoma flatworm contains many potential drug targets , such as receptors and enzymes , and appears to have cellular signaling , metabolic and neuroendocrine pathways similar to those present in higher organisms . supports the read - across hypothesis : the effects in the plant lemna gibba was seen at low g / l doses , and the molecular endpoint used ( mevalonic acid pathway biosynthesis , which is regulated by hmgr)directly related to the mode of action of the drugs tested was some 23 times more sensitive than a gross morphological endpoint ( wet weight ) . however , it is probably unrealistic to expect that a full validation of the read - across hypothesis is possible in plants , fungi , and invertebrates , in a large part due to the technical difficulties in measuring internal concentrations in very small organisms . the mode of action principle underpinning the read - across hypothesis does not preclude other effects being seen , but it is presumed that these will occur when the internal concentration exceeds the cmax . therefore , any studies where significant , and perhaps toxic , effects are seen at or below the cmax , would suggest that the read - across hypothesis is not universally applicable , perhaps as a consequence of differences in detoxification mechanisms between humans and non - target organisms . the most well - known example of the effect of a pharmaceutical that does not appear to support the read - across hypothesis is the death of old world vultures ( gyps genus ) , due to kidney failure , following ingestion of diclofenac present in the cadavers of cattle on which they fed . no diclofenac was detected in cathares tissues and the estimated half - life was 6 h whereas , in the gyps vultures , diclofenac residues were found in the kidney and the estimated half - life was 1618 h. thus , the diclofenac and vultures story , while representing the only example where impact at a population level can be attributed to the presence of a pharmaceutical in the environment , does not invalidate the read - across hypothesis . there is probably a need to recognize that drugs act over a range of doses , and in assessing whether a non - target organism displays greater sensitivity than a human , the no observable therapeutic effect level should be considered . in terms of assessing the potential impact of environmental exposures , the conclusions drawn would differ depending on which scenario was correct , and there is , therefore , a need for these studies to be replicated before too much emphasis can be placed on them . there are a large number of studies where extremely high doses ( mg / l ) of pharmaceuticals have been administered , or are required , to elicit an effect . if polymorphic variation is also relevant for other organisms , we may need to be more aware of the potential side effects of drugs could these become a major effect in some species , or perhaps more likely , some individuals within each species ? clearly , the aim of the research being carried out under the pharmaceuticals in the environment banner is to aid decision making with regards to the risks drugs may pose to non - target organisms exposed via the environment . it is unrealistic for experimental data to be obtained for all pharmaceuticals in use , or on all possible species receiving exposure , but the read - across hypothesis could be tested with a number of drugs , representing different modes of action , and the data obtained used to derive an improved model for predicting likely environmental impact of different drugs , or classes of drugs . the read - across hypothesis relies on a conserved mode of action , and it would therefore be expected that all drugs of a certain class , having the same mode of action , could contribute to an effect . in the case of benzafibrate , there are variations in the log kow values generated by different algorithms ; the american chemical development ( acd ) prediction gives values lower than those generated by other algorithms ( 2.5 compared to 4.25 ) , and is the only one to provide a log d7.4 value , which at 1.09 would suggest that no bioaccumulation of this drug would occur . difficulties in reliably measuring plasma concentrations of pharmaceuticals may perhaps have contributed to this variation , but the nature of the exposure matrix , such as the presence of colloids or surfactants and diurnal variation in the exposure concentrations , may also affect uptake parameters . in fish , the presence of sex - steroid - binding globulin ( ssbg ) in the gills is believed to explain the very effective uptake of steroids , resulting in plasma concentrations 1000-fold higher than the surrounding environment and exceeding the cmax . while exposure of fathead minnows to 1 g / l beclomethasone dipropionate resulted in mode of action - relevant biological effects , an exposure concentration of 500 g / l dexamethasone the explanation for these results is most likely related to the differing uptake of the two glucocorticoids ; although the studies did not measure plasma concentrations , modeling of the bioconcentration of beclomethasone diproprionate ( the formulation used by kugathas and sumpter ) , and dexamethasone indicates that only beclomethasone diproprionate would bioconcentrate to any significant extent ( table 2 ) . the read - across hypothesis can be viewed simply as what we see in one species is likely to be reflected in others , that conserve the same target , at the level of a pharmacological response ( mode of action ) ; that is , we see the same end result . although conservation of the molecular target is fundamental to the hypothesis , we do not understand the mechanism of action for most pharmaceuticals at a sufficiently detailed level for the molecular target to be a robust endpoint . in terms of evidencing the read - across hypothesis , we should be able to test the intended therapeutic end point , or a major secondary endpoint . in principle , read - across would be expected to hold true for most , but not all , classes of drugs ( and indeed other chemicals ) . further , antibodies will not be present in the environment , as they are unlikely to be excreted by patients , and even if they were , their poor stability outside the human body and large molecular size would prevent uptake into fish or other organisms . thus , understanding the pharmacokinetics in fish may be important , especially if the expected therapeutic end points are seen at plasma concentrations below human therapeutic concentrations . although the read - across hypothesis is generally accepted , this review has shown that there is currently scant evidence for it . this is not through a lack of research effort , as attested to by the large number of publications , but rather that most studies have been found wanting in some respects , mostly in relation to measurements of the pharmaceuticals in water and the exposed organism . in order to ensure that environmental policy and regulations are not informed by studies that are unlikely to be repeatable , a consortium - based approach , as well as work within and between experienced , well - resourced laboratories , may be a way forward , ensuring that complete studies ( table 1 ) that will generate robust data are undertaken . it would be beneficial to agree standard operating protocols and blind analysis of samples presently , interpretation and comparisons of results are difficult and often impossible because of varying experimental conditions ( exposure length , age of fish , different species ) ; endpoints that are related or unrelated to mode of action ; lack of information about exposure and internal concentrations ; and even the particular formulation of the drug used is not always provided . the publication process can be used to encourage more complete studies , as it is only on the basis of robust data that we can develop predictive models , which in our view , is the only feasible approach to protecting the environment .

owing to the scientific advancement and technology a drastic change is seen in the criminal scenario , road traffic / rail / aviation accidents , mass disasters , wars ; and the bodies which are found are beyond recognition . if the whole skeleton is available , the accuracy of sex determination may be of the order of ninety - eight percent as the pelvic bones alone provide accuracy of around 95% . however , the problem arises only when segments of the body or cranial cavity or isolated teeth are obtained from the site . in such scenario , teeth , being the hardest substances in the human body , potentially can survive most of insults and consequences encountered at death and during decomposition . tooth pulp remains protected in a hard tissue casing made up of dentin and enamel . the present study was conducted on pulp tissue derived from individuals belonging to age range of 16 - 55 years . the aim of this study was to evaluate the diagnostic significance of x ( barr body [ bb ] ) and y ( f body [ fb ] ) chromosomes observed in dental pulp tissue for gender determination of an individual , and to study the influence , if any , of age and the time interval on the demonstration of the sex chromatin ( i.e. , the percentage of bb or fb ) . this study was conducted on a total of 100 ( 50 male and 50 female ) teeth , which were extracted following orthodontic , pulpal or periodontal indications . thus , along with the healthy teeth , teeth with exposed pulp , or with attrition / abrasion were also included . the sample was divided in ten groups with ten teeth in each group ( 5 male and 5 female cases ) . the teeth were kept at room temperature without any preservation and were processed at various intervals as per schedule of the study [ table 1 ] . groups as per time interval a longitudinal groove was made with the help of an air - rotor handpiece using a cylindrical diamond bur , on the lingual surface of each tooth and was broken by striking with a chisel and mallet , at the junction of the crown and the root . the whole of the pulp tissue was separated out of the pulp cavity with the help of a probe and a # 10 broach . the pulp tissue was washed in normal saline to remove any debris or calcified particles . the pulpal tissue was then kept immersed in a fixative ( 3 methanol : 1 glacial acetic acid ) for 24 h. it was then transferred to a mortar , few drops of 20% acetic acid were added to soften the tissue . the suspension thus obtained was centrifuged ( remi rm12c micro centrifuge ) for 5 min at 6000 rpm . the supernatant was used to obtain a monolayer on chilled microscope slides , i.e. , a single drop was dropped by a pipette , and was spread by a blunt ended glass rod to get a homogenous population of cells . these slides were then air - dried and were fixed with a few drops of absolute methanol . one slide was stained with harris 's ( h and e ) hematoxylin and eosin stain to study the bb . the slide was examined under oil immersion lens ( 100 ) of light microscope for the bb . it appears as a dark spot usually lying against the nuclear membrane in the females . the second slide was stained with quinacrine dihydrochloride for the study of y chromosomes ( fb ) . after natural evaporation of the methanol , the slide was stained with 0.5% quinacrine dihydrochloride for 20 min . the slide was then washed in distilled water thrice and was kept in mcllvaine 's buffer ( a 0.2 m solution of dihydrate salt of disodium hydrogen phosphate was prepared by dissolving 22.71 g of the solute in 800 ml of distilled water . a 0.1 m solution of monohydrate salt of citric acid was prepared by dissolving 10.50 g of the solute in 500 ml of distilled water . 0.2 m solute of dihydrate salt of disodium hydrogen phosphate was slowly added to 500 ml of 0.1 m citric acid solution , and the ph of the buffer was adjusted to 5.5 ) for 3 min . . then a drop of glycerol was added and the slide was covered with a cover slip . the slide was then observed with olympus fluorescent microscope bx 53 ( sn ol399 t2 tokyo , japan ) under oil immersion in dark field at an objective of 40 and 100 , by blue - violet ( bv ) exciting method ( emitting a blue - violet color mainly at 4.047 and 4.038 ) . only those cells which contained the characteristic y chromatin or fb , i.e. , a brightly fluorescent spot attached to the nuclear membrane were counted as positive cells while those , which did not show any such fluorescent spot were labeled as negative . similarly in h and e , stained slides an individual cell with visible bb was counted . this study was conducted on a total of 100 ( 50 male and 50 female ) teeth , which were extracted following orthodontic , pulpal or periodontal indications . thus , along with the healthy teeth , teeth with exposed pulp , or with attrition / abrasion were also included . the sample was divided in ten groups with ten teeth in each group ( 5 male and 5 female cases ) . the teeth were kept at room temperature without any preservation and were processed at various intervals as per schedule of the study [ table 1 ] . a longitudinal groove was made with the help of an air - rotor handpiece using a cylindrical diamond bur , on the lingual surface of each tooth and was broken by striking with a chisel and mallet , at the junction of the crown and the root . the whole of the pulp tissue was separated out of the pulp cavity with the help of a probe and a # 10 broach . the pulp tissue was washed in normal saline to remove any debris or calcified particles . the pulpal tissue was then kept immersed in a fixative ( 3 methanol : 1 glacial acetic acid ) for 24 h. it was then transferred to a mortar , few drops of 20% acetic acid were added to soften the tissue . the suspension thus obtained was centrifuged ( remi rm12c micro centrifuge ) for 5 min at 6000 rpm . the supernatant was used to obtain a monolayer on chilled microscope slides , i.e. , a single drop was dropped by a pipette , and was spread by a blunt ended glass rod to get a homogenous population of cells . these slides were then air - dried and were fixed with a few drops of absolute methanol . one slide was stained with harris 's ( h and e ) hematoxylin and eosin stain to study the bb . the slide was examined under oil immersion lens ( 100 ) of light microscope for the bb . it appears as a dark spot usually lying against the nuclear membrane in the females . the second slide was stained with quinacrine dihydrochloride for the study of y chromosomes ( fb ) . after natural evaporation of the methanol , the slide was stained with 0.5% quinacrine dihydrochloride for 20 min . the slide was then washed in distilled water thrice and was kept in mcllvaine 's buffer ( a 0.2 m solution of dihydrate salt of disodium hydrogen phosphate was prepared by dissolving 22.71 g of the solute in 800 ml of distilled water . a 0.1 m solution of monohydrate salt of citric acid was prepared by dissolving 10.50 g of the solute in 500 ml of distilled water . 0.2 m solute of dihydrate salt of disodium hydrogen phosphate was slowly added to 500 ml of 0.1 m citric acid solution , and the ph of the buffer was adjusted to 5.5 ) for 3 min . . then a drop of glycerol was added and the slide was covered with a cover slip . the slide was then observed with olympus fluorescent microscope bx 53 ( sn ol399 t2 tokyo , japan ) under oil immersion in dark field at an objective of 40 and 100 , by blue - violet ( bv ) exciting method ( emitting a blue - violet color mainly at 4.047 and 4.038 ) . only those cells which contained the characteristic y chromatin or fb , i.e. , a brightly fluorescent spot attached to the nuclear membrane were counted as positive cells while those , which did not show any such fluorescent spot were labeled as negative . similarly in h and e , stained slides an individual cell with visible bb was counted . the presence of a cell with fluorescent fb was considered positive for male [ figures 1 and 2 ] , and with visible bb was considered positive for female [ figures 3 and 4 ] . the mean , standard deviation and range of fb and bb were calculated for males and for females [ table 2 ] . photomicrograph of histologic section of female dental pulp stained with h and e , ( original magnification , 100 ) . the arrows indicate the characteristic condensation of sexual chromatin ( barr body ) photomicrograph of histologic section of male dental pulp stained with h and e , original magnification , 100 ; showing absence of barr bodies photomicrograph of male dental pulp smear stained with quinacrine dihydrochloride , ( original magnification , 100 ) . the arrows indicate the nucleus with fluorescent f bodies photomicrograph of female dental pulp smear stained with quinacrine dihydrochloride , ( original magnification , 100 ) ; showing absence of fluorescence incidence of sex chromatin ( f - body and barr body ) in male and female cases from the period 0 - 12 h to 42 - 49 days in females , the mean percentage of bb was 34.6 5.41 in the 0 - 12 h group . the percentage decreased with increasing post - extraction period and fell to 13.20 2.39 over the period of 42 - 49 days , i.e. , 7 weeks . the percentage of fb was in the range of 0 - 8 with mean to be 2.56 2.31 . in males , the mean percentage of fb was found to be 59.8 6.06 in the freshly extracted teeth which decreased gradually with increasing time interval . the range of bb in male subjects was found to be 0 - 14 with a mean of 5.16 3.05 . these figures were found to be statistically significant with p value 0.000 , [ table 3 ] . the range of fb in male teeth was found to be 7 - 69% with an overlap of 2% , and the range of the bb in female teeth was found to be 10 - 41% with an overlap of 5% . canonical discriminant function coefficients were derived from these data : y = ( 0.927 ) + ( number of fb ) ( 0.05 ) + ( number of bb ) 0.14 ( male : y < 0 ; female : y > 0 ) where y provides us the discriminant score of any person , whose fb and bb scores are known . in case of males , the value of y will be around 1.752 , and in case of females it will be around + 1.752 . thus , by substituting the number of fb and bb in the equation the sex of the individual to whom the tooth belonged could be determined . with the help of this model , 50 women were correctly classified as women and 50 men were correctly classified as men thus giving a total of 100% correct predictions [ table 4 ] . various features of teeth , such as morphology , crown size , and root length ; deoxyribonucleic acid ( dna ) amplification , x / y chromosomes from the pulp tissue etc . due to continuous wearing of the teeth along occlusal and proximal surfaces , the accuracy and reliability of the sex determination based on the morphology of the tooth is questionable , especially , after 22 - 25 years of age . sex identification by dna amplification shows high accuracy ; however , requires more complex work and time to complete the analysis and needs special equipment . study of x and y chromosomes in the pulpal cells , which are not undergoing active division , is considered to be an easily accessible , less expensive and reliable method . a normal human female has only one bb per somatic cell while a normal human male has none . moore , graham and barr described a simple method of chromatin testing in human skin biopsies and demonstrated the practical value of the test as a guide to chromosomal sex in ambisexual subjects . it is seen as an intensely stained body lying against the nuclear membrane and can be observed with most of the nuclear stains , such as h and e , papanicolaou , feulgen , cresyl violet , aceto - orcein , carbol - fuchsin , and fluorescence . bb and sexual identification method have been described in various tissues as bone cells , cells of retina , oral mucosal cells including dental pulp . , while using the fluorescent dyes to give banding of chromosomes , noticed particularly a bright fluorescence of the long arm of the y chromosome . the alkylating agent such as quinacrine , accumulate in dna regions rich in guanine and is responsible for the bright fluorescence of the y chromosome . bobrow and vosa used quinacrine dihydrochloride ( atebrin ) to demonstrate the y chromosome in the interphase nuclei as a characteristic fluorescent body ; thus , providing the ideal counterpart of the bb or x chromosome . in the present study , the mean percentage of male pulp cells showing fb was found to be 30.68 18.32% and in females 2.56 2.31% , which was found to be almost similar to the figures obtained by the other studies . as far as bb are concerned the mean percentage obtained in the present study was 20.12 7.4% in females and 5.16 3.05% in males , which was similar to that reported by das et al . comparison of the incidence of the sex chromatin ( barr bodies and f - bodies ) in the present study with that of the other studies in our study , we were able to differentiate sex with certainty up to 7 weeks , though the percentage of fb and bb gradually decrease as the time interval increases [ graphs 1 and 1 ] . highest level of fb in our study was 69 and of bb was 41 , which was seen in the time period of 0 - 48 h. there was a remarkable fall in the bb after 48 h , and a drop was seen in the count of bb after 14 days of the time period [ graph 1 ] . as per das et al . and whittakerwhittaker et al . , the accuracy of sex determination was 100% up to 4 weeks and 5 weeks respectively . with a decreased accuracy , the sex could be determined up to 10 weeks ( whittaker et al . ) , which is in contrast to seno and ishizu and veeraraghavan et al . , who in their respective studies could observe fluorescent fb ( 5 - 22 bodies ) even up to 5 months . ionesi found that there was a reduction in the ability of sex chromatin staining in cases of teeth , which were stored at room temperature for 1 year , as compared with that of the freshly extracted teeth . . stored samples of permanent teeth for 4 years and still could discriminate the sex through their cells , when analyzed . the discrepancy between the various studies can be due to geographical factors as weather , temperature , and humidity . made an attempt to study the effect of environment , i.e. , the role of temperature and humidity on pulp tissue after the extraction of the tooth and concluded that the process of decomposition by bacterial action generally reduces the possibility of clear observation of chromatin . effect of time interval on sex chromatin ( f and barr bodies ) effect of time interval and age on sex chromatin ( fb - f body ; bb - barr bodies ) the age of the subject did not show any significant effect on the percentage of fb observed in male - dental pulp and bb observed in female dental pulp [ table 6 , graph 2 ] . as the age increases the teeth undergo several regressive changes as attrition , abrasion , thermal / mechanical / chemical trauma . these factors have a certain effect on the tooth as deposition of secondary dentin , reversible / irreversible pulpitis . in the present study , along with the healthy teeth , teeth with exposed pulp , or with attrition / abrasion it was observed that the sex chromatin could be efficiently recognized and counted even in teeth with acute and chronic inflammatory processes , and also in those with the regressive alterations as in attrition and abrasion . these teeth did not give any discrepancy as in the percentage of fb or bb as compared to the healthy teeth in their respective age groups . in their respective studies , larson and knapp have concluded that the barr chromatin could be differentiated in polymorphonuclear neutrophils in teeth with acute and chronic inflammatory processes . in the present study , the percentage of fb has also proved to be unaffected by inflammation or regressive alteration . effect of age on the incidence of sex chromatin ( fb and bb ) in the study of bb , suazo gi embedded the pulp tissue in the paraffin wax block to obtain the histological sections of 5 m thickness and examined for the bb . the appearance of bb in histologic section may be less frequent as the bb tend to hide behind or in front of the nucleoplasm and may not be very evident . in the present study , the method given by das et al . the mean percentage of bb did not show much discrepancy among the three studies [ table 5 ] . to consider a positive barr chromatin test , one cell in the sheet has to form chromatin condensation at the nuclear periphery , which gets stained intensely [ figure 1 ] . certain cells , wherein the intensely stained chromatin condensation fails to lie against the nuclear membrane , tend to get counted as negative for bb . unlike fb , which can be easily identified by their fluorescence , identification and counting of bb seems to be difficult . thus , it is stated that the frequency of the bb will be definitely more per hundred cells than reflected in our results . bb is found only in those cases in which more than one x chromosome is present and thus it is not found in male cells . in normal men , no bb are reported , and in 46xx women , one bb in cell nuclei is observed . however , in certain individuals with abnormal chromosomal levels [ table 7 ] gender can not be correctly identified using the bb technique ; for example 47xxy men will have a bb , and 47xxx women will have two . the procedure for quinacrine dihydrochloride staining is very technique sensitive , with each step right from the separation of pulp from the dental hard tissue until the counting of the brightly fluorescent spots attached to the nucleus . it is necessary to get a homogenous monolayer of cells from the suspension on the slide ; thus , preventing the masking of the fluorescent fb by fluorescent debris or by another cell . further , the bacteria , dead cells , and putrefied cellular debris may give false positive fluorescence particularly in case of female cells . barr body level abnormalities in certain conditions further , as the number of both fb and bb decrease with time interval [ graph 1 ] a cutoff value can not be given either for fb or bb . taking into consideration these limitations and the overlap in the count of x and y chromatin , a combined search for the presence or absence of bb and fb should be made to establish the sex from human pulp tissue . in the present study , the mean percentage of male pulp cells showing fb was found to be 30.68 18.32% and in females 2.56 2.31% , which was found to be almost similar to the figures obtained by the other studies . as far as bb are concerned the mean percentage obtained in the present study was 20.12 7.4% in females and 5.16 3.05% in males , which was similar to that reported by das et al . comparison of the incidence of the sex chromatin ( barr bodies and f - bodies ) in the present study with that of the other studies in our study , we were able to differentiate sex with certainty up to 7 weeks , though the percentage of fb and bb gradually decrease as the time interval increases [ graphs 1 and 1 ] . highest level of fb in our study was 69 and of bb was 41 , which was seen in the time period of 0 - 48 h. there was a remarkable fall in the bb after 48 h , and a drop was seen in the count of bb after 14 days of the time period [ graph 1 ] . , the accuracy of sex determination was 100% up to 4 weeks and 5 weeks respectively . with a decreased accuracy , the sex could be determined up to 10 weeks ( whittaker et al . ) , which is in contrast to seno and ishizu and veeraraghavan et al . , who in their respective studies could observe fluorescent fb ( 5 - 22 bodies ) even up to 5 months . ionesi found that there was a reduction in the ability of sex chromatin staining in cases of teeth , which were stored at room temperature for 1 year , as compared with that of the freshly extracted teeth . stored samples of permanent teeth for 4 years and still could discriminate the sex through their cells , when analyzed . the discrepancy between the various studies can be due to geographical factors as weather , temperature , and humidity . made an attempt to study the effect of environment , i.e. , the role of temperature and humidity on pulp tissue after the extraction of the tooth and concluded that the process of decomposition by bacterial action generally reduces the possibility of clear observation of chromatin . effect of time interval on sex chromatin ( f and barr bodies ) effect of time interval and age on sex chromatin ( fb - f body ; bb - barr bodies ) the age of the subject did not show any significant effect on the percentage of fb observed in male - dental pulp and bb observed in female dental pulp [ table 6 , graph 2 ] . as the age increases the teeth undergo several regressive changes as attrition , abrasion , thermal / mechanical / chemical trauma . these factors have a certain effect on the tooth as deposition of secondary dentin , reversible / irreversible pulpitis . in the present study , along with the healthy teeth , teeth with exposed pulp , or with attrition / abrasion it was observed that the sex chromatin could be efficiently recognized and counted even in teeth with acute and chronic inflammatory processes , and also in those with the regressive alterations as in attrition and abrasion . these teeth did not give any discrepancy as in the percentage of fb or bb as compared to the healthy teeth in their respective age groups . in their respective studies , larson and knapp have concluded that the barr chromatin could be differentiated in polymorphonuclear neutrophils in teeth with acute and chronic inflammatory processes . in the present study , the percentage of fb has also proved to be unaffected by inflammation or regressive alteration . in the study of bb , suazo gi embedded the pulp tissue in the paraffin wax block to obtain the histological sections of 5 m thickness and examined for the bb . the appearance of bb in histologic section may be less frequent as the bb tend to hide behind or in front of the nucleoplasm and may not be very evident . in the present study , the method given by das et al . the mean percentage of bb did not show much discrepancy among the three studies [ table 5 ] . to consider a positive barr chromatin test , one cell in the sheet has to form chromatin condensation at the nuclear periphery , which gets stained intensely [ figure 1 ] . certain cells , wherein the intensely stained chromatin condensation fails to lie against the nuclear membrane , tend to get counted as negative for bb . unlike fb , which can be easily identified by their fluorescence , identification and counting of bb seems to be difficult . thus , it is stated that the frequency of the bb will be definitely more per hundred cells than reflected in our results . bb is found only in those cases in which more than one x chromosome is present and thus it is not found in male cells . in normal men , no bb are reported , and in 46xx women , one bb in cell nuclei is observed . however , in certain individuals with abnormal chromosomal levels [ table 7 ] gender can not be correctly identified using the bb technique ; for example 47xxy men will have a bb , and 47xxx women will have two . the procedure for quinacrine dihydrochloride staining is very technique sensitive , with each step right from the separation of pulp from the dental hard tissue until the counting of the brightly fluorescent spots attached to the nucleus . it is necessary to get a homogenous monolayer of cells from the suspension on the slide ; thus , preventing the masking of the fluorescent fb by fluorescent debris or by another cell . further , the bacteria , dead cells , and putrefied cellular debris may give false positive fluorescence particularly in case of female cells . barr body level abnormalities in certain conditions further , as the number of both fb and bb decrease with time interval [ graph 1 ] a cutoff value can not be given either for fb or bb . taking into consideration these limitations and the overlap in the count of x and y chromatin , a combined search for the presence or absence of bb and fb should be made to establish the sex from human pulp tissue . the percentage of fb and bb gradually decrease as the time interval increases . in certain disasters such as high impact accidents , explosions and fragmentation of thermal trauma , natural calamities wherein traditional methods like determination of sex from skeleton fail , this method based on dental pulp may give a more confirmatory result ; as the teeth are a stable part of skeleton and the pulp tissue is well - protected .

objective : to evaluate the diagnostic performance of x ( barr body [ bb ] ) and y ( f body [ fb ] ) chromosomes observed in dental pulp tissue for gender determination of an individual.materials and methods : the study was carried out on 100 teeth ( 50 male and 50 female ) , which were indicated for extraction . the teeth were sectioned at various intervals ( within 12 h to 49 days post - extraction ) , and the pulpal tissue was obtained . two slides for each pulp tissue were prepared , one for 5% quinacrine dihydrochloride stain ( fb ) and the other for hemotoxylin and eosin stain ( bb ) . the slides were then observed under the fluorescent microscope for fb and under the light microscope for the bb respectively.results:gender determination from human pulp is possible up to 7 weeks . the percentage of fb and bb decrease gradually as the time interval increases . further , an equation was derived from the data based on the canonical discriminant function coefficients.conclusion:the determination of gender based on a joint search for the presence or absence of x ( bb ) and y ( fb ) chromosome is a reliable and cost - effective technique .

Introduction Materials and Methods Sample collection Sectioning of teeth Staining pulpal cells Results Discussion Determination of FB and BB Effect of time interval Effect of age of the subject on the percentage of the sex chromatin Technique Limitations Conclusion

owing to the scientific advancement and technology a drastic change is seen in the criminal scenario , road traffic / rail / aviation accidents , mass disasters , wars ; and the bodies which are found are beyond recognition . if the whole skeleton is available , the accuracy of sex determination may be of the order of ninety - eight percent as the pelvic bones alone provide accuracy of around 95% . however , the problem arises only when segments of the body or cranial cavity or isolated teeth are obtained from the site . the present study was conducted on pulp tissue derived from individuals belonging to age range of 16 - 55 years . the aim of this study was to evaluate the diagnostic significance of x ( barr body [ bb ] ) and y ( f body [ fb ] ) chromosomes observed in dental pulp tissue for gender determination of an individual , and to study the influence , if any , of age and the time interval on the demonstration of the sex chromatin ( i.e. , the percentage of bb or fb ) . this study was conducted on a total of 100 ( 50 male and 50 female ) teeth , which were extracted following orthodontic , pulpal or periodontal indications . the teeth were kept at room temperature without any preservation and were processed at various intervals as per schedule of the study [ table 1 ] . groups as per time interval a longitudinal groove was made with the help of an air - rotor handpiece using a cylindrical diamond bur , on the lingual surface of each tooth and was broken by striking with a chisel and mallet , at the junction of the crown and the root . the whole of the pulp tissue was separated out of the pulp cavity with the help of a probe and a # 10 broach . the pulp tissue was washed in normal saline to remove any debris or calcified particles . the pulpal tissue was then kept immersed in a fixative ( 3 methanol : 1 glacial acetic acid ) for 24 h. it was then transferred to a mortar , few drops of 20% acetic acid were added to soften the tissue . these slides were then air - dried and were fixed with a few drops of absolute methanol . one slide was stained with harris 's ( h and e ) hematoxylin and eosin stain to study the bb . the slide was examined under oil immersion lens ( 100 ) of light microscope for the bb . the second slide was stained with quinacrine dihydrochloride for the study of y chromosomes ( fb ) . after natural evaporation of the methanol , the slide was stained with 0.5% quinacrine dihydrochloride for 20 min . 0.2 m solute of dihydrate salt of disodium hydrogen phosphate was slowly added to 500 ml of 0.1 m citric acid solution , and the ph of the buffer was adjusted to 5.5 ) for 3 min . the slide was then observed with olympus fluorescent microscope bx 53 ( sn ol399 t2 tokyo , japan ) under oil immersion in dark field at an objective of 40 and 100 , by blue - violet ( bv ) exciting method ( emitting a blue - violet color mainly at 4.047 and 4.038 ) . similarly in h and e , stained slides an individual cell with visible bb was counted . this study was conducted on a total of 100 ( 50 male and 50 female ) teeth , which were extracted following orthodontic , pulpal or periodontal indications . the teeth were kept at room temperature without any preservation and were processed at various intervals as per schedule of the study [ table 1 ] . a longitudinal groove was made with the help of an air - rotor handpiece using a cylindrical diamond bur , on the lingual surface of each tooth and was broken by striking with a chisel and mallet , at the junction of the crown and the root . the whole of the pulp tissue was separated out of the pulp cavity with the help of a probe and a # 10 broach . the pulp tissue was washed in normal saline to remove any debris or calcified particles . the pulpal tissue was then kept immersed in a fixative ( 3 methanol : 1 glacial acetic acid ) for 24 h. it was then transferred to a mortar , few drops of 20% acetic acid were added to soften the tissue . , a single drop was dropped by a pipette , and was spread by a blunt ended glass rod to get a homogenous population of cells . these slides were then air - dried and were fixed with a few drops of absolute methanol . one slide was stained with harris 's ( h and e ) hematoxylin and eosin stain to study the bb . the slide was examined under oil immersion lens ( 100 ) of light microscope for the bb . the second slide was stained with quinacrine dihydrochloride for the study of y chromosomes ( fb ) . after natural evaporation of the methanol , the slide was stained with 0.5% quinacrine dihydrochloride for 20 min . 0.2 m solute of dihydrate salt of disodium hydrogen phosphate was slowly added to 500 ml of 0.1 m citric acid solution , and the ph of the buffer was adjusted to 5.5 ) for 3 min . then a drop of glycerol was added and the slide was covered with a cover slip . the slide was then observed with olympus fluorescent microscope bx 53 ( sn ol399 t2 tokyo , japan ) under oil immersion in dark field at an objective of 40 and 100 , by blue - violet ( bv ) exciting method ( emitting a blue - violet color mainly at 4.047 and 4.038 ) . similarly in h and e , stained slides an individual cell with visible bb was counted . the presence of a cell with fluorescent fb was considered positive for male [ figures 1 and 2 ] , and with visible bb was considered positive for female [ figures 3 and 4 ] . the mean , standard deviation and range of fb and bb were calculated for males and for females [ table 2 ] . the arrows indicate the characteristic condensation of sexual chromatin ( barr body ) photomicrograph of histologic section of male dental pulp stained with h and e , original magnification , 100 ; showing absence of barr bodies photomicrograph of male dental pulp smear stained with quinacrine dihydrochloride , ( original magnification , 100 ) . the arrows indicate the nucleus with fluorescent f bodies photomicrograph of female dental pulp smear stained with quinacrine dihydrochloride , ( original magnification , 100 ) ; showing absence of fluorescence incidence of sex chromatin ( f - body and barr body ) in male and female cases from the period 0 - 12 h to 42 - 49 days in females , the mean percentage of bb was 34.6 5.41 in the 0 - 12 h group . the percentage decreased with increasing post - extraction period and fell to 13.20 2.39 over the period of 42 - 49 days , i.e. , 7 weeks . the percentage of fb was in the range of 0 - 8 with mean to be 2.56 2.31 . in males , the mean percentage of fb was found to be 59.8 6.06 in the freshly extracted teeth which decreased gradually with increasing time interval . the range of fb in male teeth was found to be 7 - 69% with an overlap of 2% , and the range of the bb in female teeth was found to be 10 - 41% with an overlap of 5% . canonical discriminant function coefficients were derived from these data : y = ( 0.927 ) + ( number of fb ) ( 0.05 ) + ( number of bb ) 0.14 ( male : y < 0 ; female : y > 0 ) where y provides us the discriminant score of any person , whose fb and bb scores are known . thus , by substituting the number of fb and bb in the equation the sex of the individual to whom the tooth belonged could be determined . various features of teeth , such as morphology , crown size , and root length ; deoxyribonucleic acid ( dna ) amplification , x / y chromosomes from the pulp tissue etc . due to continuous wearing of the teeth along occlusal and proximal surfaces , the accuracy and reliability of the sex determination based on the morphology of the tooth is questionable , especially , after 22 - 25 years of age . study of x and y chromosomes in the pulpal cells , which are not undergoing active division , is considered to be an easily accessible , less expensive and reliable method . bb and sexual identification method have been described in various tissues as bone cells , cells of retina , oral mucosal cells including dental pulp . the alkylating agent such as quinacrine , accumulate in dna regions rich in guanine and is responsible for the bright fluorescence of the y chromosome . bobrow and vosa used quinacrine dihydrochloride ( atebrin ) to demonstrate the y chromosome in the interphase nuclei as a characteristic fluorescent body ; thus , providing the ideal counterpart of the bb or x chromosome . in the present study , the mean percentage of male pulp cells showing fb was found to be 30.68 18.32% and in females 2.56 2.31% , which was found to be almost similar to the figures obtained by the other studies . as far as bb are concerned the mean percentage obtained in the present study was 20.12 7.4% in females and 5.16 3.05% in males , which was similar to that reported by das et al . comparison of the incidence of the sex chromatin ( barr bodies and f - bodies ) in the present study with that of the other studies in our study , we were able to differentiate sex with certainty up to 7 weeks , though the percentage of fb and bb gradually decrease as the time interval increases [ graphs 1 and 1 ] . highest level of fb in our study was 69 and of bb was 41 , which was seen in the time period of 0 - 48 h. there was a remarkable fall in the bb after 48 h , and a drop was seen in the count of bb after 14 days of the time period [ graph 1 ] . with a decreased accuracy , the sex could be determined up to 10 weeks ( whittaker et al . ) , which is in contrast to seno and ishizu and veeraraghavan et al . , who in their respective studies could observe fluorescent fb ( 5 - 22 bodies ) even up to 5 months . ionesi found that there was a reduction in the ability of sex chromatin staining in cases of teeth , which were stored at room temperature for 1 year , as compared with that of the freshly extracted teeth . effect of time interval on sex chromatin ( f and barr bodies ) effect of time interval and age on sex chromatin ( fb - f body ; bb - barr bodies ) the age of the subject did not show any significant effect on the percentage of fb observed in male - dental pulp and bb observed in female dental pulp [ table 6 , graph 2 ] . as the age increases the teeth undergo several regressive changes as attrition , abrasion , thermal / mechanical / chemical trauma . in the present study , along with the healthy teeth , teeth with exposed pulp , or with attrition / abrasion it was observed that the sex chromatin could be efficiently recognized and counted even in teeth with acute and chronic inflammatory processes , and also in those with the regressive alterations as in attrition and abrasion . these teeth did not give any discrepancy as in the percentage of fb or bb as compared to the healthy teeth in their respective age groups . in the present study , the percentage of fb has also proved to be unaffected by inflammation or regressive alteration . effect of age on the incidence of sex chromatin ( fb and bb ) in the study of bb , suazo gi embedded the pulp tissue in the paraffin wax block to obtain the histological sections of 5 m thickness and examined for the bb . the appearance of bb in histologic section may be less frequent as the bb tend to hide behind or in front of the nucleoplasm and may not be very evident . the mean percentage of bb did not show much discrepancy among the three studies [ table 5 ] . to consider a positive barr chromatin test , one cell in the sheet has to form chromatin condensation at the nuclear periphery , which gets stained intensely [ figure 1 ] . thus , it is stated that the frequency of the bb will be definitely more per hundred cells than reflected in our results . bb is found only in those cases in which more than one x chromosome is present and thus it is not found in male cells . in normal men , no bb are reported , and in 46xx women , one bb in cell nuclei is observed . however , in certain individuals with abnormal chromosomal levels [ table 7 ] gender can not be correctly identified using the bb technique ; for example 47xxy men will have a bb , and 47xxx women will have two . the procedure for quinacrine dihydrochloride staining is very technique sensitive , with each step right from the separation of pulp from the dental hard tissue until the counting of the brightly fluorescent spots attached to the nucleus . it is necessary to get a homogenous monolayer of cells from the suspension on the slide ; thus , preventing the masking of the fluorescent fb by fluorescent debris or by another cell . further , the bacteria , dead cells , and putrefied cellular debris may give false positive fluorescence particularly in case of female cells . barr body level abnormalities in certain conditions further , as the number of both fb and bb decrease with time interval [ graph 1 ] a cutoff value can not be given either for fb or bb . taking into consideration these limitations and the overlap in the count of x and y chromatin , a combined search for the presence or absence of bb and fb should be made to establish the sex from human pulp tissue . in the present study , the mean percentage of male pulp cells showing fb was found to be 30.68 18.32% and in females 2.56 2.31% , which was found to be almost similar to the figures obtained by the other studies . as far as bb are concerned the mean percentage obtained in the present study was 20.12 7.4% in females and 5.16 3.05% in males , which was similar to that reported by das et al . comparison of the incidence of the sex chromatin ( barr bodies and f - bodies ) in the present study with that of the other studies in our study , we were able to differentiate sex with certainty up to 7 weeks , though the percentage of fb and bb gradually decrease as the time interval increases [ graphs 1 and 1 ] . highest level of fb in our study was 69 and of bb was 41 , which was seen in the time period of 0 - 48 h. there was a remarkable fall in the bb after 48 h , and a drop was seen in the count of bb after 14 days of the time period [ graph 1 ] . , the accuracy of sex determination was 100% up to 4 weeks and 5 weeks respectively . with a decreased accuracy , the sex could be determined up to 10 weeks ( whittaker et al . ) , which is in contrast to seno and ishizu and veeraraghavan et al . , who in their respective studies could observe fluorescent fb ( 5 - 22 bodies ) even up to 5 months . ionesi found that there was a reduction in the ability of sex chromatin staining in cases of teeth , which were stored at room temperature for 1 year , as compared with that of the freshly extracted teeth . effect of time interval on sex chromatin ( f and barr bodies ) effect of time interval and age on sex chromatin ( fb - f body ; bb - barr bodies ) the age of the subject did not show any significant effect on the percentage of fb observed in male - dental pulp and bb observed in female dental pulp [ table 6 , graph 2 ] . as the age increases the teeth undergo several regressive changes as attrition , abrasion , thermal / mechanical / chemical trauma . in the present study , along with the healthy teeth , teeth with exposed pulp , or with attrition / abrasion it was observed that the sex chromatin could be efficiently recognized and counted even in teeth with acute and chronic inflammatory processes , and also in those with the regressive alterations as in attrition and abrasion . these teeth did not give any discrepancy as in the percentage of fb or bb as compared to the healthy teeth in their respective age groups . in the present study , the percentage of fb has also proved to be unaffected by inflammation or regressive alteration . in the study of bb , suazo gi embedded the pulp tissue in the paraffin wax block to obtain the histological sections of 5 m thickness and examined for the bb . the appearance of bb in histologic section may be less frequent as the bb tend to hide behind or in front of the nucleoplasm and may not be very evident . the mean percentage of bb did not show much discrepancy among the three studies [ table 5 ] . to consider a positive barr chromatin test , one cell in the sheet has to form chromatin condensation at the nuclear periphery , which gets stained intensely [ figure 1 ] . unlike fb , which can be easily identified by their fluorescence , identification and counting of bb seems to be difficult . thus , it is stated that the frequency of the bb will be definitely more per hundred cells than reflected in our results . bb is found only in those cases in which more than one x chromosome is present and thus it is not found in male cells . in normal men , no bb are reported , and in 46xx women , one bb in cell nuclei is observed . however , in certain individuals with abnormal chromosomal levels [ table 7 ] gender can not be correctly identified using the bb technique ; for example 47xxy men will have a bb , and 47xxx women will have two . the procedure for quinacrine dihydrochloride staining is very technique sensitive , with each step right from the separation of pulp from the dental hard tissue until the counting of the brightly fluorescent spots attached to the nucleus . it is necessary to get a homogenous monolayer of cells from the suspension on the slide ; thus , preventing the masking of the fluorescent fb by fluorescent debris or by another cell . further , the bacteria , dead cells , and putrefied cellular debris may give false positive fluorescence particularly in case of female cells . barr body level abnormalities in certain conditions further , as the number of both fb and bb decrease with time interval [ graph 1 ] a cutoff value can not be given either for fb or bb . taking into consideration these limitations and the overlap in the count of x and y chromatin , a combined search for the presence or absence of bb and fb should be made to establish the sex from human pulp tissue . the percentage of fb and bb gradually decrease as the time interval increases . in certain disasters such as high impact accidents , explosions and fragmentation of thermal trauma , natural calamities wherein traditional methods like determination of sex from skeleton fail , this method based on dental pulp may give a more confirmatory result ; as the teeth are a stable part of skeleton and the pulp tissue is well - protected .

over the past years , it became clear that frontotemporal lobar degeneration ( ftld ) and amyotrophic lateral sclerosis ( als ) constitute the opposite ends of a disease continuum of overlapping disease phenotypes ( 1 , 2 ) . ftld is a presenile dementia characterized by selective atrophy of the frontal and anterior temporal lobes of the brain ( 3 ) . clinically , patients develop progressive behavioral changes , language impairment and/or executive dysfunction ( 4 , 5 ) . als is an incurable , severely disabling condition in which both upper and lower motor neurons degenerate ( 68 ) . disease progression is characterized by progressive muscle weakening evolving into paralysis with respiratory failure leading to death within 15 years after disease onset ( 8 , 9 ) . the comorbidity of als and ftld syndromes in patients is estimated to occur in 50% of the patients ( 1013 ) . currently , there is no effective treatment available for either ftld or als . in addition to the overlapping clinical symptomatology , the common pathological hallmark in the majority of these patients consists of tar dna - binding protein 43 ( tdp-43 ) , the major protein within ubiquitinated cytoplasmic inclusions . tdp-43 aggregates are present in a spectrum of distinctive neurodegenerative disorders suggesting a key role for tdp-43 in disease pathogenesis ( 1416 ) . furthermore , secondary tdp-43 accumulation occurs in multiple other neurodegenerative disorders ( 1 , 17 ) and is also observed in brains of control subjects over 65 years ( 18 ) . since the discovery of tdp-43 in 2006 ( 14 , 15 ) , major efforts have been directed to unravel its physiological functions in normal and disease brain . to date , tdp-43 is known as a highly conserved , nuclear rna - binding protein ( rbp ) involved in transcription and splicing regulation ( 1922 ) ( reviewed in 23 ) . subsequently , identification of causal mutations in tardbp , the gene encoding tdp-43 ( 2426 ) ( reviewed in 27 ) , mechanistically linked neurodegeneration to the occurrence of tdp-43 aggregates . causal tardbp mutations were first observed in families segregating autosomal dominant als identifying tardbp as a new als gene . few mutations were also described in ftld als or ftld patients ( 2831 ) , although their pathogenicity was not always convincing . in addition to tardbp , mutations were identified in other genes that had been associated with tdp-43 pathology . mutations are observed at variable frequencies in als , ftld als or ftld patients in progranulin ( grn ) ( 32 , 33 ) , angiogenin ( ang ) ( 34 ) , heterogeneous nuclear ribonucleoprotein a1 and a2/b1 ( hnrnpa1 and hnrnpa2/b1 ) ( 35 ) , optineurin ( optn ) ( 36 ) , ubiquilin 2 ( ubqln2 ) ( 37 ) , sequestosome 1 ( sqstm1 ) ( 38 , 39 ) and valosin - containing protein ( vcp ) ( 40 , 41 ) ( table 1 ) . a large number of these genes is thought to be implicated in rna processing and protein degradation pathways , suggesting that impairment of these processes might be central to the disease cascade ( 42 , 43 ) . the majority of the encoded proteins have been identified as components of the pathological inclusions , emphasizing the heterogeneous molecular basis of both ftld and als . the discovery of a pathogenic ggggcc ( g4c2 ) repeat expansion mutation in the gene c9orf72 further stressed the involvement of altered rna pathways in ftld / als pathogenesis ( table 1 ) . not only became c9orf72 the most frequently mutated gene in both als and ftld , the sharing of the ( g4c2 ) repeat expansion mutation linked both disorders into one disease continuum of overlapping clinical symptoms and tdp-43 pathology ( 4446 ) . understanding the molecular basis of c9orf72 associated diseases might provide important insights into common biological mechanisms . table 1.genetics of the ftld and als spectrum related to tdp-43 proteinopathygeneproteinlocusinheritanceclinical phenotypemutation typeref.rna metabolismtardbptdp-431p36auto . ( ftld ) modifier ( als)ftdmissense , nonsense , deletion , frameshift , splice site(32 , 33)auto . rec . , autosomal recessive ; ftd , frontotemporal dementia ; ibmpfd , inclusion body myopathy with early - onset paget disease and frontotemporal dementia data from ad & ftd mutation database ( http://www.molgen.ua.ac.be/ftdmutations accessed 29 july 2013 ) ( 164 ) . rec . , autosomal recessive ; ftd , frontotemporal dementia ; ibmpfd , inclusion body myopathy with early - onset paget disease and frontotemporal dementia data from ad & ftd mutation database ( http://www.molgen.ua.ac.be/ftdmutations accessed 29 july 2013 ) ( 164 ) . in contrast to ftld , a large proportion of als patients do not have documented family histories of disease but express disease due to a complex interplay of genetic and environmental risk factors . genome - wide genetic and animal model screens have identified several putative susceptibility genes and modifiers of tdp-43 toxicity that were strongly associated with als including ephrin type - a receptor 4 precursor ( epha4 ) ( 47 ) , rna lariat debranching enzyme ( dbr1 ) ( 48 ) , elongator protein 3 ( elp3 ) ( 49 ) and intermediate - length polyq expansions in ataxin 2 ( atxn2 ) ( table 1 ) ( 50 , 51 ) . the common tdp-43 pathology in als and ftld patients suggests that pathways disrupting tdp-43 integrity might be shared between patients with a different clinical , pathological and genetic etiology . the question remains , however , which disease processes are essential to drive tdp-43-related pathogenesis . in this review paper , we highlight the research outcomes that contributed to valuable insights into tdp-43-related pathomechanisms . as suggested by the molecular genetic findings , multiple pathways related to rna processing , repeat expansions , protein aggregation and proteostasis are likely contributing to the multifactorial nature of ftld / als disorders processing of rna molecules in the nervous system is an elaborate and remarkably complex network to maintain a functional environment in neurons . hence , mutations in tardbp and fus indicate that rbps might exert a central role in the pathogenesis of ftld / als - related disorders . tdp-43 and fus / tls are typically accumulating in the majority of these disorders ( 55 , 56 ) . likewise , genetic studies identified mutations in additional rbps such as taf15 , ewsr1 and in hnnrpa1 and hnrnpa2/b1 , demonstrating that rbps might contribute generally to als and/or fltd ( 35 , 5759 ) ( tables 1 and 2 ) . table 2.cellular protein functions and molecular pathology of ftld- and als - related genesproteinabbreviationsuggested protein functionmolecular pathologyref.rna-binding proteinstar dna - binding protein 43tdp-43transcription and splicing regulation microrna biogenesisrna transport and stabilization ( member of hnrnp protein family)tdp-43(14 , 15)fused in sarcoma / translocated in liposarcomafus / tlstranscription and splicing regulation microrna processingmaintenance of genomic integrity ( member of fet proteins)fus / tls(165 , 166)tata - binding protein - associated factor 15taf15rna polymerase ii componenttranscription initiation ( member of fet proteins)fus / tls(57)ewing sarcoma breakpoint region 1ewsr1transcriptional repressor(member of fet protein family)fus / tls(58)angiogeninangrna processing and trna modificationvascularizationassembly of stress granulestdp-43(34 , 167)heterogeneous nuclear ribonucleoproteinhnrnpa1hnrnpa2/b1hnrnpa3packing and transport of mrna ( member of hnrnp protein family)n.d.(35 , 95)repeat expansionschromosome 9 open reading frame 72c9orf72unknown protein function ( related to denn proteins)tdp-43 , ups(4446)ataxin 2atxn2regulator of egfr traffickingtdp-43(50 , 168)protein homeostasisvalosin - containing proteinvcpmembrane fusionprotein degradation ( er , proteasome and autophagy - associated)tdp-43(40 , 41)ubiquilin 2ubqln2proteasome - mediated protein degradationtdp-43(37)sequestosome 1 p62sqstm1autophagic degradationregulator of nf-b signaling pathwayinvolved in immune responsen.d.(38)optineurinoptngolgi maintenanceexocytosisvesicular traffickingtdp-43(36)growth factorprogranulingrnmultifunctional growth factorinflammationwound repairtdp-43(32 , 33 , 169)fet proteins , fus ewsr1taf15 dna / rna - binding proteins ; egfr , epidermal growth factor receptor ; er , endoplasmatic reticulum ; denn protein , differentially expressed in normal and neoplastic cells ; n.d . cellular protein functions and molecular pathology of ftld- and als - related genes fet proteins , fus ewsr1taf15 dna / rna - binding proteins ; egfr , epidermal growth factor receptor ; er , endoplasmatic reticulum ; denn protein , differentially expressed in normal and neoplastic cells ; n.d . , not determined ; ups , ubiquitin proteasome system . as a member of the hnrnp family , tdp-43 is involved in multiple steps of gene expression regulation , including rna splicing and transport ( 22 , 60 , 61 ) ( fig . 1 ) . loss of spliceosome integrity was reported in motor neurons of als patients ( 62 ) . a mouse model expressing mutant tdp-43 induced significant splicing alterations accompanying motor neuron disease in the absence of tdp-43 aggregation or nuclear clearing ( 63 ) . to unravel the biological functions of tdp-43 , tdp-43 rna targets were characterized in cultured cells , mouse brain , and in human brain of ftld and als patients . tdp-43 was shown to bind to 30% of the mouse transcriptome , highlighting the versatility and importance of tdp-43 for splicing regulation ( 64 , 65 ) . preferentially , tdp-43 bound long clusters of ug - rich sequences , mostly at intronic regions . pre - mrnas with exceptionally long introns ( > 100 kb ) are a characteristic feature of brain - enriched transcripts which might explain neuronal vulnerability observed in patients ( 64 , 66 ) . also , proteins encoded by the rna targets were enriched for genes involved in synaptic function , neuronal development and rna metabolism . a number of these proteins are implicated in neurological diseases including sortilin ( sort1 ) , fus / tls and grn , providing a speculative connection between disease mutations and pathology ( 64 , 65 , 67 ) . expression of a muscle - specific actin binding protein filamin c , one of the identified genes regulated by tdp-43 , was increased in frontal cortex of ftld patients ( 68 ) . figure 1.schematic representation of tdp-43 with its protein domain structures and localization of disease - associated mutations . tdp-43 comprises an nls and nes , respectively , 2 rna - recognition motifs ( rrm1 and rrm2 ) and a c - terminal glycine - rich region ( grr ) . numerous mutations in tardbp have been identified in sporadic and familial als patients and rarely in ftld patients . furthermore , experimental evidence also suggested that different domain structures of tdp-43 are involved in the aggregation process . abbreviations : del , deletion ; ins , insertion ; fl - tdp , full - length tdp-43 ; grr , glycine - rich region ; utr , untranslated region . schematic representation of tdp-43 with its protein domain structures and localization of disease - associated mutations . tdp-43 comprises an nls and nes , respectively , 2 rna - recognition motifs ( rrm1 and rrm2 ) and a c - terminal glycine - rich region ( grr ) . numerous mutations in tardbp have been identified in sporadic and familial als patients and rarely in ftld patients . furthermore , experimental evidence also suggested that different domain structures of tdp-43 are involved in the aggregation process . abbreviations : del , deletion ; ins , insertion ; fl - tdp , full - length tdp-43 ; grr , glycine - rich region ; utr , untranslated region . similar to other hnrnps , tdp-43 auto - regulates its expression levels through a negative - feedback loop involving alternative polya site selection and exosome or mirna - regulated degradation mechanisms ( fig . the c - terminal region of tdp-43 , harboring most of the pathogenic disease mutations , is required for self - regulation ( fig . 1 ) ( 69 ) . tight regulation of tdp-43 expression levels was further illustrated in animal models where human tdp-43 overexpression reduced endogenous protein levels . because of the lack of tdp-43 aggregates in these models , down - regulation of tdp-43 has been suggested to be toxic ( 7274 ) . however , this is questionable taken the strong homology ( i.e. 96% sequence identity ) of human and mouse tdp-43 ( 20 ) and the toxicity of mouse tdp-43 overexpression ( 75 , 76 ) . tdp-43 overexpression might directly alter splicing or stability of rna targets as shown by dose - dependent toxicity in mice ( 63 , 76 , 77 ) . increased tdp-43 levels have rarely been described in patients ( 78 , 79 ) , but cell stress and pathogenic als mutations might indirectly increase tdp-43 levels ( 8082 ) . figure 2.overview of putative mechanisms involved in tdp-43 proteinopathy observed in als and ftld patients . physiological tdp-43 shuttles between the nucleus and the cytoplasm to exert its cellular functions ranging from gene expression regulation at the transcription and splicing level to mrna transport and stabilization . upon cellular stress , mutations in several genes ( table 1 ) have been observed in als and ftld patients with tdp-43 proteinopathy , demonstrating that tdp-43 has a key role in the neurodegeneration process . tdp-43 proteinopathy is characterized by pathological modifications including aggregation , c - terminal cleavage into ctfs , hyperphosphorylation and ubiquitination of tdp-43 . furthermore , extensive research on tdp-43-related pathomechanisms suggests that different putative mechanisms might contribute to tdp-43 aggregation , including impaired protein degradation , alterations of tdp-43-associated splicing events , nuclear transport defects , loss of tdp-43 autoregulation and enhanced self - interaction of tdp-43 . / , importin / ; ub , ubiquitin ; p , phosphorylation ; star * , mutant protein . overview of putative mechanisms involved in tdp-43 proteinopathy observed in als and ftld patients . physiological tdp-43 shuttles between the nucleus and the cytoplasm to exert its cellular functions ranging from gene expression regulation at the transcription and splicing level to mrna transport and stabilization . upon cellular stress mutations in several genes ( table 1 ) have been observed in als and ftld patients with tdp-43 proteinopathy , demonstrating that tdp-43 has a key role in the neurodegeneration process . tdp-43 proteinopathy is characterized by pathological modifications including aggregation , c - terminal cleavage into ctfs , hyperphosphorylation and ubiquitination of tdp-43 . furthermore , extensive research on tdp-43-related pathomechanisms suggests that different putative mechanisms might contribute to tdp-43 aggregation , including impaired protein degradation , alterations of tdp-43-associated splicing events , nuclear transport defects , loss of tdp-43 autoregulation and enhanced self - interaction of tdp-43 . abbreviations : / , importin / ; ub , ubiquitin ; p , phosphorylation ; star * , mutant protein . the wide range of tdp-43 targets makes it difficult to pinpoint the exact disease culprit(s ) . also , it is inconceivable that a single tdp-43 target acts as a sole trigger of neurodegeneration . therefore , determining the key targets and their altered function in disease are a major goal . ftld and als constitute the opposite ends of a broad disease continuum , but also co - occur in the same patient or segregate within the same family . a substantial fraction of the families with concomitant ftld and als were explained by the co - segregation of a g4c2 repeat expansion mutation in c9orf72 located at chromosome 9p21 ( 4446 , 8385 ) . in unaffected , non - expanded repeat carriers the repeat length does not exceed 24 units , while in patients the pathological expansions range from 700 to 4400 repeat units ( 86 , 87 ) . at present , little or no information exists about the physiological functions of the c9orf72 protein nevertheless , several g4c2-related pathomechanisms have been suggested , involving both loss - of - function and toxic gain - of - function , which might not be mutually exclusive . based on homology searches , c9orf72 appears distantly related to denn ( differentially expressed in normal and neoplastic cells ) domain - containing proteins which are regulators of membrane trafficking ( 88 ) . denn domain - containing proteins and impaired trafficking have been linked to alzheimer 's disease ( ad ) and als ( 89 , 90 ) . primary model systems have demonstrated that the pathological expansion of the g4c2 repeat is sufficient to induce neurodegeneration ( 91 ) . as the g4c2 repeat is located between two transcription initiation sites , c9orf72 expression might be disrupted by aberrant binding of regulatory elements or altered methylation . cpg hypermethylation near the repeat and loss - of - transcription have been reported ( 44 , 46 , 87 , 92 ) . the reduced expression , however , has not yet been confirmed on the protein level due to lack of proper c9orf72 antibodies . supporting evidence was obtained for decreasing transcriptional activity when intermediate repeat alleles with increasing numbers of repeat units were expressed in cultured cells ( 93 ) . entrapment of rbps and other rna molecules by the expanded g4c2 repeat was suggested to induce rna toxicity through the formation of rna foci ( 46 ) . however , this was not a universal finding in all c9orf72 studies ( 94 ) . nonetheless , hnrnpa3 was shown to bind the g4c2 motif and was deposited in the enigmatic tdp-43-negative neuronal inclusions ( 95 ) . another interesting candidate is hnnrpa2/b1 which is known to directly interact with tdp-43 and forms rna foci by binding c / g - rich repeats in certain ataxias ( 61 , 96 ) . apart from the pathognomonic tdp-43 inclusions , tdp43 negative / p62-positive inclusions were also observed in the hippocampus and cerebellum of c9orf72 g4c2 expansion carriers ( 9799 ) . two studies reported the co - localization of aggregation - prone dipeptide repeat proteins ( dprs ) with these tdp-43 negative / p62-positive inclusions ( 100 , 101 ) . these dprs are generated by translation of the expanded g4c2 repeat through repeat - associated non - atg ( ran ) translation ( 102 ) . since hairpin formation is essential to initiate ran translation , formation of stable rna g - quadruplexes by the g4c2 repeat might trigger initiation of translation of the expanded repeat sequences ( 103 , 104 ) . while different putative mechanisms have emerged for c9orf72 , the key step will be to confirm a pathogenic nature for these mechanisms . appropriate disease models as well as specific antibodies will be crucial to clarify disease pathogenesis in c9orf72 repeat mutation carriers . formation of tdp-43 pathology is a distinguishing feature in a wide range of neurodegenerative disorders including ftld and als disorders , and to a lesser extent in , for example , alzheimer 's and huntington disease ( 1 , 17 ) . however , the pathogenicity of tdp-43 aggregates and the accompanying protein modifications , including hyperphosphorylation , ubiquitination and cleavage into c - terminal fragments ( ctfs ) , remain poorly understood ( 16 ) . few als and ftld patients carry coding mutations in tdp-43 implying that in most patients the aggregation and concomitant nuclear depletion is of wild - type tdp-43 . in these patients , tdp-43 proteinopathy is associated with mutations in other genes like grn and vcp that are underlying the ftld extensive research is ongoing on how these mutated proteins contribute to tdp-43 proteinopathy leading to novel insights in the molecular pathogenesis underlying neurodegeneration in these diseases . primary studies within different model systems found that at least one als mutation ( p.a315 t ) in tardbp , or a disruption of the nuclear localization signal ( nls ) enhanced cytoplasmic mislocalization increasing toxicity in the absence of tdp-43 aggregates ( 72 , 105111 ) . given the diversity of rna targets directly bound by tdp-43 ( 64 , 65 ) , it is not inconceivable that loss of nuclear tdp-43 has detrimental effects on neuronal function . in contrast to fus / tls , however , solid evidence for nuclear transport deficits related to tdp-43 mutations is lacking as none of the mutations cluster around the nls or directly affect its sequence ( 112 ) . nonetheless , inhibition of the importin / pathway and altered transcription profiles of genes active in import processes were associated with sporadic ftld / als and increased aging ( fig . , these data indicated that cytoplasmic mislocalization might contribute to disease pathogenesis , although additional hits are probably required such as cellular stress or genetic / environmental risk factors to induce full blown tdp-43 pathology ( 112 ) . currently , most of the available data points towards increased aggregation propensity of tdp-43 in the presence of pathogenic tardbp mutations , although variable results have been obtained for different mutations ( fig . purified full - length tdp-43 is intrinsically aggregation prone and requires the c - terminal domain ( 114 , 116 , 117 ) . the n - terminus and both rna - recognition motifs of tdp-43 have been implicated in regulating inclusion formation ( 118120 ) . this suggested that rna binding in addition to aggregation is a component of tdp-43 toxicity ( fig . 1 ) ( 121 ) . more specifically , the c - terminal region harbors a glutamine / asparagine - rich ( q / n - rich ) domain that shares similarities with yeast prions ( fig . 1 ) ( 122 , 123 ) . prion proteins exhibit ordered , self - perpetuating aggregation and are thought to transmit from an affected cell to its progeny ( 124 , 125 ) . the importance of the q / n - rich prion - like domain was further exemplified by the recurrent detection of this domain in other rbps using algorithmic searches ( 57 , 126 , 127 ) . several of these rbps , including taf15 , ewsr1 , hnrnpa3 , hnrnpa2/b1 and hnrnpa1 ( table 2 ) , were also found associated with als and/or ftld either by the identification of pathogenic mutations or as component of the proteinaceous inclusions ( 35 , 57 , 58 , 95 ) . these results indicate that perturbed rna - binding might be crucial in the neurodegeneration process ( 42 ) . therefore , it is presumed that prionoid properties of tdp-43 might explain the progressive spread of tdp-43 pathology observed in als patients ( 128 , 129 ) . in contrast to known prions whose aggregates have amyloidogenic properties , conflicting results exist for pathological tdp-43 aggregates ( 130133 ) . one study suggested that the tdp-43 prion domain is involved in physiological mechanisms controlling splicing and protein stability ( 134 ) . regulated protein aggregation is reported for rbps in the formation of rna and stress granules ( sgs ) ( 135 , 136 ) . hence , one of the remaining key questions is how regulated tdp-43 aggregation evolves into pathological structures . as a stress - responsive protein , tdp-43 is recruited to sgs following different forms of cellular stress ( 137140 ) . cytoplasmic mislocalization was a common prerequisite for sgs recruitment of fus / tls and tdp-43 ( 141 ) . consequently , it was suggested that sgs might seed pathological aggregates in a prion - like manner under prolonged cellular stress conditions or by disease - associated mutations in sg - related proteins ( 136 , 142 ) . additionally , disease - associated tdp-43 mutations might also impair protein stability inducing pathological cleavage and aggregation ( 134 ) . it is important to note that at least two hits are required to establish ctf aggregation ( 143 ) . despite the increasing evidence for a role of aggregates in disease , although numerous models overexpressing both wild - type and mutant tdp-43 have been generated , cytoplasmic tdp-43 aggregates were rarely observed , despite the development of an aggressive als - like phenotype in nearly all models . this might imply that pathological aggregates are not a prerequisite for toxicity ( 63 , 76 , 144 ) . once formed , however , tdp-43 aggregates could hasten disease pathogenesis as has been observed in sporadic als patients ( 145 ) . it has become clear that tdp-43 is a dosage - sensitive protein ( 76 , 77 ) that plays an important role during development ( 146148 ) and in adulthood ( 149 , 150 ) . despite lack of clear evidence for tdp-43 overexpression in als and ftld patients , certain pathogenic mutations in tdp-43 showed longer half - lives of mutant protein which correlated with an earlier disease onset ( 82 , 151 ) . furthermore , stabilized proteins were shown to provoke toxicity through protein cleavage and insolubility together with proteasomal impairment and deregulation of mrna levels ( 151 ) . besides animal models , human induced pluripotent stem ( ips ) cell lines have been generated from patients carrying a grn or tardbp mutation recapitulating different disease aspects ( 152 , 153 ) . using this technology , neuronal cell models can be studied in the context of the same genetic background of the patient . the abundant accumulation of ubiquitinated proteins present in many neurodegenerative disorders strongly suggests that components of the protein degradation machinery are defective ( 154 ) . proteolysis is a complex process , which requires molecular chaperones , the ubiquitin proteasome system ( ups ) and the autophagy lysosome system ( hereafter called autophagy ) to monitor protein quality and protect cells from dysfunctional or misfolded proteins ( unfolded protein response , upr ) ( 155 ) . evidence that these systems are impaired in ftld als spectrum diseases came from the identification of mutations in genes involved in proteostasis such as ubqln2 , sqstm , vcp and optn ( table 1 ) ( 43 , 156 ) . these genes have different cellular functions but all share a link to protein degradation . more specifically , the majority operates as adaptor proteins linking ubiquitinated proteins to either the ups or the autophagy system , necessary to initiate proteolysis . a detailed description of the individual gene functions falls outside of the scope of this review , but we summarized the essentials in table 2 . inhibition of either the ups or autophagy results in increased tdp-43 aggregation and enhanced toxicity ( fig . the autophagy receptor protein p62 , encoded by the gene sqstm1 , has gained increasing interest , since it was identified as a component of tdp-43-negative , ubiquitin - positive inclusions in c9orf72-related patients ( 9799 ) . overexpression of p62 has been shown to reduce tdp-43 aggregation in both an autophagy- and proteasome - dependent manner ( 159 ) moreover , p62 directly interacts with tdp-43 and this interaction was disrupted in ftld brains ( 160 ) . motor neuron - specific disruption of the proteasome subunit rpt3 in mice induced motor neuron loss and tdp-43 aggregation , whereas disrupted autophagy did not ( 161 ) . these findings suggested that motor neurons might have a greater sensitivity to proteasome failure and that defective proteolysis might also play a causal role in disease . in addition to ups and autophagy pathways , dysfunction of the upr is also implicated in tdp-43 pathogenesis as mutant tdp-43 failed to up - regulate chaperons due to depletion of x - box - binding protein 1 ( xbp1 ) , a key component of the upr ( 162 ) and regulator of stress resistance and longevity ( 163 ) . impaired proteostasis and concomitant accumulation of rbps holds only for tdp-43 , because fus / tls abnormalities in response to autophagy / proteasome failure are rarely observed ( 43 ) . taken together , these findings indicate that both the ups and autophagy play a key role in tdp-43 turnover . substantial progress has been made in elucidating the biology of tdp-43 proteinopathy associated with neurodegeneration in ftld and als , though the molecular and cellular mechanisms leading to disease remain largely elusive . based on genetic and pathological evidence , different disease pathways are currently emerging which involve impaired rna processing , protein homeostasis , tdp-43 autoregulation and enhanced self - interaction of tdp-43 . hence , disease etiology of als and ftld is very likely to be multifactorial with different pathways converging into a common feature , i.e. tdp-43 misprocessing . the broad functionality of physiological tdp-43 impedes the identification of disease - related abnormalities and consequently also the development of valuable therapeutic treatments . currently , no effective therapies are available that cure or delay disease progression of both ftld and als . animal models mimicking als and ftld disorders are and will be very informative to gain novel insights into the tdp-43 biology and pathogenesis . advances in ips cell technology have been very instructive and may create new opportunities to study tdp-43-related neurodegeneration processes as well as therapeutic strategies . the research in the authors ' group is in part funded by the interuniversity attraction poles program of the belgian science policy office , the medical foundation queen elisabeth , the foundation alzheimer research ( sao - fra ) , the flemish government initiated methusalem excellence program , the research foundation flanders ( fwo ) , the agency for innovation by science and technology ( iwt ) , the university of antwerp research fund , belgium ; and the metlife foundation medical research award to c.v.b . funding to pay the open access publication charges for this article was provided by the agency for innovation by science and technology ( iwt ) flanders , belgium .

aggregation of misfolded tar dna - binding protein 43 ( tdp-43 ) is a striking hallmark of neurodegenerative processes that are observed in several neurological disorders , and in particular in most patients diagnosed with frontotemporal lobar degeneration ( ftld ) or amyotrophic lateral sclerosis ( als ) . a direct causal link with tdp-43 brain proteinopathy was provided by the identification of pathogenic mutations in tardbp , the gene encoding tdp-43 , in als families . however , tdp-43 proteinopathy has also been observed in carriers of mutations in several other genes associated with both als and ftld demonstrating a key role for tdp-43 in neurodegeneration . to date , and despite substantial research into the biology of tdp-43 , its functioning in normal brain and in neurodegeneration processes remains largely elusive . nonetheless , breakthroughs using cellular and animal models have provided valuable insights into als and ftld pathogenesis . accumulating evidence has redirected the research focus towards a major role for impaired rna metabolism and protein homeostasis . at the same time , the concept that toxic tdp-43 protein aggregates promote neurodegeneration is losing its credibility . this review aims at highlighting and discussing the current knowledge on tdp-43 driven pathomechanisms leading to neurodegeneration as observed in tdp-43 proteinopathies . based on the complexity of the associated neurological diseases , a clear understanding of the essential pathological modifications will be crucial for further therapeutic interventions .

INTRODUCTION EMERGING ROLE FOR ALTERED RNA PROCESSING AND RBPS None TDP-43 AGGREGATION VERSUS NEURODEGENERATION CO-OCCURRENCE OF IMPAIRMENTS IN PROTEIN DEGRADATION MACHINERY CONCLUDING REMARKS FUNDING

over the past years , it became clear that frontotemporal lobar degeneration ( ftld ) and amyotrophic lateral sclerosis ( als ) constitute the opposite ends of a disease continuum of overlapping disease phenotypes ( 1 , 2 ) . ftld is a presenile dementia characterized by selective atrophy of the frontal and anterior temporal lobes of the brain ( 3 ) . the comorbidity of als and ftld syndromes in patients is estimated to occur in 50% of the patients ( 1013 ) . in addition to the overlapping clinical symptomatology , the common pathological hallmark in the majority of these patients consists of tar dna - binding protein 43 ( tdp-43 ) , the major protein within ubiquitinated cytoplasmic inclusions . tdp-43 aggregates are present in a spectrum of distinctive neurodegenerative disorders suggesting a key role for tdp-43 in disease pathogenesis ( 1416 ) . since the discovery of tdp-43 in 2006 ( 14 , 15 ) , major efforts have been directed to unravel its physiological functions in normal and disease brain . to date , tdp-43 is known as a highly conserved , nuclear rna - binding protein ( rbp ) involved in transcription and splicing regulation ( 1922 ) ( reviewed in 23 ) . subsequently , identification of causal mutations in tardbp , the gene encoding tdp-43 ( 2426 ) ( reviewed in 27 ) , mechanistically linked neurodegeneration to the occurrence of tdp-43 aggregates . in addition to tardbp , mutations were identified in other genes that had been associated with tdp-43 pathology . mutations are observed at variable frequencies in als , ftld als or ftld patients in progranulin ( grn ) ( 32 , 33 ) , angiogenin ( ang ) ( 34 ) , heterogeneous nuclear ribonucleoprotein a1 and a2/b1 ( hnrnpa1 and hnrnpa2/b1 ) ( 35 ) , optineurin ( optn ) ( 36 ) , ubiquilin 2 ( ubqln2 ) ( 37 ) , sequestosome 1 ( sqstm1 ) ( 38 , 39 ) and valosin - containing protein ( vcp ) ( 40 , 41 ) ( table 1 ) . the majority of the encoded proteins have been identified as components of the pathological inclusions , emphasizing the heterogeneous molecular basis of both ftld and als . not only became c9orf72 the most frequently mutated gene in both als and ftld , the sharing of the ( g4c2 ) repeat expansion mutation linked both disorders into one disease continuum of overlapping clinical symptoms and tdp-43 pathology ( 4446 ) . understanding the molecular basis of c9orf72 associated diseases might provide important insights into common biological mechanisms . ( ftld ) modifier ( als)ftdmissense , nonsense , deletion , frameshift , splice site(32 , 33)auto . genome - wide genetic and animal model screens have identified several putative susceptibility genes and modifiers of tdp-43 toxicity that were strongly associated with als including ephrin type - a receptor 4 precursor ( epha4 ) ( 47 ) , rna lariat debranching enzyme ( dbr1 ) ( 48 ) , elongator protein 3 ( elp3 ) ( 49 ) and intermediate - length polyq expansions in ataxin 2 ( atxn2 ) ( table 1 ) ( 50 , 51 ) . the common tdp-43 pathology in als and ftld patients suggests that pathways disrupting tdp-43 integrity might be shared between patients with a different clinical , pathological and genetic etiology . the question remains , however , which disease processes are essential to drive tdp-43-related pathogenesis . in this review paper , we highlight the research outcomes that contributed to valuable insights into tdp-43-related pathomechanisms . hence , mutations in tardbp and fus indicate that rbps might exert a central role in the pathogenesis of ftld / als - related disorders . likewise , genetic studies identified mutations in additional rbps such as taf15 , ewsr1 and in hnnrpa1 and hnrnpa2/b1 , demonstrating that rbps might contribute generally to als and/or fltd ( 35 , 5759 ) ( tables 1 and 2 ) . table 2.cellular protein functions and molecular pathology of ftld- and als - related genesproteinabbreviationsuggested protein functionmolecular pathologyref.rna-binding proteinstar dna - binding protein 43tdp-43transcription and splicing regulation microrna biogenesisrna transport and stabilization ( member of hnrnp protein family)tdp-43(14 , 15)fused in sarcoma / translocated in liposarcomafus / tlstranscription and splicing regulation microrna processingmaintenance of genomic integrity ( member of fet proteins)fus / tls(165 , 166)tata - binding protein - associated factor 15taf15rna polymerase ii componenttranscription initiation ( member of fet proteins)fus / tls(57)ewing sarcoma breakpoint region 1ewsr1transcriptional repressor(member of fet protein family)fus / tls(58)angiogeninangrna processing and trna modificationvascularizationassembly of stress granulestdp-43(34 , 167)heterogeneous nuclear ribonucleoproteinhnrnpa1hnrnpa2/b1hnrnpa3packing and transport of mrna ( member of hnrnp protein family)n.d.(35 , 95)repeat expansionschromosome 9 open reading frame 72c9orf72unknown protein function ( related to denn proteins)tdp-43 , ups(4446)ataxin 2atxn2regulator of egfr traffickingtdp-43(50 , 168)protein homeostasisvalosin - containing proteinvcpmembrane fusionprotein degradation ( er , proteasome and autophagy - associated)tdp-43(40 , 41)ubiquilin 2ubqln2proteasome - mediated protein degradationtdp-43(37)sequestosome 1 p62sqstm1autophagic degradationregulator of nf-b signaling pathwayinvolved in immune responsen.d. (38)optineurinoptngolgi maintenanceexocytosisvesicular traffickingtdp-43(36)growth factorprogranulingrnmultifunctional growth factorinflammationwound repairtdp-43(32 , 33 , 169)fet proteins , fus ewsr1taf15 dna / rna - binding proteins ; egfr , epidermal growth factor receptor ; er , endoplasmatic reticulum ; denn protein , differentially expressed in normal and neoplastic cells ; n.d . cellular protein functions and molecular pathology of ftld- and als - related genes fet proteins , fus ewsr1taf15 dna / rna - binding proteins ; egfr , epidermal growth factor receptor ; er , endoplasmatic reticulum ; denn protein , differentially expressed in normal and neoplastic cells ; n.d . as a member of the hnrnp family , tdp-43 is involved in multiple steps of gene expression regulation , including rna splicing and transport ( 22 , 60 , 61 ) ( fig . a mouse model expressing mutant tdp-43 induced significant splicing alterations accompanying motor neuron disease in the absence of tdp-43 aggregation or nuclear clearing ( 63 ) . to unravel the biological functions of tdp-43 , tdp-43 rna targets were characterized in cultured cells , mouse brain , and in human brain of ftld and als patients . tdp-43 was shown to bind to 30% of the mouse transcriptome , highlighting the versatility and importance of tdp-43 for splicing regulation ( 64 , 65 ) . also , proteins encoded by the rna targets were enriched for genes involved in synaptic function , neuronal development and rna metabolism . expression of a muscle - specific actin binding protein filamin c , one of the identified genes regulated by tdp-43 , was increased in frontal cortex of ftld patients ( 68 ) . numerous mutations in tardbp have been identified in sporadic and familial als patients and rarely in ftld patients . furthermore , experimental evidence also suggested that different domain structures of tdp-43 are involved in the aggregation process . numerous mutations in tardbp have been identified in sporadic and familial als patients and rarely in ftld patients . the c - terminal region of tdp-43 , harboring most of the pathogenic disease mutations , is required for self - regulation ( fig . tight regulation of tdp-43 expression levels was further illustrated in animal models where human tdp-43 overexpression reduced endogenous protein levels . because of the lack of tdp-43 aggregates in these models , down - regulation of tdp-43 has been suggested to be toxic ( 7274 ) . figure 2.overview of putative mechanisms involved in tdp-43 proteinopathy observed in als and ftld patients . upon cellular stress , mutations in several genes ( table 1 ) have been observed in als and ftld patients with tdp-43 proteinopathy , demonstrating that tdp-43 has a key role in the neurodegeneration process . tdp-43 proteinopathy is characterized by pathological modifications including aggregation , c - terminal cleavage into ctfs , hyperphosphorylation and ubiquitination of tdp-43 . overview of putative mechanisms involved in tdp-43 proteinopathy observed in als and ftld patients . upon cellular stress mutations in several genes ( table 1 ) have been observed in als and ftld patients with tdp-43 proteinopathy , demonstrating that tdp-43 has a key role in the neurodegeneration process . tdp-43 proteinopathy is characterized by pathological modifications including aggregation , c - terminal cleavage into ctfs , hyperphosphorylation and ubiquitination of tdp-43 . a substantial fraction of the families with concomitant ftld and als were explained by the co - segregation of a g4c2 repeat expansion mutation in c9orf72 located at chromosome 9p21 ( 4446 , 8385 ) . at present , little or no information exists about the physiological functions of the c9orf72 protein nevertheless , several g4c2-related pathomechanisms have been suggested , involving both loss - of - function and toxic gain - of - function , which might not be mutually exclusive . based on homology searches , c9orf72 appears distantly related to denn ( differentially expressed in normal and neoplastic cells ) domain - containing proteins which are regulators of membrane trafficking ( 88 ) . the reduced expression , however , has not yet been confirmed on the protein level due to lack of proper c9orf72 antibodies . entrapment of rbps and other rna molecules by the expanded g4c2 repeat was suggested to induce rna toxicity through the formation of rna foci ( 46 ) . however , this was not a universal finding in all c9orf72 studies ( 94 ) . nonetheless , hnrnpa3 was shown to bind the g4c2 motif and was deposited in the enigmatic tdp-43-negative neuronal inclusions ( 95 ) . apart from the pathognomonic tdp-43 inclusions , tdp43 negative / p62-positive inclusions were also observed in the hippocampus and cerebellum of c9orf72 g4c2 expansion carriers ( 9799 ) . these dprs are generated by translation of the expanded g4c2 repeat through repeat - associated non - atg ( ran ) translation ( 102 ) . since hairpin formation is essential to initiate ran translation , formation of stable rna g - quadruplexes by the g4c2 repeat might trigger initiation of translation of the expanded repeat sequences ( 103 , 104 ) . while different putative mechanisms have emerged for c9orf72 , the key step will be to confirm a pathogenic nature for these mechanisms . appropriate disease models as well as specific antibodies will be crucial to clarify disease pathogenesis in c9orf72 repeat mutation carriers . formation of tdp-43 pathology is a distinguishing feature in a wide range of neurodegenerative disorders including ftld and als disorders , and to a lesser extent in , for example , alzheimer 's and huntington disease ( 1 , 17 ) . however , the pathogenicity of tdp-43 aggregates and the accompanying protein modifications , including hyperphosphorylation , ubiquitination and cleavage into c - terminal fragments ( ctfs ) , remain poorly understood ( 16 ) . few als and ftld patients carry coding mutations in tdp-43 implying that in most patients the aggregation and concomitant nuclear depletion is of wild - type tdp-43 . in these patients , tdp-43 proteinopathy is associated with mutations in other genes like grn and vcp that are underlying the ftld extensive research is ongoing on how these mutated proteins contribute to tdp-43 proteinopathy leading to novel insights in the molecular pathogenesis underlying neurodegeneration in these diseases . primary studies within different model systems found that at least one als mutation ( p.a315 t ) in tardbp , or a disruption of the nuclear localization signal ( nls ) enhanced cytoplasmic mislocalization increasing toxicity in the absence of tdp-43 aggregates ( 72 , 105111 ) . in contrast to fus / tls , however , solid evidence for nuclear transport deficits related to tdp-43 mutations is lacking as none of the mutations cluster around the nls or directly affect its sequence ( 112 ) . nonetheless , inhibition of the importin / pathway and altered transcription profiles of genes active in import processes were associated with sporadic ftld / als and increased aging ( fig . currently , most of the available data points towards increased aggregation propensity of tdp-43 in the presence of pathogenic tardbp mutations , although variable results have been obtained for different mutations ( fig . this suggested that rna binding in addition to aggregation is a component of tdp-43 toxicity ( fig . the importance of the q / n - rich prion - like domain was further exemplified by the recurrent detection of this domain in other rbps using algorithmic searches ( 57 , 126 , 127 ) . several of these rbps , including taf15 , ewsr1 , hnrnpa3 , hnrnpa2/b1 and hnrnpa1 ( table 2 ) , were also found associated with als and/or ftld either by the identification of pathogenic mutations or as component of the proteinaceous inclusions ( 35 , 57 , 58 , 95 ) . these results indicate that perturbed rna - binding might be crucial in the neurodegeneration process ( 42 ) . therefore , it is presumed that prionoid properties of tdp-43 might explain the progressive spread of tdp-43 pathology observed in als patients ( 128 , 129 ) . once formed , however , tdp-43 aggregates could hasten disease pathogenesis as has been observed in sporadic als patients ( 145 ) . it has become clear that tdp-43 is a dosage - sensitive protein ( 76 , 77 ) that plays an important role during development ( 146148 ) and in adulthood ( 149 , 150 ) . despite lack of clear evidence for tdp-43 overexpression in als and ftld patients , certain pathogenic mutations in tdp-43 showed longer half - lives of mutant protein which correlated with an earlier disease onset ( 82 , 151 ) . using this technology , neuronal cell models can be studied in the context of the same genetic background of the patient . the abundant accumulation of ubiquitinated proteins present in many neurodegenerative disorders strongly suggests that components of the protein degradation machinery are defective ( 154 ) . proteolysis is a complex process , which requires molecular chaperones , the ubiquitin proteasome system ( ups ) and the autophagy lysosome system ( hereafter called autophagy ) to monitor protein quality and protect cells from dysfunctional or misfolded proteins ( unfolded protein response , upr ) ( 155 ) . evidence that these systems are impaired in ftld als spectrum diseases came from the identification of mutations in genes involved in proteostasis such as ubqln2 , sqstm , vcp and optn ( table 1 ) ( 43 , 156 ) . more specifically , the majority operates as adaptor proteins linking ubiquitinated proteins to either the ups or the autophagy system , necessary to initiate proteolysis . a detailed description of the individual gene functions falls outside of the scope of this review , but we summarized the essentials in table 2 . the autophagy receptor protein p62 , encoded by the gene sqstm1 , has gained increasing interest , since it was identified as a component of tdp-43-negative , ubiquitin - positive inclusions in c9orf72-related patients ( 9799 ) . in addition to ups and autophagy pathways , dysfunction of the upr is also implicated in tdp-43 pathogenesis as mutant tdp-43 failed to up - regulate chaperons due to depletion of x - box - binding protein 1 ( xbp1 ) , a key component of the upr ( 162 ) and regulator of stress resistance and longevity ( 163 ) . impaired proteostasis and concomitant accumulation of rbps holds only for tdp-43 , because fus / tls abnormalities in response to autophagy / proteasome failure are rarely observed ( 43 ) . taken together , these findings indicate that both the ups and autophagy play a key role in tdp-43 turnover . substantial progress has been made in elucidating the biology of tdp-43 proteinopathy associated with neurodegeneration in ftld and als , though the molecular and cellular mechanisms leading to disease remain largely elusive . based on genetic and pathological evidence , different disease pathways are currently emerging which involve impaired rna processing , protein homeostasis , tdp-43 autoregulation and enhanced self - interaction of tdp-43 . hence , disease etiology of als and ftld is very likely to be multifactorial with different pathways converging into a common feature , i.e. the broad functionality of physiological tdp-43 impedes the identification of disease - related abnormalities and consequently also the development of valuable therapeutic treatments . animal models mimicking als and ftld disorders are and will be very informative to gain novel insights into the tdp-43 biology and pathogenesis . the research in the authors ' group is in part funded by the interuniversity attraction poles program of the belgian science policy office , the medical foundation queen elisabeth , the foundation alzheimer research ( sao - fra ) , the flemish government initiated methusalem excellence program , the research foundation flanders ( fwo ) , the agency for innovation by science and technology ( iwt ) , the university of antwerp research fund , belgium ; and the metlife foundation medical research award to c.v.b . funding to pay the open access publication charges for this article was provided by the agency for innovation by science and technology ( iwt ) flanders , belgium .

patients aged 60 years were recruited from the outpatient clinic of the seoul national university bundang hospital ( snubh ) from may to june 2009 . block randomization was used to assign each patient to one of three groups : routine care ( control group ) , self - monitored blood glucose ( smbg ) group , or u - healthcare group with wired telephone - connected glucometer plus mobile phone , in which the glucometer data were technically transmitted by wired telephone through public switched telephone network ( pstn ) . all enrolled participants had been diagnosed with type 2 diabetes for at least 1 year , and their a1c level was 6.510.5% . the study excluded patients with severe diabetes complications ( e.g. , diabetic foot or severe diabetic retinopathy ) , liver dysfunction ( aspartate aminotransferase or alanine aminotransferase > 2.5 times the reference level ) , or renal dysfunction ( serum creatinine > 132 mol / l [ 1.7 mg / dl ] ) , or other medical problems that could affect study results or trial participation . the study screened 180 patients and 26 were excluded : 19 because of the exclusion criteria , and 5 refused to participate after being informed of the study protocol , which required frequent self - blood glucose monitoring or the u - healthcare service . the study finally enrolled 154 individuals , comprising 51 in u - healthcare , 51 in smbg , and 52 in control , and 144 ( 93.5% ) completed the study . the snubh institutional review board approved the study , and all patients gave their written , informed consent . we provided pertinent diabetes education , including a therapeutic lifestyle change program , to standardize every patient s education level and practice of diabetes management . after the education , individuals in the control group did not receive an intervention and were advised to follow - up according to their current medical care . the smbg group was advised to measure their blood glucose level at least 8 times a week ( 3 at fasting , 3 postprandial , and 2 bedtimes ) . the u - healthcare group was educated to use pstn - connected glucometer to measure their blood glucose level at the same frequency as the smbg group and to start short message service ( sms ) on their mobile phone to receive messages from the cdss rule engine server . the pstn - connected glucometer was designed to be elderly - friendly and thus was appropriate for use in this study : the control buttons on glucometer and the screen size of liquid - crystal display ( lcd ) displaying the glucose results were bigger than those of other commercially available glucometers . also included in the pstn - connected glucometer was a step - by - step video instruction for use . an average of 2 to 3 h was required to educate each patient for the device and system use . a specialized diabetes management team consisting of well - trained professionals , including diabetologists , nurses , dietitians , and exercise trainers , organized and directed patient education . for participants who did not fully understand device and system use even after training , more time with simpler instructions were given by the trainers until all participants were comfortable with the glucometer and service delivery system . we also allowed the participants to have a lead - in period to ensure that they fully understood the system and were able to apply it . the study enrollment excluded patients without a text message function on their cellular phone or who were unable to use text messages for any reason . the primary end point of the study was the proportion of patients achieving an a1c level of < 7% without hypoglycemia at 6 months . for the intervention groups , the method and frequency of smbg was informed to ensure compliance of multiple home glucose testing . for those who were randomized to the u - healthcare group , patients were also instructed to telephone the research center if technical difficulties arose at home and were given help directly over the telephone or through a home visit by a technician . all patients visited the outpatient clinic every 3 months for an interview conducted by their physician and provided a blood sample . this study used glucometers ( glucodr supersensor , agm-2200 , allmedicus , korea ) that were specifically devised for u - healthcare . various testing conditions , such as fasting , postprandial , and bedtime , or with control solution , could be set to the meter at the measurement time . in addition , the measurement system adopted flavine - adenine dinucleotide glucose dehydrogenase enzyme technology so the results to be highly glucose - specific without being affected by the oxygen concentration in the blood . after glucose levels were measured , the glucodr supersensor glucometer was placed onto its own cradle , after which all of the tested data were automatically transferred through the pstn and stored in the database of the remote data collection server . cdss - generated messages were sent to the patient s mobile phone within 2 minutes of data transfer . the u - healthcare service used in this study was based on a cdss ( supplementary fig . the patient s anthropometry , blood pressure , current blood glucose and a1c levels , and current medication were simultaneously uploaded from the hospital s electronic medical record ( emr ) server to the u - healthcare server . personal information , including diet and exercise , was also collected and stored on the server to provide appropriate individualized service ( supplementary fig . information from the patient s glucometer was automatically sent to the server by the pstn , after which instructions that were appropriate and specific for each patient were generated by the cdss rule engine . the cdss rule engine is based on the clinical practice recommendations of the american diabetes association and the korean diabetes association ( 14,15 ) ( supplementary fig . in addition to providing messages as a response to the patient s glucose testing , the cdss rule engine also generated evaluation messages on each patient s the weekly and monthly average glucose levels . to ensure compliance with frequent glucose testing ( at least 8 times / week ) , evaluation messages on the total number of weekly glucose measurements were also sent on wednesdays as a reminder . the cdss - generated messages were patient - specific , for example : if the patient s fasting glucose level was < 4 mmol / l ( 72 mg / dl ) twice in a week , a message to change the patient s current diabetes medications ( 50% reduction of sulfonylurea or 2-unit reduction of insulin ) was sent to the patient . mmol / l ( 144 mg / dl ) twice in a week , a message to change the patient s lifestyle or increase current diabetes therapy ( intensification of lifestyle or 2-unit increase of insulin ) was sent to the patient . if the glucose level fell < 2.8 mmol / l ( 50 mg / dl ) , a family member whose information was given to researchers by the patient before the study initiation was also notified to ensure appropriate action to treat hypoglycemia ( supplementary fig . patients were fully educated on the type of messages and their implications to make sure patients knew exactly what to do when they received an automated message . patients were counseled regarding the symptoms of hypoglycemia ( e.g. , weakness , dizziness , shakiness , increased sweating , palpitations , or confusion ) and requested to immediately perform a finger stick glucose measurement if any symptoms occurred that might have been related to hypoglycemia . events of symptomatic hypoglycemia were analyzed as follows : minor hypoglycemia , which was defined as symptoms coexisting with capillary blood glucose levels < 3.5 mmol / l ( 63 mg / dl ) , and major hypoglycemia , which was defined as blood glucose levels < 2.8 mmol / l ( 50 mg / dl ) and an episode requiring medical intervention or exhibiting markedly depressed level of consciousness or seizure . repeated measure of anova was used to determine whether a1c differed significantly over time or among the three groups . for all tests , p < 0.05 was accepted as significant . the analysis was done using spss 11.0 software ( spss inc , chicago , il ) . patients aged 60 years were recruited from the outpatient clinic of the seoul national university bundang hospital ( snubh ) from may to june 2009 . block randomization was used to assign each patient to one of three groups : routine care ( control group ) , self - monitored blood glucose ( smbg ) group , or u - healthcare group with wired telephone - connected glucometer plus mobile phone , in which the glucometer data were technically transmitted by wired telephone through public switched telephone network ( pstn ) . all enrolled participants had been diagnosed with type 2 diabetes for at least 1 year , and their a1c level was 6.510.5% . the study excluded patients with severe diabetes complications ( e.g. , diabetic foot or severe diabetic retinopathy ) , liver dysfunction ( aspartate aminotransferase or alanine aminotransferase > 2.5 times the reference level ) , or renal dysfunction ( serum creatinine > 132 mol / l [ 1.7 mg / dl ] ) , or other medical problems that could affect study results or trial participation . the study screened 180 patients and 26 were excluded : 19 because of the exclusion criteria , and 5 refused to participate after being informed of the study protocol , which required frequent self - blood glucose monitoring or the u - healthcare service . the study finally enrolled 154 individuals , comprising 51 in u - healthcare , 51 in smbg , and 52 in control , and 144 ( 93.5% ) completed the study . the snubh institutional review board approved the study , and all patients gave their written , informed consent . we provided pertinent diabetes education , including a therapeutic lifestyle change program , to standardize every patient s education level and practice of diabetes management . after the education , individuals in the control group did not receive an intervention and were advised to follow - up according to their current medical care . the smbg group was advised to measure their blood glucose level at least 8 times a week ( 3 at fasting , 3 postprandial , and 2 bedtimes ) . the u - healthcare group was educated to use pstn - connected glucometer to measure their blood glucose level at the same frequency as the smbg group and to start short message service ( sms ) on their mobile phone to receive messages from the cdss rule engine server . the pstn - connected glucometer was designed to be elderly - friendly and thus was appropriate for use in this study : the control buttons on glucometer and the screen size of liquid - crystal display ( lcd ) displaying the glucose results were bigger than those of other commercially available glucometers . also included in the pstn - connected glucometer was a step - by - step video instruction for use . an average of 2 to 3 h was required to educate each patient for the device and system use . a specialized diabetes management team consisting of well - trained professionals , including diabetologists , nurses , dietitians , and exercise trainers , organized and directed patient education . for participants who did not fully understand device and system use even after training , more time with simpler instructions were given by the trainers until all participants were comfortable with the glucometer and service delivery system . we also allowed the participants to have a lead - in period to ensure that they fully understood the system and were able to apply it . the study enrollment excluded patients without a text message function on their cellular phone or who were unable to use text messages for any reason . the primary end point of the study was the proportion of patients achieving an a1c level of < 7% without hypoglycemia at 6 months . for the intervention groups , the method and frequency of smbg was informed to ensure compliance of multiple home glucose testing . for those who were randomized to the u - healthcare group , patients were also instructed to telephone the research center if technical difficulties arose at home and were given help directly over the telephone or through a home visit by a technician . all patients visited the outpatient clinic every 3 months for an interview conducted by their physician and provided a blood sample . this study used glucometers ( glucodr supersensor , agm-2200 , allmedicus , korea ) that were specifically devised for u - healthcare . various testing conditions , such as fasting , postprandial , and bedtime , or with control solution , could be set to the meter at the measurement time . in addition , the measurement system adopted flavine - adenine dinucleotide glucose dehydrogenase enzyme technology so the results to be highly glucose - specific without being affected by the oxygen concentration in the blood . after glucose levels were measured , the glucodr supersensor glucometer was placed onto its own cradle , after which all of the tested data were automatically transferred through the pstn and stored in the database of the remote data collection server . cdss - generated messages were sent to the patient s mobile phone within 2 minutes of data transfer . this study used glucometers ( glucodr supersensor , agm-2200 , allmedicus , korea ) that were specifically devised for u - healthcare . various testing conditions , such as fasting , postprandial , and bedtime , or with control solution , could be set to the meter at the measurement time . in addition , the measurement system adopted flavine - adenine dinucleotide glucose dehydrogenase enzyme technology so the results to be highly glucose - specific without being affected by the oxygen concentration in the blood . after glucose levels were measured , the glucodr supersensor glucometer was placed onto its own cradle , after which all of the tested data were automatically transferred through the pstn and stored in the database of the remote data collection server . cdss - generated messages were sent to the patient s mobile phone within 2 minutes of data transfer . the u - healthcare service used in this study was based on a cdss ( supplementary fig . 1 ) . the patient s anthropometry , blood pressure , current blood glucose and a1c levels , and current medication were simultaneously uploaded from the hospital s electronic medical record ( emr ) server to the u - healthcare server . personal information , including diet and exercise , was also collected and stored on the server to provide appropriate individualized service ( supplementary fig . information from the patient s glucometer was automatically sent to the server by the pstn , after which instructions that were appropriate and specific for each patient were generated by the cdss rule engine . the cdss rule engine is based on the clinical practice recommendations of the american diabetes association and the korean diabetes association ( 14,15 ) ( supplementary fig . in addition to providing messages as a response to the patient s glucose testing , the cdss rule engine also generated evaluation messages on each patient s the weekly and monthly average glucose levels . 1c ) . to ensure compliance with frequent glucose testing ( at least 8 times / week ) , evaluation messages on the total number of weekly glucose measurements the cdss - generated messages were patient - specific , for example : if the patient s fasting glucose level was < 4 mmol / l ( 72 mg / dl ) twice in a week , a message to change the patient s current diabetes medications ( 50% reduction of sulfonylurea or 2-unit reduction of insulin ) was sent to the patient . if the fasting glucose level was > 8 mmol / l ( 144 mg / dl ) twice in a week , a message to change the patient s lifestyle or increase current diabetes therapy ( intensification of lifestyle or 2-unit increase of insulin ) was sent to the patient . if the glucose level fell < 2.8 mmol / l ( 50 mg / dl ) , a family member whose information was given to researchers by the patient before the study initiation was also notified to ensure appropriate action to treat hypoglycemia ( supplementary fig . patients were fully educated on the type of messages and their implications to make sure patients knew exactly what to do when they received an automated message . patients were counseled regarding the symptoms of hypoglycemia ( e.g. , weakness , dizziness , shakiness , increased sweating , palpitations , or confusion ) and requested to immediately perform a finger stick glucose measurement if any symptoms occurred that might have been related to hypoglycemia . events of symptomatic hypoglycemia were analyzed as follows : minor hypoglycemia , which was defined as symptoms coexisting with capillary blood glucose levels < 3.5 mmol / l ( 63 mg / dl ) , and major hypoglycemia , which was defined as blood glucose levels < 2.8 mmol / l ( 50 mg / dl ) and an episode requiring medical intervention or exhibiting markedly depressed level of consciousness or seizure . anova was used to compare among the three groups . paired t tests to analyze changes of parameters before and after intervention were used . repeated measure of anova was used to determine whether a1c differed significantly over time or among the three groups . for all tests , p < the analysis was done using spss 11.0 software ( spss inc , chicago , il ) . the clinical characteristics and baseline biochemical data of the 154 participants are summarized in table 1 . no significant differences were noted in anthropometry or biochemical parameters , including fasting glucose or a1c , and prescriptions of antidiabetic agents among the three groups . baseline characteristics of participants by group data are presented as mean ( sd ) or number of participants ( % ) . at the conclusion of the study , 49 ( 96.1% ) , 47 ( 92.2% ) , and 48 ( 92.3% ) participants of the u - healthcare , smbg , and control group , respectively , completed the study . the dropout rates were not different among the groups ; two in the u - healthcare group and four in the smbg group wanted to withdraw because of inconvenience of frequent testing . four patients in control group were withdrawn because of missing laboratory follow - up tests . anthropometric and biochemical parameters after 6 months of follow - up are summarized in table 2 . fasting and postprandial glucose concentrations were significantly decreased in the u - healthcare group , but no significant changes were observed in the smbg or control groups . body weight , bmi , and ldl - cholesterol levels were significantly reduced in the u - healthcare group compared with smbg and control groups . the frequency of self - glucose monitoring was significantly increased in the u - healthcare and smbg groups compared with the control group . the proportion of patients reaching target frequency of glucose testing ( 8 times / week ) was 81.2% , 68.5% , and 31.2% in the u - healthcare , smbg , and control groups , respectively ( p < 0.01 ) . changes of anthropometrics , biochemical parameters , and frequency of self - monitoring blood glucose by the groups after 6 months data are presented as mean ( sd ) or n ( % ) . the a1c level of the control group did not change , but levels in the smbg and u - healthcare groups significantly decreased at the 3-month follow - up ( p < 0.05 ) . the u - healthcare group showed a continuous decreased level of a1c at 6 months ( p < 0.05 ) . thus , the mean a1c level of the u - healthcare group was lower than that of control at the 3-month follow - up , and was lower than that of both the smbg or control groups at 6 months . a : changes of a1c level over 6 months of study in the u - healthcare , smbg , and control groups . b : proportion of patients who achieved a1c < 7.0% without hypoglycemia at 6 months . d : number of hypoglycemia incidences study period among the u - healthcare , smbg , and control groups ( * p < 0.05 ) . the proportion of patients with a1c < 7.0% after 6 months of follow - up was not significantly different among the three groups , although there was a trend showing a greater proportion of the u - healthcare group achieved a1c the proportion of patients that achieved a1c < 7.0% without hypoglycemia , the primary end point of this study , was 30.6% in the u - healthcare group , which was significantly higher than in the smbg ( 23.4% ) or control groups ( 14.0% ; vs. smbg , p = 0.027 and vs. control p = 0.019 , respectively ; fig . before study enrollment , the participants in the three groups had experienced a similar number of hypoglycemic events . during the 6 months of this study , the proportion of patients experiencing minor hypoglycemia seemed to be higher in u - healthcare group ( 32.2% ) than in the smbg ( 24.5% ) or control groups ( 21.8% ; fig . in contrast , major and nocturnal hypoglycemia was smaller in the u - healthcare group than in the smbg or control groups ( p < 0.05 ) . the clinical characteristics and baseline biochemical data of the 154 participants are summarized in table 1 . no significant differences were noted in anthropometry or biochemical parameters , including fasting glucose or a1c , and prescriptions of antidiabetic agents among the three groups . baseline characteristics of participants by group data are presented as mean ( sd ) or number of participants ( % ) . at the conclusion of the study , 49 ( 96.1% ) , 47 ( 92.2% ) , and 48 ( 92.3% ) participants of the u - healthcare , smbg , and control group , respectively , completed the study . the dropout rates were not different among the groups ; two in the u - healthcare group and four in the smbg group wanted to withdraw because of inconvenience of frequent testing . four patients in control group were withdrawn because of missing laboratory follow - up tests . anthropometric and biochemical parameters after 6 months of follow - up are summarized in table 2 . fasting and postprandial glucose concentrations were significantly decreased in the u - healthcare group , but no significant changes were observed in the smbg or control groups . body weight , bmi , and ldl - cholesterol levels were significantly reduced in the u - healthcare group compared with smbg and control groups . the frequency of self - glucose monitoring was significantly increased in the u - healthcare and smbg groups compared with the control group . the proportion of patients reaching target frequency of glucose testing ( 8 times / week ) was 81.2% , 68.5% , and 31.2% in the u - healthcare , smbg , and control groups , respectively ( p < 0.01 ) . changes of anthropometrics , biochemical parameters , and frequency of self - monitoring blood glucose by the groups after 6 months data are presented as mean ( sd ) or n ( % ) . . the a1c level of the control group did not change , but levels in the smbg and u - healthcare groups significantly decreased at the 3-month follow - up ( p < 0.05 ) . the u - healthcare group showed a continuous decreased level of a1c at 6 months ( p < 0.05 ) . thus , the mean a1c level of the u - healthcare group was lower than that of control at the 3-month follow - up , and was lower than that of both the smbg or control groups at 6 months . a : changes of a1c level over 6 months of study in the u - healthcare , smbg , and control groups . b : proportion of patients who achieved a1c < 7.0% without hypoglycemia at 6 months . d : number of hypoglycemia incidences study period among the u - healthcare , smbg , and control groups ( * p < 0.05 ) . the proportion of patients with a1c < 7.0% after 6 months of follow - up was not significantly different among the three groups , although there was a trend showing a greater proportion of the u - healthcare group achieved a1c the proportion of patients that achieved a1c < 7.0% without hypoglycemia , the primary end point of this study , was 30.6% in the u - healthcare group , which was significantly higher than in the smbg ( 23.4% ) or control groups ( 14.0% ; vs. smbg , p = 0.027 and vs. control p = 0.019 , respectively ; fig . before study enrollment , the participants in the three groups had experienced a similar number of hypoglycemic events . during the 6 months of this study , the proportion of patients experiencing minor hypoglycemia seemed to be higher in u - healthcare group ( 32.2% ) than in the smbg ( 24.5% ) or control groups ( 21.8% ; fig . in contrast , major and nocturnal hypoglycemia was smaller in the u - healthcare group than in the smbg or control groups ( p < 0.05 ) . the cdss - based u - healthcare service achieved better glycemic control with less hypoglycemia than smbg and routine care , and may provide effective and safe diabetes management in elderly diabetic patients . the cdss - based u - healthcare system generates instant feedback on patients glucose levels by providing appropriate recommendations , including lifestyle changes and drug adjustments . in this study , we focused on the target goal of glycemic control ( a1c < 7% ) without hypoglycemia . because the participants were all elderly ( > 60 years ) , hypoglycemia may be critical in aggravating chronic complications as well as worsening glycemic control . recent large - scale studies showed that intensive glucose control had not benefited patients ; in fact , tighter control caused more harm than the conventional treatment ( 16,17 ) . this lack of benefit may be explained by the occurrence of hypoglycemia , which is particularly important in elderly individuals , who are less aware of hypoglycemic symptoms ( 18,19 ) . the mean a1c value during the study decreased significantly in the u - healthcare group compared with the control group . there were more participants with a mean value of a1c < 7.0% in the u - healthcare ( 34% ) than in the smbg ( 31.9% ) or control ( 20.4% ) group , although this was not statistically significant . compared with the change of fasting glucose level , the decrease in the postprandial 2-h glucose level was more prominent in the u - healthcare group ( by 40 mg / dl , p = 0.007 ) . these data indicate that the cdss - based u - healthcare service was effective in decreasing postprandial glucose surge by immediately alerting patients with lifestyle change recommendations . according to previous study results , postprandial glucose concentration is regarded as a risk factor for developing long - term complications ( 2022 ) . therefore , less fluctuation of glucose levels would lead to better prevention of long - term diabetes complications . in this study , glycemic control also improved in the smbg group , but this effect was attenuated over time . this implies that self - management of diabetes is difficult without close and consistent supervision . thus , prompt follow - up involving appropriate recommendations and consistent reminder messages that motivate patients may be more important than the frequency of glucose self - measurement per se . although , the frequency of smbg was directly related to the mean a1c levels , it is noteworthy to recognize its attenuating relationship at the end of the study . in addition , u - healthcare service group showed a decrease in bmi and ldl - cholesterol level . these results can be interpreted as unintended benefits resulting from adhering to lifestyle change recommendations that were generated by the cdss rule engine . thus , u - healthcare service has many other beneficial effects pertaining to healthy lifestyle changes encouraged by automated messages . several studies have used a different telehealth system in different settings . a previous study from our group confirmed the use of the glucometer with a mobile system and zigbee communication protocol and showed improved self - care in diabetes management in elderly diabetic patients ( 8) . an internet - based glucose control system used in the middle - aged type 2 diabetic patients recently showed a significant reduction of the a1c level ( 11 ) . more recently , another group from south korea reported that the combined application of a mobile device and a web - based monitoring system for 12 weeks improved various metabolic parameters in obese patients with diabetes and hypertension ( 12 ) . thus , various applications of advanced information technology in different settings and populations would be helpful in diabetes management , and as a result , a great number of studies are ongoing in this field ( 13,2325 ) . patients in this study were allowed to change their therapeutic regimen according to the text messages generated by the cdss rule engine . generally , every change in the drug regimen in korea must be certified by a doctor . this study , however , was conducted under controlled and special circumstances where the participants were educated intensively and the dose of self - adjustment was limited to a very narrow range . in addition , the investigators frequently monitored the text messages , and an active alert system also sent timely notification of adverse events . nevertheless , the issue of self - changing therapy by patients is an area of uncertainty and concern , and more clinical studies are required to support its validity . in addition , study participants were limited to elderly individuals , and only blood glucose levels were involved . further development of the cdss rule engine for patient s blood pressure or other medical condition is warranted . thus , a large - scale , long - term clinical trial using the advanced cdss rule engine for type 2 diabetic patients is required in the near future . despite more frequent testing , 96.1% of the u - healthcare group completed the study . this could be attributed to education , reminder messages generated by the glucometer , and patient satisfaction with the glucose results associated with frequent testing . the high proportion of elderly patients who completed the study is important for several reasons . the successful completion serves as an indication that even technologically challenged elderly individuals can adopt a new and advanced system . it is critical , however , to provide sufficient education and training before such system implementation , as was done in this study . another significance of this adapted population is its implication on the use of a new system in the general public . successful adoption of new technology by the general public when the u - healthcare system is widely implemented is anticipated from the results of our study . in conclusion , we have demonstrated that the 6-month application of the u - healthcare system , which is characterized by a proactive automated communication system using the cdss rule engine , helped diabetic patients achieve target glycemic control with less hypoglycemia . in the near future , we hope that the individualized u - healthcare system will contribute to diabetes management by reducing complications and improving quality of life and self - care in patients with diabetes .

objectiveto improve quality and efficiency of care for elderly patients with type 2 diabetes , we introduced elderly - friendly strategies to the clinical decision support system ( cdss)-based ubiquitous healthcare ( u - healthcare ) service , which is an individualized health management system using advanced medical information technology.research design and methodswe conducted a 6-month randomized , controlled clinical trial involving 144 patients aged > 60 years . participants were randomly assigned to receive routine care ( control , n = 48 ) , to the self - monitored blood glucose ( smbg , n = 47 ) group , or to the u - healthcare group ( n = 49 ) . the primary end point was the proportion of patients achieving a1c < 7% without hypoglycemia at 6 months . u - healthcare system refers to an individualized medical service in which medical instructions are given through the patient s mobile phone . patients receive a glucometer with a public switched telephone network - connected cradle that automatically transfers test results to a hospital - based server . once the data are transferred to the server , an automated system , the cdss rule engine , generates and sends patient - specific messages by mobile phone.resultsafter 6 months of follow - up , the mean a1c level was significantly decreased from 7.8 1.3% to 7.4 1.0% ( p < 0.001 ) in the u - healthcare group and from 7.9 1.0% to 7.7 1.0% ( p = 0.020 ) in the smbg group , compared with 7.9 0.8% to 7.8 1.0% ( p = 0.274 ) in the control group . the proportion of patients with a1c < 7% without hypoglycemia was 30.6% in the u - healthcare group , 23.4% in the smbg group ( 23.4% ) , and 14.0% in the control group ( p < 0.05).conclusionsthe cdss - based u - healthcare service achieved better glycemic control with less hypoglycemia than smbg and routine care and may provide effective and safe diabetes management in the elderly diabetic patients .

RESEARCH DESIGN AND METHODS Study participants Study design and intervention Device Blood glucose monitoring system Data transfer CDSS-based u-healthcare service Definition of hypoglycemia Statistical analysis RESULTS Baseline characteristics of participants Follow-up of study participants Effects of intervention Achievement of primary target goal (A1C <7.0% without hypoglycemia) Hypoglycemia CONCLUSIONS

block randomization was used to assign each patient to one of three groups : routine care ( control group ) , self - monitored blood glucose ( smbg ) group , or u - healthcare group with wired telephone - connected glucometer plus mobile phone , in which the glucometer data were technically transmitted by wired telephone through public switched telephone network ( pstn ) . all enrolled participants had been diagnosed with type 2 diabetes for at least 1 year , and their a1c level was 6.510.5% . the study screened 180 patients and 26 were excluded : 19 because of the exclusion criteria , and 5 refused to participate after being informed of the study protocol , which required frequent self - blood glucose monitoring or the u - healthcare service . the study finally enrolled 154 individuals , comprising 51 in u - healthcare , 51 in smbg , and 52 in control , and 144 ( 93.5% ) completed the study . after the education , individuals in the control group did not receive an intervention and were advised to follow - up according to their current medical care . the smbg group was advised to measure their blood glucose level at least 8 times a week ( 3 at fasting , 3 postprandial , and 2 bedtimes ) . the u - healthcare group was educated to use pstn - connected glucometer to measure their blood glucose level at the same frequency as the smbg group and to start short message service ( sms ) on their mobile phone to receive messages from the cdss rule engine server . the pstn - connected glucometer was designed to be elderly - friendly and thus was appropriate for use in this study : the control buttons on glucometer and the screen size of liquid - crystal display ( lcd ) displaying the glucose results were bigger than those of other commercially available glucometers . the primary end point of the study was the proportion of patients achieving an a1c level of < 7% without hypoglycemia at 6 months . for those who were randomized to the u - healthcare group , patients were also instructed to telephone the research center if technical difficulties arose at home and were given help directly over the telephone or through a home visit by a technician . in addition , the measurement system adopted flavine - adenine dinucleotide glucose dehydrogenase enzyme technology so the results to be highly glucose - specific without being affected by the oxygen concentration in the blood . after glucose levels were measured , the glucodr supersensor glucometer was placed onto its own cradle , after which all of the tested data were automatically transferred through the pstn and stored in the database of the remote data collection server . cdss - generated messages were sent to the patient s mobile phone within 2 minutes of data transfer . the u - healthcare service used in this study was based on a cdss ( supplementary fig . the patient s anthropometry , blood pressure , current blood glucose and a1c levels , and current medication were simultaneously uploaded from the hospital s electronic medical record ( emr ) server to the u - healthcare server . information from the patient s glucometer was automatically sent to the server by the pstn , after which instructions that were appropriate and specific for each patient were generated by the cdss rule engine . the cdss rule engine is based on the clinical practice recommendations of the american diabetes association and the korean diabetes association ( 14,15 ) ( supplementary fig . in addition to providing messages as a response to the patient s glucose testing , the cdss rule engine also generated evaluation messages on each patient s the weekly and monthly average glucose levels . the cdss - generated messages were patient - specific , for example : if the patient s fasting glucose level was < 4 mmol / l ( 72 mg / dl ) twice in a week , a message to change the patient s current diabetes medications ( 50% reduction of sulfonylurea or 2-unit reduction of insulin ) was sent to the patient . events of symptomatic hypoglycemia were analyzed as follows : minor hypoglycemia , which was defined as symptoms coexisting with capillary blood glucose levels < 3.5 mmol / l ( 63 mg / dl ) , and major hypoglycemia , which was defined as blood glucose levels < 2.8 mmol / l ( 50 mg / dl ) and an episode requiring medical intervention or exhibiting markedly depressed level of consciousness or seizure . block randomization was used to assign each patient to one of three groups : routine care ( control group ) , self - monitored blood glucose ( smbg ) group , or u - healthcare group with wired telephone - connected glucometer plus mobile phone , in which the glucometer data were technically transmitted by wired telephone through public switched telephone network ( pstn ) . all enrolled participants had been diagnosed with type 2 diabetes for at least 1 year , and their a1c level was 6.510.5% . the study screened 180 patients and 26 were excluded : 19 because of the exclusion criteria , and 5 refused to participate after being informed of the study protocol , which required frequent self - blood glucose monitoring or the u - healthcare service . the study finally enrolled 154 individuals , comprising 51 in u - healthcare , 51 in smbg , and 52 in control , and 144 ( 93.5% ) completed the study . we provided pertinent diabetes education , including a therapeutic lifestyle change program , to standardize every patient s education level and practice of diabetes management . after the education , individuals in the control group did not receive an intervention and were advised to follow - up according to their current medical care . the smbg group was advised to measure their blood glucose level at least 8 times a week ( 3 at fasting , 3 postprandial , and 2 bedtimes ) . the u - healthcare group was educated to use pstn - connected glucometer to measure their blood glucose level at the same frequency as the smbg group and to start short message service ( sms ) on their mobile phone to receive messages from the cdss rule engine server . the pstn - connected glucometer was designed to be elderly - friendly and thus was appropriate for use in this study : the control buttons on glucometer and the screen size of liquid - crystal display ( lcd ) displaying the glucose results were bigger than those of other commercially available glucometers . the primary end point of the study was the proportion of patients achieving an a1c level of < 7% without hypoglycemia at 6 months . for those who were randomized to the u - healthcare group , patients were also instructed to telephone the research center if technical difficulties arose at home and were given help directly over the telephone or through a home visit by a technician . in addition , the measurement system adopted flavine - adenine dinucleotide glucose dehydrogenase enzyme technology so the results to be highly glucose - specific without being affected by the oxygen concentration in the blood . cdss - generated messages were sent to the patient s mobile phone within 2 minutes of data transfer . various testing conditions , such as fasting , postprandial , and bedtime , or with control solution , could be set to the meter at the measurement time . in addition , the measurement system adopted flavine - adenine dinucleotide glucose dehydrogenase enzyme technology so the results to be highly glucose - specific without being affected by the oxygen concentration in the blood . cdss - generated messages were sent to the patient s mobile phone within 2 minutes of data transfer . the u - healthcare service used in this study was based on a cdss ( supplementary fig . the patient s anthropometry , blood pressure , current blood glucose and a1c levels , and current medication were simultaneously uploaded from the hospital s electronic medical record ( emr ) server to the u - healthcare server . information from the patient s glucometer was automatically sent to the server by the pstn , after which instructions that were appropriate and specific for each patient were generated by the cdss rule engine . the cdss rule engine is based on the clinical practice recommendations of the american diabetes association and the korean diabetes association ( 14,15 ) ( supplementary fig . in addition to providing messages as a response to the patient s glucose testing , the cdss rule engine also generated evaluation messages on each patient s the weekly and monthly average glucose levels . to ensure compliance with frequent glucose testing ( at least 8 times / week ) , evaluation messages on the total number of weekly glucose measurements the cdss - generated messages were patient - specific , for example : if the patient s fasting glucose level was < 4 mmol / l ( 72 mg / dl ) twice in a week , a message to change the patient s current diabetes medications ( 50% reduction of sulfonylurea or 2-unit reduction of insulin ) was sent to the patient . if the fasting glucose level was > 8 mmol / l ( 144 mg / dl ) twice in a week , a message to change the patient s lifestyle or increase current diabetes therapy ( intensification of lifestyle or 2-unit increase of insulin ) was sent to the patient . events of symptomatic hypoglycemia were analyzed as follows : minor hypoglycemia , which was defined as symptoms coexisting with capillary blood glucose levels < 3.5 mmol / l ( 63 mg / dl ) , and major hypoglycemia , which was defined as blood glucose levels < 2.8 mmol / l ( 50 mg / dl ) and an episode requiring medical intervention or exhibiting markedly depressed level of consciousness or seizure . at the conclusion of the study , 49 ( 96.1% ) , 47 ( 92.2% ) , and 48 ( 92.3% ) participants of the u - healthcare , smbg , and control group , respectively , completed the study . the dropout rates were not different among the groups ; two in the u - healthcare group and four in the smbg group wanted to withdraw because of inconvenience of frequent testing . four patients in control group were withdrawn because of missing laboratory follow - up tests . anthropometric and biochemical parameters after 6 months of follow - up are summarized in table 2 . fasting and postprandial glucose concentrations were significantly decreased in the u - healthcare group , but no significant changes were observed in the smbg or control groups . body weight , bmi , and ldl - cholesterol levels were significantly reduced in the u - healthcare group compared with smbg and control groups . the frequency of self - glucose monitoring was significantly increased in the u - healthcare and smbg groups compared with the control group . the proportion of patients reaching target frequency of glucose testing ( 8 times / week ) was 81.2% , 68.5% , and 31.2% in the u - healthcare , smbg , and control groups , respectively ( p < 0.01 ) . changes of anthropometrics , biochemical parameters , and frequency of self - monitoring blood glucose by the groups after 6 months data are presented as mean ( sd ) or n ( % ) . the a1c level of the control group did not change , but levels in the smbg and u - healthcare groups significantly decreased at the 3-month follow - up ( p < 0.05 ) . the u - healthcare group showed a continuous decreased level of a1c at 6 months ( p < 0.05 ) . thus , the mean a1c level of the u - healthcare group was lower than that of control at the 3-month follow - up , and was lower than that of both the smbg or control groups at 6 months . a : changes of a1c level over 6 months of study in the u - healthcare , smbg , and control groups . b : proportion of patients who achieved a1c < 7.0% without hypoglycemia at 6 months . d : number of hypoglycemia incidences study period among the u - healthcare , smbg , and control groups ( * p < 0.05 ) . the proportion of patients with a1c < 7.0% after 6 months of follow - up was not significantly different among the three groups , although there was a trend showing a greater proportion of the u - healthcare group achieved a1c the proportion of patients that achieved a1c < 7.0% without hypoglycemia , the primary end point of this study , was 30.6% in the u - healthcare group , which was significantly higher than in the smbg ( 23.4% ) or control groups ( 14.0% ; vs. smbg , p = 0.027 and vs. control p = 0.019 , respectively ; fig . during the 6 months of this study , the proportion of patients experiencing minor hypoglycemia seemed to be higher in u - healthcare group ( 32.2% ) than in the smbg ( 24.5% ) or control groups ( 21.8% ; fig . in contrast , major and nocturnal hypoglycemia was smaller in the u - healthcare group than in the smbg or control groups ( p < 0.05 ) . at the conclusion of the study , 49 ( 96.1% ) , 47 ( 92.2% ) , and 48 ( 92.3% ) participants of the u - healthcare , smbg , and control group , respectively , completed the study . the dropout rates were not different among the groups ; two in the u - healthcare group and four in the smbg group wanted to withdraw because of inconvenience of frequent testing . four patients in control group were withdrawn because of missing laboratory follow - up tests . anthropometric and biochemical parameters after 6 months of follow - up are summarized in table 2 . fasting and postprandial glucose concentrations were significantly decreased in the u - healthcare group , but no significant changes were observed in the smbg or control groups . body weight , bmi , and ldl - cholesterol levels were significantly reduced in the u - healthcare group compared with smbg and control groups . the frequency of self - glucose monitoring was significantly increased in the u - healthcare and smbg groups compared with the control group . the proportion of patients reaching target frequency of glucose testing ( 8 times / week ) was 81.2% , 68.5% , and 31.2% in the u - healthcare , smbg , and control groups , respectively ( p < 0.01 ) . changes of anthropometrics , biochemical parameters , and frequency of self - monitoring blood glucose by the groups after 6 months data are presented as mean ( sd ) or n ( % ) . the a1c level of the control group did not change , but levels in the smbg and u - healthcare groups significantly decreased at the 3-month follow - up ( p < 0.05 ) . the u - healthcare group showed a continuous decreased level of a1c at 6 months ( p < 0.05 ) . thus , the mean a1c level of the u - healthcare group was lower than that of control at the 3-month follow - up , and was lower than that of both the smbg or control groups at 6 months . a : changes of a1c level over 6 months of study in the u - healthcare , smbg , and control groups . b : proportion of patients who achieved a1c < 7.0% without hypoglycemia at 6 months . d : number of hypoglycemia incidences study period among the u - healthcare , smbg , and control groups ( * p < 0.05 ) . the proportion of patients with a1c < 7.0% after 6 months of follow - up was not significantly different among the three groups , although there was a trend showing a greater proportion of the u - healthcare group achieved a1c the proportion of patients that achieved a1c < 7.0% without hypoglycemia , the primary end point of this study , was 30.6% in the u - healthcare group , which was significantly higher than in the smbg ( 23.4% ) or control groups ( 14.0% ; vs. smbg , p = 0.027 and vs. control p = 0.019 , respectively ; fig . during the 6 months of this study , the proportion of patients experiencing minor hypoglycemia seemed to be higher in u - healthcare group ( 32.2% ) than in the smbg ( 24.5% ) or control groups ( 21.8% ; fig . in contrast , major and nocturnal hypoglycemia was smaller in the u - healthcare group than in the smbg or control groups ( p < 0.05 ) . the cdss - based u - healthcare service achieved better glycemic control with less hypoglycemia than smbg and routine care , and may provide effective and safe diabetes management in elderly diabetic patients . the cdss - based u - healthcare system generates instant feedback on patients glucose levels by providing appropriate recommendations , including lifestyle changes and drug adjustments . in this study , we focused on the target goal of glycemic control ( a1c < 7% ) without hypoglycemia . because the participants were all elderly ( > 60 years ) , hypoglycemia may be critical in aggravating chronic complications as well as worsening glycemic control . the mean a1c value during the study decreased significantly in the u - healthcare group compared with the control group . there were more participants with a mean value of a1c < 7.0% in the u - healthcare ( 34% ) than in the smbg ( 31.9% ) or control ( 20.4% ) group , although this was not statistically significant . compared with the change of fasting glucose level , the decrease in the postprandial 2-h glucose level was more prominent in the u - healthcare group ( by 40 mg / dl , p = 0.007 ) . these data indicate that the cdss - based u - healthcare service was effective in decreasing postprandial glucose surge by immediately alerting patients with lifestyle change recommendations . in this study , glycemic control also improved in the smbg group , but this effect was attenuated over time . although , the frequency of smbg was directly related to the mean a1c levels , it is noteworthy to recognize its attenuating relationship at the end of the study . in addition , u - healthcare service group showed a decrease in bmi and ldl - cholesterol level . thus , u - healthcare service has many other beneficial effects pertaining to healthy lifestyle changes encouraged by automated messages . a previous study from our group confirmed the use of the glucometer with a mobile system and zigbee communication protocol and showed improved self - care in diabetes management in elderly diabetic patients ( 8) . an internet - based glucose control system used in the middle - aged type 2 diabetic patients recently showed a significant reduction of the a1c level ( 11 ) . patients in this study were allowed to change their therapeutic regimen according to the text messages generated by the cdss rule engine . nevertheless , the issue of self - changing therapy by patients is an area of uncertainty and concern , and more clinical studies are required to support its validity . further development of the cdss rule engine for patient s blood pressure or other medical condition is warranted . thus , a large - scale , long - term clinical trial using the advanced cdss rule engine for type 2 diabetic patients is required in the near future . despite more frequent testing , 96.1% of the u - healthcare group completed the study . successful adoption of new technology by the general public when the u - healthcare system is widely implemented is anticipated from the results of our study . in conclusion , we have demonstrated that the 6-month application of the u - healthcare system , which is characterized by a proactive automated communication system using the cdss rule engine , helped diabetic patients achieve target glycemic control with less hypoglycemia . in the near future , we hope that the individualized u - healthcare system will contribute to diabetes management by reducing complications and improving quality of life and self - care in patients with diabetes .

rna molecules are not merely simple carriers of genetic information but can assemble into complex tertiary structures and even catalyze reactions . in fact , the existence of catalytic rna molecules ( ribozymes ) has led to the proposition of the rna world hypothesis . in modern cells , rna molecules catalyze just two classes of chemical reactions : modifications of phosphodiester bonds ( dna and rna cleavage , rna splicing ) and peptide bond formation . artificially designed ribozymes , however , are known to catalyze a wide range of chemical reactions . in some ribozymes , the slow opening and closing of tertiary structure ( rna breathing ) this latter process is hierarchical , first proceeding on the secondary structure level via formation of fairly stable watson crick base pairs . subsequently , secondary structure elements fold into a compact , three - dimensional structure . the folding of rna into the native tertiary fold may proceed via a complex sequence of secondary structures . formation of compact tertiary structures may require the presence of counterions , particularly divalent cations such as mg , which screen the intrinsic negative charges on the rna phosphate groups and , thereby , stabilize certain tertiary motifs . even small modifications of single nucleotides may result in different tertiary structures and hence different energy landscapes . indeed , rna sequence , structure , and function interact in a complex , not yet fully understood fashion , and the characterization of rna folding kinetics , including the pathways of secondary and tertiary structure changes , remains an intricate problem . in this work , we have investigated the conformational equilibrium and the folding pathway of the 49mer single - stranded rna ribozyme diels alderase ( dase ) using a novel hidden markov model ( hmm ) analysis of single - molecule fret data . dase catalyzes a diels alder reaction , i.e. , the [ 4 + 2 ] cycloaddition reaction between anthracene dienes and maleimide dienophiles . dase is a true multiple - turnover catalyst and shows remarkable enantioselectivity ( > 95% enantiomeric excess ) . it has a well - defined folded structure , as revealed by x - ray crystallography . the folded state consists of three helices arranged around a pseudoknot region , in which the catalytic pocket of the ribozyme is located ( figure 1b ) . a continuous sequence of stacking interactions runs from the bottom of helix ii to the top of helix iii and has been termed the spine of the folded structure . the tertiary fold is held together by a pseudoknot , in which the 5-g1-g2-a3-g4 segment bridges the unpaired strands of the asymmetric bulge ( figure 1a ) . the precise hydrogen - bond pattern in the pseudoknot region is known to be crucial both for thermal stability of the overall fold as well as for the shape of the catalytic pocket . recent experimental and computational evidence showed that cations specifically bind to certain sites that stabilize the tertiary fold , without interfering with the catalytic reaction . low mg concentrations were found to destabilize the folded conformation and to dramatically decrease the catalytic activity of the ribozyme . diels solid lines , secondary structure base pairs ; dotted lines , tertiary structure base pairs . attachment sites of the fret labels are marked by green ( donor dye cy3 at u6 in construct i and at the 5 end in construct ii ) and orange ( acceptor dye cy5 at u42 in construct i and at u30 in construct ii ) arrows . color - coding of the secondary structure elements as in panel a. attachment sites of the fret labels are indicated by green ( cy3 , donor ) and red ( cy5 , acceptor ) spheres . ( the figure has been adapted from figure 1 in ref ( 9 ) . ) single - molecule frster resonance energy transfer ( smfret ) is a powerful tool to follow conformational fluctuations of biomolecules on length scales of a few nanometers in real time . smfret measurements with surface - immobilized molecules revealed that dase is highly dynamic and can exist in substantially different conformations , which were found to interconvert on time scales of hundreds of milliseconds . the concentration of mg influences the shape or population of the accessible conformational states , as indicated by the mg dependence of the fret efficiency histograms and the apparent folding rates . consistent with conformational fluctuations , a poor resolution of dase spectra was found in subsequent nmr studies . the mg - dependent fret efficiency histograms revealed at least two conformational ensembles : ( i ) a high fret state , attributed to the folded conformation , whose population increases with increasing mg concentration , and ( ii ) a distribution of intermediate fret efficiencies , whose population decreases with increasing mg concentration . the intermediates were observed to spread out over a wide range of fret efficiency values and , presumably , comprise multiple conformations with different secondary and tertiary structures . in practice , only two or three states with significantly different fret efficiencies can be distinguished in a histogram - based analysis . consequently , stochastic fluctuations of the number of photons within a time bin ( shot noise ) significantly contribute to the widths of the fret distributions and prevent the separation of states with similar mean fret efficiencies . it completely neglects the time sequence of events in the single - molecule trajectories and , thus , discards a substantial part of the available information . in contrast , hidden markov models can distinguish states in the data by using both the differences in fret efficiency and the time sequence of events , and , thus , can decompose states with similar fret efficiencies but different kinetic properties . recent studies on single - molecule protein and rna data sets have demonstrated the power of hmms to resolve a multitude of states . hmm analysis has its intrinsic challenges , however , because ( i ) the results depend on the number of states used , ( ii ) the hmm optimization may get stuck in local minima , ( iii ) models with many states are difficult to validate , and ( iv ) the quality of the model depends crucially on the validity of the underlying likelihood function ( i.e. , the stochastic model of the measured process ) . here , we present an hmm analysis scheme that addresses these problems . at the core of this scheme is the idea that the number of states required to describe the kinetics in a hierarchical energy landscape is not fixed but depends on the time scales of interest ( figure 2 ) . directly estimating hmms with a few states often yields wrong kinetics , as they tend to prefer models whose states have clearly different fret efficiency . however , in real data , distinct and slowly interconverting conformations may have strongly overlapping fret efficiency distributions , which are difficult to separate . therefore , we construct an initial hmm with many states ( corresponding to a fine discretization of conformational space ) . the initial number of states is determined by a validation scheme , which tests reproducibility and consistency of the model with the underlying data set . the initial states are subsequently coarse - grained on the basis of their kinetics . this approach allows us to model ( coarse ) states even when they strongly overlap in their fret efficiencies and have very irregular ( e.g. , non - gaussian ) fret distributions . our hmm uses a poissonian likelihood function to model the physical process of photon emission . this approach is preferable over using gaussian likelihood functions of the fret efficiency . for a detailed discussion , see the supporting information . in addition , we have developed an approach to account for the trace - specific background noise . ( a ) hierarchical free energy landscape with various minima ( conformations ) interconverting on different time scales . ( b ) a fret efficiency versus distance curve , with the five conformations in panel a assigned to certain fret efficiencies ( distances ) . conformations with suitably long lifetimes can be distinguished by hmm analysis of fret traces but may have overlapping fret efficiencies even when they are distinct . ( c ) probability density function of finding the system at a certain value of the distance parameter . ( d ) the states found in the hmm analysis are depicted as disks located in a two - dimensional space of efficiency ( abcissa ) and lifetime ( ordinate ) . ( e , f ) some states kinetically merge on longer observation time scales indicated by the blue and red areas in panels e ( = 10 ms ) and f ( = 100 ms ) . for example , state pairs ( i , ii ) and ( iv , v ) each merge into a single apparent state for times longer than 10 ms . independent hmm analyses were carried out on two differently labeled dase constructs , referred to as constructs i and ii . altogether , four different data sets were analyzed ( dase construct i at mg concentrations of 0.0 , 5.0 , and 40.0 mm and dase construct ii at mg concentration 5.0 mm ) , yielding hmms with seven to nine conformational states . these hmms provide comprehensive models of the dynamics on millisecond time scales . we also determined relaxation times , identified the associated conformational transitions by an eigenvector / eigenvalue analysis of the transition matrix , and computed the ensemble of rna folding pathways . on the basis of their kinetics , the original states were lumped together to effective five - state ( on time scales of tens of milliseconds ) and three- or four - state models ( on time scales of hundreds of milliseconds ) . most notably , we identified consistent , characteristic features of the kinetic network of dase in all four data sets . to the best of our knowledge , these results represent the most detailed rna folding models obtained from single - molecule measurements to date . furthermore , they reveal that the transition rates in this landscape change substantially as the mg concentration is varied , while the general topology of the landscape ( position of minima , relative height of energy barriers ) is not affected . at all mg concentrations , the observed kinetic processes can be attributed to either secondary or tertiary structure rearrangements . by using a combinatorial strategy , we had earlier synthesized a set of nine dase fret constructs with dyes attached at different nucleotide positions . construct i was chosen for in - depth studies because it showed the most pronounced changes in its fret histogram with varying mg concentration . here we have also performed surface - immobilized measurements on a second variant , construct ii , because ( 1 ) its fret histogram was multimodal , suggesting that multiple states could be distinguished by the hmm , and ( 2 ) it was not too different from construct i and , therefore , could serve for validation ( see below ) . single - molecule fluorescence time traces of surface - immobilized dase were obtained for construct i ( cy3 at u6 and cy5 at u42 ) at mg concentrations of 0 , 5 , and 40 mm and for construct ii ( cy3 at the 5 end and cy5 at u30 ) at a mg concentration of 5 mm . details on the data , the experimental procedures , and the effects of surface immobilization are included in the supporting information , tables s1 and s2 and figures s2 and s3 . for each trace , the rates of the background noise , ka , bg an kd , bg , in the acceptor and donor channel , respectively , as well as the amount of spectral crosstalk , , from the donor into the acceptor channel were estimated , as described in the supporting information . we have developed an hmm analysis and associated optimization algorithms for single - molecule fret . the hmm analysis scheme has the following features:the hmm works with discrete photon counts , which are assumed to obey poissonian statistics ( figure s4 , supporting information).background noise levels of measured photon traces are taken into account explicitly by employing an appropriate emission probability.the reproducibility of the hmms is tested.the number of states of the hmm is maximized under a number of constraints , which ensures that the model reproduces physically and chemically relevant quantities.the final hmm represents a fine discretization into states that , depending on the time scale , are lumped into larger states according to kinetic proximity . the hmm works with discrete photon counts , which are assumed to obey poissonian statistics ( figure s4 , supporting information ) . background noise levels of measured photon traces are taken into account explicitly by employing an appropriate emission probability . the number of states of the hmm is maximized under a number of constraints , which ensures that the model reproduces physically and chemically relevant quantities . the final hmm represents a fine discretization into states that , depending on the time scale , are lumped into larger states according to kinetic proximity . a workflow diagram of the hmm analysis scheme is shown in figure 3 . the algorithms are described in full detail in the supporting information , and the salient characteristics of the workflow are discussed in the following sections . workflow diagram used for our hmm analysis of single - molecule fret data . consider the hypothetical energy landscape with five minima in panel a in figure 2 . each minimum corresponds to a conformational state and is associated with a mean fret efficiency , ei ( figure 2b ) , and a fractional population in equilibrium , i ( figure 2c ) , where i denotes the number of the state . using hmm analysis , these five states can be extracted from smfret traces of a molecule diffusing in this free energy landscape . we represent the main characteristics of the states of the hmms by scatter plots ( figure 2d ) : each state is marked by a disc , the position of which encodes the mean fret efficiency of the state and its lifetime , i . the area of the disc is proportional to the stationary probability i of the state , as computed from the hmm . the hmm transition matrix has eigenvalues corresponding to time scales of transitions and eigenvectors denoting states that interconvert on these time scales . , states i and ii interconvert on time scales of 10 ms ( figure 2a ) . thus , when computing a fret histogram with an averaging window much longer than 10 ms , these two states merge into a single apparent state . likewise , states iv and v kinetically merge for time scales longer than 10 ms , as depicted by the red and blue regions in figure 2e . states i and ii kinetically merge with state iii for time scales above 100 ms ( figure 2f ) . complete equilibration occurs for times longer than 1 s. in a high - dimensional energy landscape , kinetic merging may not necessarily involve only neighboring states along the fret efficiency axis . in fact , high fret efficiency states can merge kinetically with low fret efficiency states even if there are states with intermediate fret efficiencies in between . hidden markov models ( hmms ) are stochastic models , = ( t , e ) , of the observed ( measured ) trace , o = ( o1 , ... , on ) , with oi = ( na , i , nd , i ) containing the number of photons observed in the acceptor and donor channels at each time step i. in the construction of hmms , it is assumed that the observation is generated by a hidden markov chain with transition matrix t , whose states represent regions in the conformational space of the molecule . at every time step in the markov chain , an additional stochastic process , the emission probability , , describes the conditional probability of observing the signal , oi , given that the molecule is currently in conformation ( hidden state ) si . one typically chooses the same functional form of for all hidden states but uses a parameter ej to adapt it to a specific hidden state . the parameters ej form a vector e and are part of the model . the hmm optimization problem maximizes the likelihood ( i.e. , the conditional probability of observing the measured trace o , given that the molecule is accurately described by the model = ( t , e)):1over all values of ( t , e ) and all possible hidden paths . for a given number of states , n , the model consists of an n n transition matrix , t , and of a vector of observation parameters e , of length n. hmm classes differ by the way that the hidden process and the measurement process are modeled and by the way that the corresponding parameters are optimized . it is crucial to choose an emission probability , , that models the measurement process as accurately as possible . the arrival times of the photons are assumed to obey poissonian statistics , which is validated in figure s4 ( supporting information ) . n ) is a poisson distribution of variable n with rate coefficient k. the acceptor and donor photon count rates , ka and kd , are given as3where ei is the apparent fret efficiency of the current hidden state si and kmol is the detection rate of photons emitted by the labeled molecule ( through either the donor or the acceptor ) . a problem inherent in the experimental data is the presence of trace - dependent background noise , which may cause identical conformational states to display different apparent fret efficiencies in different time traces . the trace specific background rates , ka , bg and kd , bg , can be estimated from the bleached phase of the measured photon traces . given these rates , we derive a likelihood of observing ( na , nd ) photons during a time step , t , in the acceptor and donor channels , respectively ( see the supporting information ) . the emission probability has the functional form given in eq 2 , but the photon count rates are now given as4we assume that background noise may vary from trace to trace but that all other measurement errors , including spectral cross - talk and differences in the quantum yield of the chromophores , depend on the conformational state but are identical for different traces . then , e contains the apparent fret efficiencies ( without background noise ) of the hidden states . these apparent fret efficiencies can be corrected for spectral cross - talk a posteriori to obtain the true fret efficiencies ( see the supporting information ) . hmm optimization is done by using the expectation - maximization algorithm , which finds a local maximum of from an initial guess of the parameters ( t , e ) . to facilitate finding the global optimum , the hmms presented here are obtained by first running 100 explorations that optimize random starting values of ( t , e ) for a few steps only . subsequently , the parameter set with the largest likelihood is optimized to full convergence . nonetheless , the hmm algorithm might find different local maxima for different initial parameters . hence , for each mg concentration , we compute 10 hmms in the described way to test for reproducibility . two hmms are accepted as identical if their log - likelihoods differ by less than 1.0 . by a heuristic criterion , an hmm optimization is reproducible if identical maximum likelihood hmms are found in at least 2 out of the 10 trials . the number of states , n , is an input parameter for the hmm optimization algorithm . as argued in the supporting information , information - criteria - based choices of the number of states are inadequate for the present data . to determine the number of hidden states , we instead adopt a viewpoint for the construction of direct markov models that is well established in the community : rather than finding the ideal number of states to statistically classify the data consequently , the resulting discretization of state space will be fine enough that the hmms can reproduce the stationary and long - time kinetic behavior of the data . the resulting states can subsequently be grouped according to kinetic connectivity given by t , as described in refs ( 31 and 32 ) and illustrated in figure 2 . following this approach , we build hmms for a varying number of states , n = 2 , 3 , ... , and choose the largest number of states for which hmms can be constructed reproducibly . different tests were used to check whether the hmms are consistent with the data set from which they were parametrized , and whether the hidden paths obtained from the hmms are consistent with markovian dynamics . the consistency of the hmm with the underlying data set was tested by comparing fret efficiency histograms obtained from the data with histograms estimated from the hmms . for this test , we used time windows between 10 and 100 ms . as previously discussed , this approach tests both the stationary and kinetic properties of the model . the data - based distributions were obtained using the likelihood from eq 2 , as described in the supporting information . the hmm - based distributions were obtained by sampling hidden trajectories of the time window length from an equilibrium distribution , and then generating artificial photon counts using poisson statistics with the appropriate output rates ( figure 4a and figures s6a , s7a , and s8a , supporting information ) . the markovianity of individual states was tested by inspecting their lifetime distributions , which can be computed from the maximum - likelihood hidden paths , (t ) , of the hmm . a single exponential decay in these distributions is consistent with markovian dynamics ( figure 4b ) . states that failed this test were split using a newly developed bayesian model selection algorithm ( supporting information ) . the overall markovianity of the hmms was tested using the implied time scales test that is frequently used for simulation - based markov state models . to this end , the relaxation time scales , tihmm = t / ln ihmm , were computed , where ihmm are the eigenvalues of the hmm transition matrix t. these are compared to the implied time scales of a markov model t( ) constructed from the maximum likelihood hidden paths , (t ) , for different lag times . if the overall dynamics is markovian , these time scales should be independent of the lag time used to compute them , hence yielding constant functions in figure 4c . as an additional test , they should agree with the hmm time scales , tihmm . figure s9 ( supporting information ) shows fret traces colored according to the hidden states in the final model . ( a ) dependence of the fret efficiency histograms on the lengths of the time windows ( 10 , 50 , and 100 ms ) . dashed colored lines , prediction from the hidden markov model ; gray areas / dotted black lines , estimation from the smfret data set ( bootstrapping mean/95% confidence interval ) . ( b ) lifetime distributions of the individual states calculated from the maximum - likelihood paths . ( c ) implied time scales , indicating that the long - time kinetics of the hidden paths is markovian and converges to time scales similar to those found in the hmm . the divergence of the shortest time scales at larger lag times is expected and due to numerical problems . we analyzed three sets of smfret traces of dase construct i ( chromophores attached to residues 6 and 42 , see figure 1 ) , measured at different mg concentrations , 0.0 , 5.0 , and 40.0 mm . background - corrected fret efficiency distributions were calculated from these data sets by using the likelihood ( eq 2 ) and a bootstrapping procedure to estimate the uncertainty in the data ( dotted gray lines and gray areas in figure 4a ) . two ensembles of states can be visually distinguished : a broad intermediate state in the fret efficiency range 0.40.8 and a putative native state at efficiency values of 0.91.0 . with increasing mg concentration , the populations shift to states with high fret efficiency . in ref ( 9 ) , it was already hypothesized that the broad ensemble at intermediate fret efficiencies may consist of multiple conformational states with overlapping fret efficiency distributions . hmms were constructed for the smfret data sets as described in the materials and methods section . the largest number of states for which hmms could be reproducibly obtained was eight ( 0 mm mg ) , eight ( 5 mm mg ) , and seven ( 40 mm mg ) states , where we used the optimization protocol described in the materials and methods section . the eight - state models for 0 and 5 mm mg passed the validation test ( figure 4 ) . a single , weakly populated state with fret efficiency e 0 , which was assigned to an acceptor blinking state , was removed from these models a posteriori . the seven - state model at 40 mm mg required an intermediate step , in which non - markovian states were split and regrouped according to kinetic proximity , yielding a nine - state model . ( see the supporting information for a detailed description of the protocol employed . ) to test whether the remaining nonexponentiality came from an actual non - markovianity of the discrete state dynamics or just from spurious transitions generated from the estimation of the maximum likelihood , we conducted the implied time scale test as described in the materials and methods section . the results shown in figure 4c demonstrate that the maximum likelihood hidden paths , (t ) , are non - markovian in all models at short time scales but then converge to approximately constant time scale estimates at lag times of 1030 ms . the time scales agree with the time scales estimated from the hmm transition matrix , indicating that the kinetics of all three hmms are consistent with the data . note that the hmms for the three different mg concentrations were constructed independently of each other . therefore , when similar or consistent features are found across all three mg concentrations , this is a two - fold validation of an observation . the scatter plots in figure 5a ( upper row ) show the main characteristics of the ( hidden ) states of the hmms : each state is represented by a disc whose position indicates the mean fret efficiency of the state and its lifetime i = t / ln tii , where t is the time step of the hmm transition matrix and tii are the diagonal elements of this matrix . the area of the disc is proportional to the stationary probability i of the state as computed from the hmm . the states that consistently appear in construct i at different mg concentrations are depicted in the same color ( i.e. , black , blue , red , and green states ) . the purple state at 0.0 mm mg could not be matched to any state at higher mg concentrations . conformational states and subensembles found by the hmm analysis of construct i and construct ii . ( a ) first row : state parameters of the hidden markov models which are for each state i : the fret efficiency ei ( abcissa ) , the state lifetime i ( ordinate ) , and the equilibrium population i ( disc size ) . second and third rows : state decomposition for time scales of 10 and > 100 ms . ( b ) fret histograms of the subensembles of the states shown in the second row of panel a. a feature found for all mg concentrations is the black high - fret efficiency state . it has a relatively small stationary probability but a long lifetime at all mg conditions . the region of intermediate fret efficiencies is populated mostly by short - lived states ( blue , red ) , and a few long - lived states with low fret efficiencies ( green ) . remarkably , the states appearing at multiple mg concentrations show only rather subtle changes . there are two cooperative effects upon mg increase : ( i ) all states shift to slightly higher fret efficiencies , indicating that mg causes these conformations to become more compact , ( ii ) the intermediate - efficiency purple state is depopulated with increasing mg , while some substates with higher fret efficiencies ( light red state , which is split into an orange and a dark red state at 40 mm mg , as well as the dark blue state ) become more populated at high mg concentrations . the populations of the other red and blue states , as well as the black state , show surprisingly little dependence on the mg concentration , indicating that the associated conformations do not experience stabilization by mg ions . to better understand the nature of the conformational states of the hmms , we have investigated their kinetics . detailed information is presented by the networks plotted in figures s10s13 ( supporting information ) . alternatively , an eigenvector / eigenvalue analysis of the transition matrix t allows conformational states interconverting faster than the time scale of interest to be grouped ( figures s10s13 , supporting information ) . the second row of figure 5a shows a striking feature found independently for the hmms at all mg concentrations : at a few tens of milliseconds , the substates of the red subensemble as well as the substates of the blue subensemble interconvert . consequently , kinetic proximitiy and proximity on the fret axis are , in general , unrelated properties . this finding is emphasized by the fret efficiency histograms of the corresponding subsembles in figure 5b , which were constructed by partitioning the photon traces according to the associated hidden states . the blue and red subensembles are doubly peaked because they are composed of multiple hidden states . in addition , these subensembles overlap strongly , clearly showing why the present single - molecule fret data were difficult to model kinetically , and emphasizing the usefulness of a detailed hmm analysis for dissecting them . for all mg concentrations , the high - efficiency peak in the fret histograms of the blue subensemble overlaps with the high - efficiency black state , indicating that the high - fret - efficiency peak identified in ref ( 9 ) consists of two states , one of which rapidly interconverts with a state of intermediate fret efficiency ( blue ) and is stabilized by mg , and a long - lived high - efficiency state ( black ) , which is insensitive to mg . in figure 5a , the third row shows that , on time scales of a few hundred ms , the long - lived state ( black ) interconverts with the blue subsemble . the mixing time for all subensembles is on the order of seconds ( see figure 6 ) . these results indicate the presence of a hierarchical energy landscape , with different processes occurring on very different time scales , ranging from a few milliseconds to 1 s. free energy landscape and folding pathways . states are indicated by bars or discs with the same colors used in figure 5 . gray bullets indicate transition states facilitating that states or sets of states kinetically merge at longer time scales . ( b ) the complete ensemble of folding pathways from the least compact states ( green / yellow ) to the most compact state ( black ) . the states are positioned depending on their mean fret efficiency ( abcissa ) and the probability of folding ( committor , q , ordinate ) . the thickness of an arrow is proportional to the probability that a green / yellow state will fold along this pathway . on the basis of the processes depicted in figure 5 , we find fast interconversion between the open ( e 0.5 ) and closed ( e > 0.7 ) states within the blue and red subensembles , while the exchange dynamics between these subensembles happens much slower . we propose that the states within each subensemble ( with a given color in figure 5 ) have similar secondary structures yet different tertiary structures , interconverting rapidly without breaking large strands of watson crick base pairs . this proposition is supported by the fact that , at high mg concentrations , the compact parts of the red and blue subensemble are stabilized . different subensembles are proposed to correspond to different secondary structures because they are long - lived , suggesting that the stable watson crick base pairs need to be broken in order to transit to another subensemble . the connectivity between different subensembles ( and , thus , presumably different secondary structures ) is similar at all mg concentrations . the high - efficiency ( black ) state is connected to the blue subensemble in the presence of mg ( 5 and 40 mm ) directly and , at 0 mm mg , via the purple intermediate . figure 6a illustrates this connectivity , and the free energies of these conformations as well as the transition states ( see the materials and methods section ) . this connectivity suggests an ordering of subensembles from the least compact ( lowest fret efficiencies ) to the most compact ( highest fret efficiencies ) which can be found at all mg concentrations : ( 1 ) green , ( 2 ) red , ( 3 ) blue , and ( 4 ) black . the fact that they have high lifetimes and fret efficiencies that are much greater than zero suggests that they still have some secondary structure , although probably not the native one . they are therefore called misfolded . this ordering suggests to study the transition pathways from the misfolded states ( green ) to the most compact state ( black ) . transition path theory provides the basis for calculating the pathways between two subensembles . we use the protocol and equations described in ref ( 36 ) employing the implementation in the emma software . a transition pathway is defined as a series of transitions that lead from the misfolded to the native state without returning to the misfolded state . figure 6b locates the states by their fret efficiency , and by the committor value ( vertical axis ) , i.e. , the probability of the system , when being in this state , to move forward and fold toward the black state , rather than misfold back to the green state . the committor value q = 0.5 designates states in which the molecule is equally likely to go either way . note that there is a continuous shift of these transition states with increasing mg concentration . at 0 mm mg , the transition state lies between the green and red subensembles . once a molecule has reached the red subensemble , it is likely to continue folding to the black state . with increasing mg concentration , the red and blue subensembles become more and more kinetic intermediates , and lie at committor values around 0.5 for 40 mm mg . figure 6b shows the probability fluxes of transition pathways from misfolded states to the folded state . the size of the arrows indicates the probability flux , which is related to the folding rate . without mg , the folding rate kab is about 0.09 s , and increases to 0.28 s for 5 mm and 0.17 s for 40 mm mg . the strong increase in folding rate from 0 to 5 mm mg is mainly due to a lowering of the transition state energy , while the decrease in folding rate from 5 to 40 mm mg is mainly due to an increased stability of the dark blue intermediate state ( compare figure 6a and b ) . moreover , it is apparent that addition of mg increases the number of accessible pathways , making the folding process more parallel . two main mechanisms are observed at all mg concentrations : a compact folding mechanism , in which the green misfolded state refolds via the higher fret efficiency substates of red and blue toward the black state , and an close open close mechanism , in which the green state folds via the open substates , or via successive closing , opening , and closing , i.e. , involving tertiary unfolded states . both types of pathways have similar weights , with some preference for close open close pathways at low mg concentrations and a slight preference for compact pathways at high mg concentrations . to further confirm our findings , we performed a fourth independent measurement on a dase ( construct ii ) with a different set of label positions . if they do not introduce major energetic conflicts , the state probabilities , time scales , and the kinetic connectivity should remain comparable . single - molecule fret data were recorded , and an hmm was computed using the same approach as above . a seven - state model was found to pass the validation test ( see figure s14a and s14b , supporting information ) . open states at efficiencies of 0.40.6 and high - fret , closed states at efficiencies above 0.8 . as for construct i , two pairs of rapidly interconverting states , each with a low- and a high - fret state , were found . additionally , a single stable state with high efficiency was also identified . consequently , the red , blue , and black subensembles of construct ii match the corresponding subensembles in construct i and , thus , can be identified in all experimental data with high confidence ( see figure 5a ) . moreover , the time scales found in constructs i and ii are in qualitative agreement ( see figure s14c in the supporting information ) . open and closed states of the red and blue subensembles interconvert at time scales of a few milliseconds ( 10 ms in construct i , 3 ms in construct ii ) . at time scales of 100 ms to seconds , ( i ) the blue ensemble merges with the black state and ( ii ) the red and blue ensembles kinetically merge . at low mg concentrations , the blue black interconversion is several 100 ms faster than the blue red interconversion , while , at 40 mm mg , the two processes happen at about the same time scales ( figure s14c , supporting information ) . the gray states in construct ii and the green / yellow states in construct i do not have clear corresponding states in the other construct . for example , the presence of a label in a particular position may prevent certain structures from forming . in the following discussion , we will thus concentrate on those states that can be safely matched across all data sets ( red , blue , and black ) . note that , due to the reduced state lifetimes in construct ii , the partitioning of the photon traces resulted in subtraces which were too short for a histogram analysis . hence , the subensemble fret histograms could not be generated ( see figure 5b ) . a kinetic pattern is found consistently for different mg ion concentrations and for different attachment points of the chromophores : ( i ) a long - lived , high - fret - efficiency state ( black ) , ( ii ) two ensembles of states ( red , blue ) comprising rapidly interconverting open and closed states , the ratio of which depends on mg , and ( iii ) a linear connection between the three subensembles ( red , blue , black ) . the long interconversion times along this linear connection suggest that these transitions involve breaking and reforming of watson crick base pairs . to investigate whether there are secondary structures consistent with this pattern , minimum energy secondary structures of the dase were calculated using the vienna rna webserver ( see the supporting information ) . the algorithm correctly identified the secondary structure of the known folded state ( excluding the pseudoknot connectivity ) as the lowest free - energy structure . two alternative secondary structures with low free energies ( g < 1.4 kj / mol above native , i.e. , accessible at room temperature ) were also identified . these structures ( labeled 2 and 3 ) are shown along with the secondary structure of the folded state ( labeled 1 ) in figure 7 . although they are very close in energy and structurally very similar to each other , structures 2 and 3 differ from structure 1 in that helix ii is broken and helix i is prolonged by two base pairs . all other secondary structures identified by the algorithm had estimated free energy differences of g > 9.5 kj / mol with respect to structure 1 . proposed folding mechanism . the red set of states has a non - native secondary structure but includes both open ( low - fret ) tertiary structures and compact ( high - fret ) tertiary structures . the blue set of states has the native secondary structure but also includes both open and compact tertiary structures . in contrast to the compact blue state , it is additionally stabilized by the tertiary watson crick pairs that form the pseudoknot . in the absence of stabilizing tertiary interactions , secondary structures 1 , 2 , and 3 facilitate transitions between open and compact states , associated with large fluctuations in the donor therefore , they have properties matching those found for the blue and red subensembles in the hmm analysis . the black state displays exclusively high fret efficiencies in all constructs under all conditions and its long lifetime and the fact that its population does not vary strongly with the mg concentration suggest that it is stabilized by base - pairing rather than mg ions . therefore , we propose that the black state represents the tertiary folded structure including the pseudoknot topology . the pseudoknot base pairs ( g1c26 , g2c25 , a3u45 , g4c44 ; see figure 1 ) are consistent with stable interactions that do not depend on mg . their formation stabilizes an already compact structure with the correct secondary fold so as to acquire a well - defined tertiary structure . this proposal is supported by computer simulations which show that the active site of dase is distorted if mg is removed ( explaining the loss in catalytic activity ) but the overall lambda - shaped tertiary structure stays intact . since the blue subensemble acts as a precursor to the black tertiary folded structure ( linearly connected folding path , figure 6 ) , it is only logical to match the blue state with the secondary structure of the folded state ( structure 1 ) . the native secondary structure still facilitates extended and compact states . like the fully native black state , the high - efficiency blue states are compact and possess the correct native secondary structure , but in contrast to the black state , they lack the pseudoknot base pairs , which stabilize the native tertiary fold . consistently , the probability of extended versus compact blue states depends on the concentration of mg ions that are required to stabilize the compact state in the absence of tertiary base pairs . consequently , the red ensemble contains structures 2 and/or 3 , i.e. , extended and compact states with non - native secondary structure . the proposed assignment is consistent not only with the kinetic connectivity and the mg - dependent equilibrium populations but also with the observed time scales . the fluctuation between open and compact conformations within the blue and red ensembles involves no or little secondary structure change , consistent with relatively short transition time scales ( figures 5 and 6 ) . in contrast , a transition from the red to the blue subensemble involves rupture of watson crick pairs , which is consistent with slower transition time scales of hundreds of milliseconds ( figures 5 and 6 ) . likewise , the change of tertiary base - pairing is consistent with long transition time scales between the blue and black states and the long lifetime of the black native state . the kinetic model found here and our proposed folding mechanism exhibits a number of features consistent with previous findings or hypotheses for other rna systems . in particular , secondary and tertiary structure formation has been proposed to be kinetically decoupled , such that secondary structure elements can exist without further stabilization by specific tertiary interactions . for the tetrahymena thermophila ribosome , metastable structures with a partially misfolded secondary structure have been described , lending credibility to the present assignment of the red subensemble to structures 2 and/or 3 . in addition , other rnas have been proposed to fold via multiple parallel pathways . to the best of our knowledge , we have presented the most detailed experimentally derived model of an rna folding mechanism , providing a kinetic model connecting different secondary and tertiary stabilized structures , and showing how they are orchestrated during the folding pathways . the multitude of time scales found in the data provide direct evidence that the rna folding landscape is hierarchical and that secondary and tertiary structure formation occur on different time scales . the techniques described here also facilitate detailed kinetic models to be derived for other macromolecular systems . as yet , the field is still lacking an experiment that could simultaneously resolve kinetics and the structures of the individual states in detail . unfortunately , computational approaches can not step in here . with folding times on the order of seconds , the dynamics are as yet out of reach for direct molecular dynamics ( md ) simulation . over time , however , enhanced sampling strategies may help access these processes . however , molecular modeling and md simulation may be useful for exploring the local dynamics within individual states , and by using new biophysical techniques , the distribution of measurable fret values can be computed and compared to the subensemble distributions shown in figure 5b . on the experimental side , using multicolor - fret or the systematic reconciliation of multiple dual - color - fret experiments may provide distance constraints to resolve the structures in more detail . finally , the combination of fret and site - specific fluorescence quenching may also be employed to disentangle the tertiary dynamics from secondary structure formation .

we have developed a hidden markov model and optimization procedure for photon - based single - molecule fret data , which takes into account the trace - dependent background intensities . this analysis technique reveals an unprecedented amount of detail in the folding kinetics of the diels alderase ribozyme . we find a multitude of extended ( low - fret ) and compact ( high - fret ) states . five states were consistently and independently identified in two fret constructs and at three mg2 + concentrations . structures generally tend to become more compact upon addition of mg2 + . some compact structures are observed to significantly depend on mg2 + concentration , suggesting a tertiary fold stabilized by mg2 + ions . one compact structure was observed to be mg2 + -independent , consistent with stabilization by tertiary watson crick base pairing found in the folded diels alderase structure . a hierarchy of time scales was discovered , including dynamics of 10 ms or faster , likely due to tertiary structure fluctuations , and slow dynamics on the seconds time scale , presumably associated with significant changes in secondary structure . the folding pathways proceed through a series of intermediate secondary structures . there exist both compact pathways and more complex ones , which display tertiary unfolding , then secondary refolding , and , subsequently , again tertiary refolding .

Introduction Materials and Methods Results and Discussion Discussion

in some ribozymes , the slow opening and closing of tertiary structure ( rna breathing ) this latter process is hierarchical , first proceeding on the secondary structure level via formation of fairly stable watson crick base pairs . the folding of rna into the native tertiary fold may proceed via a complex sequence of secondary structures . formation of compact tertiary structures may require the presence of counterions , particularly divalent cations such as mg , which screen the intrinsic negative charges on the rna phosphate groups and , thereby , stabilize certain tertiary motifs . indeed , rna sequence , structure , and function interact in a complex , not yet fully understood fashion , and the characterization of rna folding kinetics , including the pathways of secondary and tertiary structure changes , remains an intricate problem . in this work , we have investigated the conformational equilibrium and the folding pathway of the 49mer single - stranded rna ribozyme diels alderase ( dase ) using a novel hidden markov model ( hmm ) analysis of single - molecule fret data . the precise hydrogen - bond pattern in the pseudoknot region is known to be crucial both for thermal stability of the overall fold as well as for the shape of the catalytic pocket . attachment sites of the fret labels are marked by green ( donor dye cy3 at u6 in construct i and at the 5 end in construct ii ) and orange ( acceptor dye cy5 at u42 in construct i and at u30 in construct ii ) arrows . color - coding of the secondary structure elements as in panel a. attachment sites of the fret labels are indicated by green ( cy3 , donor ) and red ( cy5 , acceptor ) spheres . consistent with conformational fluctuations , a poor resolution of dase spectra was found in subsequent nmr studies . the mg - dependent fret efficiency histograms revealed at least two conformational ensembles : ( i ) a high fret state , attributed to the folded conformation , whose population increases with increasing mg concentration , and ( ii ) a distribution of intermediate fret efficiencies , whose population decreases with increasing mg concentration . the intermediates were observed to spread out over a wide range of fret efficiency values and , presumably , comprise multiple conformations with different secondary and tertiary structures . it completely neglects the time sequence of events in the single - molecule trajectories and , thus , discards a substantial part of the available information . in contrast , hidden markov models can distinguish states in the data by using both the differences in fret efficiency and the time sequence of events , and , thus , can decompose states with similar fret efficiencies but different kinetic properties . recent studies on single - molecule protein and rna data sets have demonstrated the power of hmms to resolve a multitude of states . hmm analysis has its intrinsic challenges , however , because ( i ) the results depend on the number of states used , ( ii ) the hmm optimization may get stuck in local minima , ( iii ) models with many states are difficult to validate , and ( iv ) the quality of the model depends crucially on the validity of the underlying likelihood function ( i.e. in addition , we have developed an approach to account for the trace - specific background noise . ( d ) the states found in the hmm analysis are depicted as disks located in a two - dimensional space of efficiency ( abcissa ) and lifetime ( ordinate ) . ( e , f ) some states kinetically merge on longer observation time scales indicated by the blue and red areas in panels e ( = 10 ms ) and f ( = 100 ms ) . these hmms provide comprehensive models of the dynamics on millisecond time scales . we also determined relaxation times , identified the associated conformational transitions by an eigenvector / eigenvalue analysis of the transition matrix , and computed the ensemble of rna folding pathways . on the basis of their kinetics , the original states were lumped together to effective five - state ( on time scales of tens of milliseconds ) and three- or four - state models ( on time scales of hundreds of milliseconds ) . here we have also performed surface - immobilized measurements on a second variant , construct ii , because ( 1 ) its fret histogram was multimodal , suggesting that multiple states could be distinguished by the hmm , and ( 2 ) it was not too different from construct i and , therefore , could serve for validation ( see below ) . single - molecule fluorescence time traces of surface - immobilized dase were obtained for construct i ( cy3 at u6 and cy5 at u42 ) at mg concentrations of 0 , 5 , and 40 mm and for construct ii ( cy3 at the 5 end and cy5 at u30 ) at a mg concentration of 5 mm . details on the data , the experimental procedures , and the effects of surface immobilization are included in the supporting information , tables s1 and s2 and figures s2 and s3 . for each trace , the rates of the background noise , ka , bg an kd , bg , in the acceptor and donor channel , respectively , as well as the amount of spectral crosstalk , , from the donor into the acceptor channel were estimated , as described in the supporting information . we have developed an hmm analysis and associated optimization algorithms for single - molecule fret . the hmm analysis scheme has the following features:the hmm works with discrete photon counts , which are assumed to obey poissonian statistics ( figure s4 , supporting information).background noise levels of measured photon traces are taken into account explicitly by employing an appropriate emission probability.the reproducibility of the hmms is tested.the number of states of the hmm is maximized under a number of constraints , which ensures that the model reproduces physically and chemically relevant quantities.the final hmm represents a fine discretization into states that , depending on the time scale , are lumped into larger states according to kinetic proximity . the final hmm represents a fine discretization into states that , depending on the time scale , are lumped into larger states according to kinetic proximity . the algorithms are described in full detail in the supporting information , and the salient characteristics of the workflow are discussed in the following sections . workflow diagram used for our hmm analysis of single - molecule fret data . each minimum corresponds to a conformational state and is associated with a mean fret efficiency , ei ( figure 2b ) , and a fractional population in equilibrium , i ( figure 2c ) , where i denotes the number of the state . , states i and ii interconvert on time scales of 10 ms ( figure 2a ) . hidden markov models ( hmms ) are stochastic models , = ( t , e ) , of the observed ( measured ) trace , o = ( o1 , ... , on ) , with oi = ( na , i , nd , i ) containing the number of photons observed in the acceptor and donor channels at each time step i. in the construction of hmms , it is assumed that the observation is generated by a hidden markov chain with transition matrix t , whose states represent regions in the conformational space of the molecule . a problem inherent in the experimental data is the presence of trace - dependent background noise , which may cause identical conformational states to display different apparent fret efficiencies in different time traces . the emission probability has the functional form given in eq 2 , but the photon count rates are now given as4we assume that background noise may vary from trace to trace but that all other measurement errors , including spectral cross - talk and differences in the quantum yield of the chromophores , depend on the conformational state but are identical for different traces . as argued in the supporting information , information - criteria - based choices of the number of states are inadequate for the present data . the hmm - based distributions were obtained by sampling hidden trajectories of the time window length from an equilibrium distribution , and then generating artificial photon counts using poisson statistics with the appropriate output rates ( figure 4a and figures s6a , s7a , and s8a , supporting information ) . the overall markovianity of the hmms was tested using the implied time scales test that is frequently used for simulation - based markov state models . to this end , the relaxation time scales , tihmm = t / ln ihmm , were computed , where ihmm are the eigenvalues of the hmm transition matrix t. these are compared to the implied time scales of a markov model t( ) constructed from the maximum likelihood hidden paths , (t ) , for different lag times . ( a ) dependence of the fret efficiency histograms on the lengths of the time windows ( 10 , 50 , and 100 ms ) . ( c ) implied time scales , indicating that the long - time kinetics of the hidden paths is markovian and converges to time scales similar to those found in the hmm . the divergence of the shortest time scales at larger lag times is expected and due to numerical problems . the largest number of states for which hmms could be reproducibly obtained was eight ( 0 mm mg ) , eight ( 5 mm mg ) , and seven ( 40 mm mg ) states , where we used the optimization protocol described in the materials and methods section . to test whether the remaining nonexponentiality came from an actual non - markovianity of the discrete state dynamics or just from spurious transitions generated from the estimation of the maximum likelihood , we conducted the implied time scale test as described in the materials and methods section . the time scales agree with the time scales estimated from the hmm transition matrix , indicating that the kinetics of all three hmms are consistent with the data . ( a ) first row : state parameters of the hidden markov models which are for each state i : the fret efficiency ei ( abcissa ) , the state lifetime i ( ordinate ) , and the equilibrium population i ( disc size ) . ( b ) fret histograms of the subensembles of the states shown in the second row of panel a. a feature found for all mg concentrations is the black high - fret efficiency state . there are two cooperative effects upon mg increase : ( i ) all states shift to slightly higher fret efficiencies , indicating that mg causes these conformations to become more compact , ( ii ) the intermediate - efficiency purple state is depopulated with increasing mg , while some substates with higher fret efficiencies ( light red state , which is split into an orange and a dark red state at 40 mm mg , as well as the dark blue state ) become more populated at high mg concentrations . the populations of the other red and blue states , as well as the black state , show surprisingly little dependence on the mg concentration , indicating that the associated conformations do not experience stabilization by mg ions . alternatively , an eigenvector / eigenvalue analysis of the transition matrix t allows conformational states interconverting faster than the time scale of interest to be grouped ( figures s10s13 , supporting information ) . in addition , these subensembles overlap strongly , clearly showing why the present single - molecule fret data were difficult to model kinetically , and emphasizing the usefulness of a detailed hmm analysis for dissecting them . for all mg concentrations , the high - efficiency peak in the fret histograms of the blue subensemble overlaps with the high - efficiency black state , indicating that the high - fret - efficiency peak identified in ref ( 9 ) consists of two states , one of which rapidly interconverts with a state of intermediate fret efficiency ( blue ) and is stabilized by mg , and a long - lived high - efficiency state ( black ) , which is insensitive to mg . on the basis of the processes depicted in figure 5 , we find fast interconversion between the open ( e 0.5 ) and closed ( e > 0.7 ) states within the blue and red subensembles , while the exchange dynamics between these subensembles happens much slower . we propose that the states within each subensemble ( with a given color in figure 5 ) have similar secondary structures yet different tertiary structures , interconverting rapidly without breaking large strands of watson crick base pairs . different subensembles are proposed to correspond to different secondary structures because they are long - lived , suggesting that the stable watson crick base pairs need to be broken in order to transit to another subensemble . the connectivity between different subensembles ( and , thus , presumably different secondary structures ) is similar at all mg concentrations . the high - efficiency ( black ) state is connected to the blue subensemble in the presence of mg ( 5 and 40 mm ) directly and , at 0 mm mg , via the purple intermediate . a transition pathway is defined as a series of transitions that lead from the misfolded to the native state without returning to the misfolded state . with increasing mg concentration , the red and blue subensembles become more and more kinetic intermediates , and lie at committor values around 0.5 for 40 mm mg . the size of the arrows indicates the probability flux , which is related to the folding rate . two main mechanisms are observed at all mg concentrations : a compact folding mechanism , in which the green misfolded state refolds via the higher fret efficiency substates of red and blue toward the black state , and an close open close mechanism , in which the green state folds via the open substates , or via successive closing , opening , and closing , i.e. single - molecule fret data were recorded , and an hmm was computed using the same approach as above . consequently , the red , blue , and black subensembles of construct ii match the corresponding subensembles in construct i and , thus , can be identified in all experimental data with high confidence ( see figure 5a ) . moreover , the time scales found in constructs i and ii are in qualitative agreement ( see figure s14c in the supporting information ) . open and closed states of the red and blue subensembles interconvert at time scales of a few milliseconds ( 10 ms in construct i , 3 ms in construct ii ) . note that , due to the reduced state lifetimes in construct ii , the partitioning of the photon traces resulted in subtraces which were too short for a histogram analysis . a kinetic pattern is found consistently for different mg ion concentrations and for different attachment points of the chromophores : ( i ) a long - lived , high - fret - efficiency state ( black ) , ( ii ) two ensembles of states ( red , blue ) comprising rapidly interconverting open and closed states , the ratio of which depends on mg , and ( iii ) a linear connection between the three subensembles ( red , blue , black ) . to investigate whether there are secondary structures consistent with this pattern , minimum energy secondary structures of the dase were calculated using the vienna rna webserver ( see the supporting information ) . these structures ( labeled 2 and 3 ) are shown along with the secondary structure of the folded state ( labeled 1 ) in figure 7 . the red set of states has a non - native secondary structure but includes both open ( low - fret ) tertiary structures and compact ( high - fret ) tertiary structures . in contrast to the compact blue state , it is additionally stabilized by the tertiary watson crick pairs that form the pseudoknot . in the absence of stabilizing tertiary interactions , secondary structures 1 , 2 , and 3 facilitate transitions between open and compact states , associated with large fluctuations in the donor therefore , they have properties matching those found for the blue and red subensembles in the hmm analysis . since the blue subensemble acts as a precursor to the black tertiary folded structure ( linearly connected folding path , figure 6 ) , it is only logical to match the blue state with the secondary structure of the folded state ( structure 1 ) . like the fully native black state , the high - efficiency blue states are compact and possess the correct native secondary structure , but in contrast to the black state , they lack the pseudoknot base pairs , which stabilize the native tertiary fold . consistently , the probability of extended versus compact blue states depends on the concentration of mg ions that are required to stabilize the compact state in the absence of tertiary base pairs . the fluctuation between open and compact conformations within the blue and red ensembles involves no or little secondary structure change , consistent with relatively short transition time scales ( figures 5 and 6 ) . in contrast , a transition from the red to the blue subensemble involves rupture of watson crick pairs , which is consistent with slower transition time scales of hundreds of milliseconds ( figures 5 and 6 ) . likewise , the change of tertiary base - pairing is consistent with long transition time scales between the blue and black states and the long lifetime of the black native state . in particular , secondary and tertiary structure formation has been proposed to be kinetically decoupled , such that secondary structure elements can exist without further stabilization by specific tertiary interactions . to the best of our knowledge , we have presented the most detailed experimentally derived model of an rna folding mechanism , providing a kinetic model connecting different secondary and tertiary stabilized structures , and showing how they are orchestrated during the folding pathways . the multitude of time scales found in the data provide direct evidence that the rna folding landscape is hierarchical and that secondary and tertiary structure formation occur on different time scales .

the field of exercise - oncology has burgeoned dramatically in scope and impact since publication of the first scientific papers in the mid to late 1980s.1 several systematic reviews and meta - analyses have evaluated the efficacy of structured exercise treatment in cancer.26 for example , speck et al.7 identified a total of 66 studies reported as high - quality that examined the impact of exercise treatment in a broad array of oncology scenarios ( e.g. differences in histological primary site , stage and treatment ) on a total of 60 different primary end points in adults with cancer . intriguingly , despite the degree of heterogeneity in how exercise was being utilized to manipulate physiological adaptation , the nature of the exercise prescription in the vast majority of studies was similar . more specifically , almost all prescriptions followed traditional guidelines consisting of either supervised or home - based endurance ( aerobic ) training or endurance training combined with resistance training , prescribed at a moderate - intensity ( 5075% of a predetermined physiological parameter , typically age - predicted heart rate maximum or reserve ) , for two to three sessions per week , for 10 to 60 min per exercise session , for 12 to 15 weeks . despite the adoption of a relatively homogeneous prescription approach , exercise training was , for the most part , associated with benefit across a diverse range of end points , largely irrespective of the oncology setting.7 on this evidence , it could be argued that a standardized , largely homogeneous exercise prescription that adopts a conventional approach is safe , efficacious , and therefore sufficient . this has resulted in a limited perceived need to elucidate the optimal dose , sequencing , or combination of different training stresses to specifically alter a desired physiological end point in any clinical population , including oncology . however , the significant benefit of generically dosed exercise treatment on heterogeneous end points rather reflects the remarkable pleiotropic physiologic impact of exercise . moreover , the use of generic exercise prescriptions ( irrespective of clinical population or primary end point ) is masking the full therapeutic promise of exercise treatment . indeed , for more than half a century , exercise training has been continually refined , precisely dosed , and scheduled to minimize injury and optimize human / athletic performance ; the basis of all training prescriptions in this arena adheres to fundamental tenets of human exercise physiology known as the principles of training . these tenets are rarely applied or even considered when designing exercise trials in clinical populations.8,9 accordingly , the purpose of this opinion paper is to provide an overview of the application of these principles in the design and conduct of clinical trials in exercise - oncology research . this paper will focus primarily on application of these principles to aerobic - based training , although the concepts also apply to resistance training or combination training programs . stage 1 objective assessment of response to exercise : an essential prerequisite in the design of all exercise training trials is the objective assessment of baseline physiological functioning of the systems that will be primarily targeted . this generally refers to either the cardiopulmonary responses to exercise ( in the context of aerobic training ) or the assessment of maximal muscular strength or endurance ( in the context of resistance training ) . the relative strengths , weaknesses , and conduct of the various different assessment tools available to researchers have been reviewed previously.1012 in the context of aerobic training studies , the preferred assessment tool is a cardiopulmonary exercise test ( cpet ) . the use of a cpet offers a number of distinct advantages compared with other assessments . of these , arguably the most important is that cpet provides specific information on patients ' peak rate of oxygen consumption ( vo2peak ) as well as their submaximal cardiopulmonary responses to exercise , permitting precise tailoring of training to the individual patient . in settings in which cpet is not available , investigators can also use a maximal incremental exercise tolerance test ( ett ) ( without direct vo2 measurement ) to determine peak workload and peak exercise heart rate.10,13 in addition , both cpets and etts are performed with 12-lead ecg and blood pressure monitoring , thus , they also provide important pre - exercise training clearance information on the detection of any exercise - induced impairments ( e.g. ischemia ) or symptom limitations at both submaximal and maximal exercise tolerance.14 the latter information is of critical importance when considering the inclusion of high - intensity ( 75% of vo2peak ) aerobic training sessions in the prescription . stage 2 application of the principles of training : perhaps the most essential principles of training are individualization , specificity , progressive overload , and rest / recovery ( figure 1 ) . individualization : the concept of individualization is defined as the customized application of training towards the physiological status of the patient/ subject.15,16 clearly , even within carefully selected homogenous clinical trial cohorts , considerable heterogeneity likely still exists in cardiopulmonary function , lifestyle behaviour , age , prior treatment , concomitant comorbidities , and , of course , genetic predisposition . thus , application of a generic prescription that fails to consider such parameters will most often result in either an under - dosing or over - dosing of exercise treatment . fortunately , there is a broad array of parameters available on which to individualize training . the preferred approach is individualization on the basis of workloads ( e.g. treadmill speed and power output ) corresponding to a specific percent of vo2peak or peak workload ( e.g. 55% , 65% ) elicited during each individual patient 's pre - randomization cpet or ett . the corresponding heart rate , blood pressure , and rating of perceived exertion ( rpe ) directly measured at each percent workload is used to ensure that subjects are training at , or close to , the prescribed intensity during subsequent exercise sessions ( figure 2).17 it is important to note that although the absolute training intensity may be different between individual patients , the relative intensity of training is similar , ensuring standardization across patients a critical aspect of clinical trial conduct.specificity : it is widely acknowledged that the physiological adaptations elicited by aerobic training are unique from those caused by resistance training . lesser appreciated , however , is that even within the same exercise modality , there is enormous capacity to manipulate the nature and configuration of the elements of exercise prescription ( i.e. frequency , intensity , duration , and time ) to confer remarkably distinct physiological adaptations.18,19 the concept of specificity addresses the notion that the selected exercise stress must be specific and targeted to the primary underlying system(s ) or pathway(s ) known or postulated to underpin the primary end point of interest . for example , in a trial designed to examine the impact of aerobic training on vo2peak in a particular cancer cohort , a critical prerequisite for the design of the optimal exercise prescription is determination of the primary limitation ( determinant ) to vo2peak ; in other words , is there a cardiovascular , ventilatory , and/or skeletal muscle limitation to exercise?20 for example , if the primary limitation is cardiovascular exercise training emphasizing moderate intensity , aerobic training ( i.e. 50 to 70% of vo2peak ) for longer duration ( 45 min ) is indicated to initially enhance plasma volume and structural changes in the heart and blood vessels.21 in contrast , if more general deconditioning is observed and skeletal muscle adaptation is the desired outcome , aerobic training at a higher relative intensity ( i.e. > 70% of vo2peak ) that induces enzymatic adaptation and increased capillarization and mitochondrial biogenesis may be emphasized ( after a period of more general training that acclimatizes the patient to exercise).2224 progressive overload : aerobic training provides a potent physiological stimulus that perturbs the equilibrium of multiple organ systems.18 perturbations in the cellular and systemic environments create biological stress , which challenges homeostasis . chronic ( repeated ) disturbance of homeostasis triggers a highly preserved , multi - organ stress - response leading to physiologic adaptation ( i.e. the concept of hormesis25 or supercompensation ) , wherein the host can withstand greater future system perturbations , meaning that a greater stress stimulus is required to further perturb homeostasis . by definition therefore , a requirement of effective exercise prescriptions is optimization and progressive increase in stress to confer continued physiological adaptation.26 of equal importance is recognition that increasing exercise stress pushed beyond the homeostasis will result in chronic overreaching or overtraining , leading to fatigue , maladaptation , or even illness and injury.27 accordingly , optimization of progressive overload requires appropriate increase in training stress across the training prescription and monitoring of the individual 's physiological response or readiness to receive the increased exercise load . rest and recovery : the tenet that is most frequently underemphasized or commonly neglected in the design of exercise prescriptions is the principle of rest and recovery . central to this principle is the availability of nutrients and rest ( or reduced training load ) in order to permit necessary biological resynthesis to replace the required constituents of the impacted system(s ) . quantifying optimal recovery is challenging28 because of the multidimensional application of the exercise stress.29 conversely , an extended period without adequate stress will result in a loss of adaptation and a down - regulation of the entire system.30 clearly , a balance between the principles of progressive overload and recovery are necessary to elicit optimal physiological adaptations.31 a training stress must perturb homeostasis with enough impulse to lead to chronic supercompensatory adaptations ; however , the appropriate volume of recovery must be prescribed in concert with this perturbation in order to optimize adaptation . there are many techniques in the prescription of an exercise training program that ensure adequate and optimal rest and recovery ; this is most commonly achieved by altering the frequency and duration of training ( while maintaining intensity ) or sequencing the training stress across each week in order to reduce the accumulated fatigue that may amass through repeated , consecutive high - intensity bouts of training.32,33 this is especially critical in clinical populations in which demographic and medical characteristics ( e.g. cardiopulmonary function , immune status , comorbidities , and current therapies ) further complicate optimal adaptation . with appropriate balance , positive adaptations will continue , permitting increases in exercise stresses to be applied and further physiologic conditioning towards the targeted end point . individualization : the concept of individualization is defined as the customized application of training towards the physiological status of the patient/ subject.15,16 clearly , even within carefully selected homogenous clinical trial cohorts , considerable heterogeneity likely still exists in cardiopulmonary function , lifestyle behaviour , age , prior treatment , concomitant comorbidities , and , of course , genetic predisposition . thus , application of a generic prescription that fails to consider such parameters will most often result in either an under - dosing or over - dosing of exercise treatment . fortunately , there is a broad array of parameters available on which to individualize training . the preferred approach is individualization on the basis of workloads ( e.g. treadmill speed and power output ) corresponding to a specific percent of vo2peak or peak workload ( e.g. 55% , 65% ) elicited during each individual patient 's pre - randomization cpet or ett . the corresponding heart rate , blood pressure , and rating of perceived exertion ( rpe ) directly measured at each percent workload is used to ensure that subjects are training at , or close to , the prescribed intensity during subsequent exercise sessions ( figure 2).17 it is important to note that although the absolute training intensity may be different between individual patients , the relative intensity of training is similar , ensuring standardization across patients a critical aspect of clinical trial conduct . specificity : it is widely acknowledged that the physiological adaptations elicited by aerobic training are unique from those caused by resistance training . lesser appreciated , however , is that even within the same exercise modality , there is enormous capacity to manipulate the nature and configuration of the elements of exercise prescription ( i.e. frequency , intensity , duration , and time ) to confer remarkably distinct physiological adaptations.18,19 the concept of specificity addresses the notion that the selected exercise stress must be specific and targeted to the primary underlying system(s ) or pathway(s ) known or postulated to underpin the primary end point of interest . for example , in a trial designed to examine the impact of aerobic training on vo2peak in a particular cancer cohort , a critical prerequisite for the design of the optimal exercise prescription is determination of the primary limitation ( determinant ) to vo2peak ; in other words , is there a cardiovascular , ventilatory , and/or skeletal muscle limitation to exercise?20 for example , if the primary limitation is cardiovascular exercise training emphasizing moderate intensity , aerobic training ( i.e. 50 to 70% of vo2peak ) for longer duration ( 45 min ) is indicated to initially enhance plasma volume and structural changes in the heart and blood vessels.21 in contrast , if more general deconditioning is observed and skeletal muscle adaptation is the desired outcome , aerobic training at a higher relative intensity ( i.e. > 70% of vo2peak ) that induces enzymatic adaptation and increased capillarization and mitochondrial biogenesis may be emphasized ( after a period of more general training that acclimatizes the patient to exercise).2224 progressive overload : aerobic training provides a potent physiological stimulus that perturbs the equilibrium of multiple organ systems.18 perturbations in the cellular and systemic environments create biological stress , which challenges homeostasis . chronic ( repeated ) disturbance of homeostasis triggers a highly preserved , multi - organ stress - response leading to physiologic adaptation ( i.e. the concept of hormesis25 or supercompensation ) , wherein the host can withstand greater future system perturbations , meaning that a greater stress stimulus is required to further perturb homeostasis . by definition therefore , a requirement of effective exercise prescriptions is optimization and progressive increase in stress to confer continued physiological adaptation.26 of equal importance is recognition that increasing exercise stress pushed beyond the homeostasis will result in chronic overreaching or overtraining , leading to fatigue , maladaptation , or even illness and injury.27 accordingly , optimization of progressive overload requires appropriate increase in training stress across the training prescription and monitoring of the individual 's physiological response or readiness to receive the increased exercise load . rest and recovery : the tenet that is most frequently underemphasized or commonly neglected in the design of exercise prescriptions is the principle of rest and recovery . central to this principle is the availability of nutrients and rest ( or reduced training load ) in order to permit necessary biological resynthesis to replace the required constituents of the impacted system(s ) . quantifying optimal recovery is challenging28 because of the multidimensional application of the exercise stress.29 conversely , an extended period without adequate stress will result in a loss of adaptation and a down - regulation of the entire system.30 clearly , a balance between the principles of progressive overload and recovery are necessary to elicit optimal physiological adaptations.31 a training stress must perturb homeostasis with enough impulse to lead to chronic supercompensatory adaptations ; however , the appropriate volume of recovery must be prescribed in concert with this perturbation in order to optimize adaptation . there are many techniques in the prescription of an exercise training program that ensure adequate and optimal rest and recovery ; this is most commonly achieved by altering the frequency and duration of training ( while maintaining intensity ) or sequencing the training stress across each week in order to reduce the accumulated fatigue that may amass through repeated , consecutive high - intensity bouts of training.32,33 this is especially critical in clinical populations in which demographic and medical characteristics ( e.g. cardiopulmonary function , immune status , comorbidities , and current therapies ) further complicate optimal adaptation . with appropriate balance , positive adaptations will continue , permitting increases in exercise stresses to be applied and further physiologic conditioning towards the targeted end point . stage 3 design of the exercise prescription : exercise prescriptions are most often operationalized using the following parameters : frequency ( sessions per week ) , intensity ( how hard per session ) , time ( session duration ) , and type ( modality ) or f.i.t.t.34 in the next section , we will outline the design of an exercise prescription ( using f.i.t.t ) that adopts either a conventional approach ( figure 3a ) or an alternative approach that systematically adheres to the principles of training ( figure 3b ) . to facilitate practical understanding , mock clinical trial vignette : a randomized trial to determine the efficacy of supervised exercise training on cardiac function in older ( 65 years ) women following completion of anthracycline - containing adjuvant therapy for early - stage breast cancer . the principles of training . oxygen consumption and ventilatory responses to incremental treadmill exercise in a 65 year - old woman with early - stage breast cancer . ( a ) increasing workloads during the cardiopulmonary exercise test causes linear increases in oxygen consumption ( vo2 in ml / kg / min ) to the point of volitional fatigue at a vo2peak of 18.9 ml / kg / min . ( b ) a graphical representation of alveolar ventilation ( ve in l / min ) demonstrate two exponential breakpoints in ventilation corresponding to ventilatory threshold 1 ( vt1 ) and ventilatory threshold 2 ( vt2 ) . these thresholds demarcate the transition of low , medium , and high exercise intensity , and correspond to specific parameters that may be used for identification of relative intensity for exercise prescription and monitoring . these intensities and the corresponding ranges of physiological identification thereof ( heart rate , blood pressure , and rating ( rtg ) of perceived exertion on a 620 scale ) each bar represents a training session at prescribed workloads based on the absolute or relative intensity . ( a ) the conventional ( linear ) approach utilizes standard intensity , frequency , and duration parameters after an initial lead in period , with static increases in session duration ( i.e. 20 to 45 min ) . ( b ) the alternative non - linear approach considers the principles of exercise training in order to optimize the adaptations to the exercise stimulus . sessions are tailored to an individual 's relative intensity , based on cardiopulmonary exercise testing or exercise tolerance testing , and specified to address a particular endpoint . sessions and weeks progress over the course of the prescription and vary between low intensity ( e.g. 55% vo2peak ; white bars ) and moderate ( e.g. 75% ; grey bars ) and high intensity ( e.g. 100% vo2peak ; black bars ) training in order to target various physiological systems involved in the cardiopulmonary response to exercise . session intensity is inversely related to session duration , that is , sessions involving high relative intensity workloads are conducted in shorter bouts through short duration training sessions and are less frequent to ensure recovery between sessions . vo2peak , peak rate of oxygen consumption . in the conventional approach ( figure 3a ) , exercise training intensity is prescribed using the heart rate reserve technique ( a method based on chronological age and resting heart rate ) without consideration of the patients ' functional limitations or the primary study end point . this is the most common method of individualizing training in the oncology setting.7 such an approach is problematic , however , given the 10 to 12beat - per - minute variation in maximal heart rate in normal subjects.35,36 there may be even greater variation in cancer patients , given the documented impact of current or previous systemic therapy on autonomic function.37 this aerobic training intensity prescribed at 75% heart rate reserve ( for example ) may elicit very different physiologic adaptations between patients ( because of the inaccuracy of age - predicted maximum heart rate and the decreased heart rate reserve due to elevated resting heart rate ) . further , the majority of sessions within this prescription are performed at the same intensity and duration , as determined by their initial cpet or ett ; the workload prescribed to correspond with their percent heart rate reserve will no longer be appropriate after an initial adaptation period . in this instance , training volume remains constant and does not progress across the entire intervention . this is problematic considering that as cardiorespiratory fitness improves , the adaptation from an identical exercise stimulus diminishes ; therefore , there is an insufficient stimulus to induce further physiologic adaptation . as such , this prescription fails to consider three important principles of training , namely individualization , specificity , and progressive overload . contrastingly , in the alternative exercise prescription approach , the use of appropriate baseline testing ( i.e. cpet ) permits aerobic training to be tailored to a patients baseline vo2peak ( figure 2 ) , and thereby adhering to the principle of individualization . second , the intensity , duration , and occasionally , the frequency of training sessions are sequenced in such a fashion that training volume is continually increased across the entire program ( i.e. the principles of specificity and progressive overload ) . this approach also adds important variety to the prescription that not only continually alters the exercise third , training intensity is sequenced , such that higher intensity or higher volume training is followed by lower intensity ( recovery ) training and rest days to optimize adaptation ( i.e. the principle of rest and recovery).38 finally , although not specifically outlined in figure 3b , the principle of reversibility may be a particularly important consideration in aerobic training trials conducted in oncology populations because patients may be forced to temporarily discontinue training because of therapy - induced toxicity and/or disease progression.39 detraining effects can occur rapidly ( within days to weeks ) , thus , subsequent training sessions may need to be resumed at reduced duration and intensity than initially planned . despite the proven efficacy in the arena of sports / athletic performance , consideration of the principles of training has not been translated into the design of exercise prescriptions in clinical populations . indeed , to our knowledge , only one trial to date has compared the efficacy of an exercise prescription following a non - linear approach vs. a traditional linear approach in any clinical population . specifically , klijn et al . compared the efficacy of non - linear periodized training with that of traditionally - prescribed linear combined aerobic and resistance training in 110 patients with severe chronic obstructive pulmonary disease.43 exercise training in both arms was performed three times a week for 10 weeks . results indicated that non - linear exercise training was associated with superior improvements in cycling endurance and health - related quality of life compared with linearly prescribed training . it is important to state that a common perception is that studies examining the efficacy of high - intensity interval training ( hitt ) also adhere to the principles of training / non - linear approach . however , if studies exclusively test hitt ( i.e. all exercise sessions are hitt ) and proceed without appropriate progression , then these programs are also linear in design and do not adhere to the principles of training . in the oncology setting , approximately six studies to date have examined the safety , tolerability , and preliminary efficacy of non - linear aerobic training , compared with a usual care ( no exercise training ) control group . as presented in table 1 , exercise prescriptions adhering to a non - linear approach appear to be safe ( low adverse event rate ) , tolerable ( mean adherence 75% of prescribed sessions both during and after primary adjuvant therapy ) , and efficacious , conferring favourable improvements in vo2peak , quality of life , and other physiological outcomes . on the basis of this data , our group is comparing the efficacy of either non - linear periodized training or traditionally prescribed linear aerobic training with an attention control group ( i.e. supervised progressive stretching ) in 174 women completing primary therapy for early - stage breast cancer . aerobic training in both arms is being performed four times a week for 16 weeks . the primary end point is vo2peak.44 exercise training studies adopting a non - linear approach in exercise - oncology research ( chronological order ) itt , intention to treat analysis ; ppa , per protocol analysis ; wmax , maximal work rate ; vo2peak , peak oxygen consumption ; ac , doxorubicin plus cyclophosphamide ; cpet , cardiopulmonary exercise test ; vt , ventilatory threshold ; mwd , minute walk distance . vt determined by a systematic increase in the ve / vo2 ratio , whereas ve / vco2 remained constant . the purpose of this commentary was to provide an overview of the application of the fundamental principles of training in the design and conduct of clinical trials in exercise - oncology research . it is hoped that attention to these issues will provide the platform for constructive dialogue with the view towards the development of best practice guidelines to optimize exercise training in the oncology setting . application of these guidelines will ensure continued progress in the field by producing the high - quality evidence base necessary to convince oncology professionals that exercise training is an integral aspect of the therapeutic armamentarium in the treatment and control of cancer . j.f.c . is supported by research grants from trygfonden , the novo nordic foundation , the danish cancer society , and the beckett foundation . l.w.j . is supported in part by research grants from the nci and aktiv against cancer .

the field of exercise - oncology has increased dramatically over the past two decades , with close to 100 published studies investigating the efficacy of structured exercise training interventions in patients with cancer . of interest , despite considerable differences in study population and primary study end point , the vast majority of studies have tested the efficacy of an exercise prescription that adhered to traditional guidelines consisting of either supervised or home - based endurance ( aerobic ) training or endurance training combined with resistance training , prescribed at a moderate intensity ( 5075% of a predetermined physiological parameter , typically age - predicted heart rate maximum or reserve ) , for two to three sessions per week , for 10 to 60 min per exercise session , for 12 to 15 weeks . the use of generic exercise prescriptions may , however , be masking the full therapeutic potential of exercise treatment in the oncology setting . against this background , this opinion paper provides an overview of the fundamental tenets of human exercise physiology known as the principles of training , with specific application of these principles in the design and conduct of clinical trials in exercise - oncology research . we contend that the application of these guidelines will ensure continued progress in the field while optimizing the safety and efficacy of exercise treatment following a cancer diagnosis .

Introduction Exercise intervention design considerations Efficacy of exercise prescriptions adhering to the principles of training in oncology Conclusion Funding Conflict of interest

the field of exercise - oncology has burgeoned dramatically in scope and impact since publication of the first scientific papers in the mid to late 1980s.1 several systematic reviews and meta - analyses have evaluated the efficacy of structured exercise treatment in cancer.26 for example , speck et al.7 identified a total of 66 studies reported as high - quality that examined the impact of exercise treatment in a broad array of oncology scenarios ( e.g. differences in histological primary site , stage and treatment ) on a total of 60 different primary end points in adults with cancer . intriguingly , despite the degree of heterogeneity in how exercise was being utilized to manipulate physiological adaptation , the nature of the exercise prescription in the vast majority of studies was similar . more specifically , almost all prescriptions followed traditional guidelines consisting of either supervised or home - based endurance ( aerobic ) training or endurance training combined with resistance training , prescribed at a moderate - intensity ( 5075% of a predetermined physiological parameter , typically age - predicted heart rate maximum or reserve ) , for two to three sessions per week , for 10 to 60 min per exercise session , for 12 to 15 weeks . despite the adoption of a relatively homogeneous prescription approach , exercise training was , for the most part , associated with benefit across a diverse range of end points , largely irrespective of the oncology setting.7 on this evidence , it could be argued that a standardized , largely homogeneous exercise prescription that adopts a conventional approach is safe , efficacious , and therefore sufficient . this has resulted in a limited perceived need to elucidate the optimal dose , sequencing , or combination of different training stresses to specifically alter a desired physiological end point in any clinical population , including oncology . however , the significant benefit of generically dosed exercise treatment on heterogeneous end points rather reflects the remarkable pleiotropic physiologic impact of exercise . moreover , the use of generic exercise prescriptions ( irrespective of clinical population or primary end point ) is masking the full therapeutic promise of exercise treatment . indeed , for more than half a century , exercise training has been continually refined , precisely dosed , and scheduled to minimize injury and optimize human / athletic performance ; the basis of all training prescriptions in this arena adheres to fundamental tenets of human exercise physiology known as the principles of training . these tenets are rarely applied or even considered when designing exercise trials in clinical populations.8,9 accordingly , the purpose of this opinion paper is to provide an overview of the application of these principles in the design and conduct of clinical trials in exercise - oncology research . this paper will focus primarily on application of these principles to aerobic - based training , although the concepts also apply to resistance training or combination training programs . stage 1 objective assessment of response to exercise : an essential prerequisite in the design of all exercise training trials is the objective assessment of baseline physiological functioning of the systems that will be primarily targeted . this generally refers to either the cardiopulmonary responses to exercise ( in the context of aerobic training ) or the assessment of maximal muscular strength or endurance ( in the context of resistance training ) . the relative strengths , weaknesses , and conduct of the various different assessment tools available to researchers have been reviewed previously.1012 in the context of aerobic training studies , the preferred assessment tool is a cardiopulmonary exercise test ( cpet ) . the use of a cpet offers a number of distinct advantages compared with other assessments . of these , arguably the most important is that cpet provides specific information on patients ' peak rate of oxygen consumption ( vo2peak ) as well as their submaximal cardiopulmonary responses to exercise , permitting precise tailoring of training to the individual patient . in settings in which cpet is not available , investigators can also use a maximal incremental exercise tolerance test ( ett ) ( without direct vo2 measurement ) to determine peak workload and peak exercise heart rate.10,13 in addition , both cpets and etts are performed with 12-lead ecg and blood pressure monitoring , thus , they also provide important pre - exercise training clearance information on the detection of any exercise - induced impairments ( e.g. ischemia ) or symptom limitations at both submaximal and maximal exercise tolerance.14 the latter information is of critical importance when considering the inclusion of high - intensity ( 75% of vo2peak ) aerobic training sessions in the prescription . stage 2 application of the principles of training : perhaps the most essential principles of training are individualization , specificity , progressive overload , and rest / recovery ( figure 1 ) . individualization : the concept of individualization is defined as the customized application of training towards the physiological status of the patient/ subject.15,16 clearly , even within carefully selected homogenous clinical trial cohorts , considerable heterogeneity likely still exists in cardiopulmonary function , lifestyle behaviour , age , prior treatment , concomitant comorbidities , and , of course , genetic predisposition . thus , application of a generic prescription that fails to consider such parameters will most often result in either an under - dosing or over - dosing of exercise treatment . the corresponding heart rate , blood pressure , and rating of perceived exertion ( rpe ) directly measured at each percent workload is used to ensure that subjects are training at , or close to , the prescribed intensity during subsequent exercise sessions ( figure 2).17 it is important to note that although the absolute training intensity may be different between individual patients , the relative intensity of training is similar , ensuring standardization across patients a critical aspect of clinical trial conduct.specificity : it is widely acknowledged that the physiological adaptations elicited by aerobic training are unique from those caused by resistance training . lesser appreciated , however , is that even within the same exercise modality , there is enormous capacity to manipulate the nature and configuration of the elements of exercise prescription ( i.e. frequency , intensity , duration , and time ) to confer remarkably distinct physiological adaptations.18,19 the concept of specificity addresses the notion that the selected exercise stress must be specific and targeted to the primary underlying system(s ) or pathway(s ) known or postulated to underpin the primary end point of interest . for example , in a trial designed to examine the impact of aerobic training on vo2peak in a particular cancer cohort , a critical prerequisite for the design of the optimal exercise prescription is determination of the primary limitation ( determinant ) to vo2peak ; in other words , is there a cardiovascular , ventilatory , and/or skeletal muscle limitation to exercise?20 for example , if the primary limitation is cardiovascular exercise training emphasizing moderate intensity , aerobic training ( i.e. 50 to 70% of vo2peak ) for longer duration ( 45 min ) is indicated to initially enhance plasma volume and structural changes in the heart and blood vessels.21 in contrast , if more general deconditioning is observed and skeletal muscle adaptation is the desired outcome , aerobic training at a higher relative intensity ( i.e. by definition therefore , a requirement of effective exercise prescriptions is optimization and progressive increase in stress to confer continued physiological adaptation.26 of equal importance is recognition that increasing exercise stress pushed beyond the homeostasis will result in chronic overreaching or overtraining , leading to fatigue , maladaptation , or even illness and injury.27 accordingly , optimization of progressive overload requires appropriate increase in training stress across the training prescription and monitoring of the individual 's physiological response or readiness to receive the increased exercise load . rest and recovery : the tenet that is most frequently underemphasized or commonly neglected in the design of exercise prescriptions is the principle of rest and recovery . quantifying optimal recovery is challenging28 because of the multidimensional application of the exercise stress.29 conversely , an extended period without adequate stress will result in a loss of adaptation and a down - regulation of the entire system.30 clearly , a balance between the principles of progressive overload and recovery are necessary to elicit optimal physiological adaptations.31 a training stress must perturb homeostasis with enough impulse to lead to chronic supercompensatory adaptations ; however , the appropriate volume of recovery must be prescribed in concert with this perturbation in order to optimize adaptation . there are many techniques in the prescription of an exercise training program that ensure adequate and optimal rest and recovery ; this is most commonly achieved by altering the frequency and duration of training ( while maintaining intensity ) or sequencing the training stress across each week in order to reduce the accumulated fatigue that may amass through repeated , consecutive high - intensity bouts of training.32,33 this is especially critical in clinical populations in which demographic and medical characteristics ( e.g. with appropriate balance , positive adaptations will continue , permitting increases in exercise stresses to be applied and further physiologic conditioning towards the targeted end point . individualization : the concept of individualization is defined as the customized application of training towards the physiological status of the patient/ subject.15,16 clearly , even within carefully selected homogenous clinical trial cohorts , considerable heterogeneity likely still exists in cardiopulmonary function , lifestyle behaviour , age , prior treatment , concomitant comorbidities , and , of course , genetic predisposition . thus , application of a generic prescription that fails to consider such parameters will most often result in either an under - dosing or over - dosing of exercise treatment . the corresponding heart rate , blood pressure , and rating of perceived exertion ( rpe ) directly measured at each percent workload is used to ensure that subjects are training at , or close to , the prescribed intensity during subsequent exercise sessions ( figure 2).17 it is important to note that although the absolute training intensity may be different between individual patients , the relative intensity of training is similar , ensuring standardization across patients a critical aspect of clinical trial conduct . specificity : it is widely acknowledged that the physiological adaptations elicited by aerobic training are unique from those caused by resistance training . lesser appreciated , however , is that even within the same exercise modality , there is enormous capacity to manipulate the nature and configuration of the elements of exercise prescription ( i.e. frequency , intensity , duration , and time ) to confer remarkably distinct physiological adaptations.18,19 the concept of specificity addresses the notion that the selected exercise stress must be specific and targeted to the primary underlying system(s ) or pathway(s ) known or postulated to underpin the primary end point of interest . for example , in a trial designed to examine the impact of aerobic training on vo2peak in a particular cancer cohort , a critical prerequisite for the design of the optimal exercise prescription is determination of the primary limitation ( determinant ) to vo2peak ; in other words , is there a cardiovascular , ventilatory , and/or skeletal muscle limitation to exercise?20 for example , if the primary limitation is cardiovascular exercise training emphasizing moderate intensity , aerobic training ( i.e. 50 to 70% of vo2peak ) for longer duration ( 45 min ) is indicated to initially enhance plasma volume and structural changes in the heart and blood vessels.21 in contrast , if more general deconditioning is observed and skeletal muscle adaptation is the desired outcome , aerobic training at a higher relative intensity ( i.e. > 70% of vo2peak ) that induces enzymatic adaptation and increased capillarization and mitochondrial biogenesis may be emphasized ( after a period of more general training that acclimatizes the patient to exercise).2224 progressive overload : aerobic training provides a potent physiological stimulus that perturbs the equilibrium of multiple organ systems.18 perturbations in the cellular and systemic environments create biological stress , which challenges homeostasis . the concept of hormesis25 or supercompensation ) , wherein the host can withstand greater future system perturbations , meaning that a greater stress stimulus is required to further perturb homeostasis . by definition therefore , a requirement of effective exercise prescriptions is optimization and progressive increase in stress to confer continued physiological adaptation.26 of equal importance is recognition that increasing exercise stress pushed beyond the homeostasis will result in chronic overreaching or overtraining , leading to fatigue , maladaptation , or even illness and injury.27 accordingly , optimization of progressive overload requires appropriate increase in training stress across the training prescription and monitoring of the individual 's physiological response or readiness to receive the increased exercise load . rest and recovery : the tenet that is most frequently underemphasized or commonly neglected in the design of exercise prescriptions is the principle of rest and recovery . quantifying optimal recovery is challenging28 because of the multidimensional application of the exercise stress.29 conversely , an extended period without adequate stress will result in a loss of adaptation and a down - regulation of the entire system.30 clearly , a balance between the principles of progressive overload and recovery are necessary to elicit optimal physiological adaptations.31 a training stress must perturb homeostasis with enough impulse to lead to chronic supercompensatory adaptations ; however , the appropriate volume of recovery must be prescribed in concert with this perturbation in order to optimize adaptation . there are many techniques in the prescription of an exercise training program that ensure adequate and optimal rest and recovery ; this is most commonly achieved by altering the frequency and duration of training ( while maintaining intensity ) or sequencing the training stress across each week in order to reduce the accumulated fatigue that may amass through repeated , consecutive high - intensity bouts of training.32,33 this is especially critical in clinical populations in which demographic and medical characteristics ( e.g. with appropriate balance , positive adaptations will continue , permitting increases in exercise stresses to be applied and further physiologic conditioning towards the targeted end point . stage 3 design of the exercise prescription : exercise prescriptions are most often operationalized using the following parameters : frequency ( sessions per week ) , intensity ( how hard per session ) , time ( session duration ) , and type ( modality ) or f.i.t.t.34 in the next section , we will outline the design of an exercise prescription ( using f.i.t.t ) that adopts either a conventional approach ( figure 3a ) or an alternative approach that systematically adheres to the principles of training ( figure 3b ) . to facilitate practical understanding , mock clinical trial vignette : a randomized trial to determine the efficacy of supervised exercise training on cardiac function in older ( 65 years ) women following completion of anthracycline - containing adjuvant therapy for early - stage breast cancer . the principles of training . ( b ) the alternative non - linear approach considers the principles of exercise training in order to optimize the adaptations to the exercise stimulus . sessions and weeks progress over the course of the prescription and vary between low intensity ( e.g. 100% vo2peak ; black bars ) training in order to target various physiological systems involved in the cardiopulmonary response to exercise . in the conventional approach ( figure 3a ) , exercise training intensity is prescribed using the heart rate reserve technique ( a method based on chronological age and resting heart rate ) without consideration of the patients ' functional limitations or the primary study end point . this is the most common method of individualizing training in the oncology setting.7 such an approach is problematic , however , given the 10 to 12beat - per - minute variation in maximal heart rate in normal subjects.35,36 there may be even greater variation in cancer patients , given the documented impact of current or previous systemic therapy on autonomic function.37 this aerobic training intensity prescribed at 75% heart rate reserve ( for example ) may elicit very different physiologic adaptations between patients ( because of the inaccuracy of age - predicted maximum heart rate and the decreased heart rate reserve due to elevated resting heart rate ) . further , the majority of sessions within this prescription are performed at the same intensity and duration , as determined by their initial cpet or ett ; the workload prescribed to correspond with their percent heart rate reserve will no longer be appropriate after an initial adaptation period . this is problematic considering that as cardiorespiratory fitness improves , the adaptation from an identical exercise stimulus diminishes ; therefore , there is an insufficient stimulus to induce further physiologic adaptation . as such , this prescription fails to consider three important principles of training , namely individualization , specificity , and progressive overload . contrastingly , in the alternative exercise prescription approach , the use of appropriate baseline testing ( i.e. second , the intensity , duration , and occasionally , the frequency of training sessions are sequenced in such a fashion that training volume is continually increased across the entire program ( i.e. the principles of specificity and progressive overload ) . this approach also adds important variety to the prescription that not only continually alters the exercise third , training intensity is sequenced , such that higher intensity or higher volume training is followed by lower intensity ( recovery ) training and rest days to optimize adaptation ( i.e. the principle of rest and recovery).38 finally , although not specifically outlined in figure 3b , the principle of reversibility may be a particularly important consideration in aerobic training trials conducted in oncology populations because patients may be forced to temporarily discontinue training because of therapy - induced toxicity and/or disease progression.39 detraining effects can occur rapidly ( within days to weeks ) , thus , subsequent training sessions may need to be resumed at reduced duration and intensity than initially planned . despite the proven efficacy in the arena of sports / athletic performance , consideration of the principles of training has not been translated into the design of exercise prescriptions in clinical populations . indeed , to our knowledge , only one trial to date has compared the efficacy of an exercise prescription following a non - linear approach vs. a traditional linear approach in any clinical population . compared the efficacy of non - linear periodized training with that of traditionally - prescribed linear combined aerobic and resistance training in 110 patients with severe chronic obstructive pulmonary disease.43 exercise training in both arms was performed three times a week for 10 weeks . it is important to state that a common perception is that studies examining the efficacy of high - intensity interval training ( hitt ) also adhere to the principles of training / non - linear approach . all exercise sessions are hitt ) and proceed without appropriate progression , then these programs are also linear in design and do not adhere to the principles of training . in the oncology setting , approximately six studies to date have examined the safety , tolerability , and preliminary efficacy of non - linear aerobic training , compared with a usual care ( no exercise training ) control group . as presented in table 1 , exercise prescriptions adhering to a non - linear approach appear to be safe ( low adverse event rate ) , tolerable ( mean adherence 75% of prescribed sessions both during and after primary adjuvant therapy ) , and efficacious , conferring favourable improvements in vo2peak , quality of life , and other physiological outcomes . on the basis of this data , our group is comparing the efficacy of either non - linear periodized training or traditionally prescribed linear aerobic training with an attention control group ( i.e. the primary end point is vo2peak.44 exercise training studies adopting a non - linear approach in exercise - oncology research ( chronological order ) itt , intention to treat analysis ; ppa , per protocol analysis ; wmax , maximal work rate ; vo2peak , peak oxygen consumption ; ac , doxorubicin plus cyclophosphamide ; cpet , cardiopulmonary exercise test ; vt , ventilatory threshold ; mwd , minute walk distance . the purpose of this commentary was to provide an overview of the application of the fundamental principles of training in the design and conduct of clinical trials in exercise - oncology research . it is hoped that attention to these issues will provide the platform for constructive dialogue with the view towards the development of best practice guidelines to optimize exercise training in the oncology setting . application of these guidelines will ensure continued progress in the field by producing the high - quality evidence base necessary to convince oncology professionals that exercise training is an integral aspect of the therapeutic armamentarium in the treatment and control of cancer .

in multi - trauma patients , the acute respiratory distress syndrome ( ards ) is a commonly seen complication . important risk factors for the development of ards after trauma are severe injury [ injury severity score ( iss ) > 25 ] and pulmonary contusion [ 1 , 2 ] . although the occurrence of ards in multi - trauma patients increases considerably with severity of pulmonary contusion , the incidence is surprisingly low in patients with isolated chest injury [ 24 ] . an excessive innate immune response to tissue injury is considered to be the cause of ards . due to the heterogeneity of injuries present in multi - trauma patients , the specific role of chest injury in the pathogenesis of ards is not well known . in this study we investigated the effect of isolated chest injury on the systemic innate immune response to test whether this increased risk of ards is caused by priming of the circulating neutrophils or we focused on differences in the phenotype of circulating neutrophils during the first 24 h after chest injury . neutrophils are important effector cells of the final common pathway of the innate immune response . a massive release of radical oxygen species ( ros ) and proteases by neutrophils can cause damage to the parenchyma , which can eventually result in organ dysfunction ( ards ) . systemic inflammation is characterized by the activation of circulating neutrophils [ 6 , 7 ] and the mobilization of young neutrophils from the bone marrow . surface receptors of circulating neutrophils have been used to determine the activation of circulating neutrophils in order to discriminate the severity of inflammation and to predict the occurrence of organ failure [ 69 ] . systemic neutrophil activation is typically characterized by the shedding of l - selectin ( cd62l ) and the upregulation of expression of m integrin ( cd11b ) [ 1013 ] . decreased l - selectin and increased m expression have been shown to correlate with iss and to be associated with the development of posttraumatic complications such as ards [ 1420 ] . a previous study by our group showed a significant decreased responsiveness after in vitro stimulation of active fcrii ( cd32 ) , the main igg receptor on neutrophils , in multi - trauma patients . decreased responsiveness of active fcrii was more pronounced in patients who developed ards or acute lung injury ( ali ) compared to patients without complications . expression of active fcrii is under the control of inside - out signals induced by both chemoattractants and cytokines , and seems to be more sensitive to priming stimuli compared with m / cd11b . the responsiveness of active fcrii was found to correlate better with outcome than other neutrophil receptor expressions in preceding observational studies in trauma patients [ 11 , 20 ] . we investigated whether isolated chest injury leads to a systemic innate immune response quantified by the activation of circulating neutrophils . furthermore , the release of interleukin ( il)-6 was measured as an additional marker for inflammation . patients suffering from chest injury with an abbreviated injury score ( ais ) of 2 or more who were admitted to the trauma department of the university medical centre utrecht were included . patients with injuries with an ais > 2 in other regions than the thorax were excluded to reduce systemic inflammation caused by tissue damage outside the thorax . other exclusion criteria included age < 18 or > 70 years , death within 24 h after admission , and patients with altered immunological status ( e.g. , corticosteroid use or chemotherapy ) . at admission , injury severity score ( iss ) , new injury severity score ( niss ) , apache ii score , and leukocyte count the presence of ards was assessed according to their clinical criteria , as determined in the consensus conferences on ards . blood samples ware taken at approximately 3 ( 24 ) , 9 ( 810 ) and 24 ( 2226 ) h after the accident to investigate the relationship between chest injury and systemic neutrophil activation . in an in vivo human inflammation model , we saw previously that systemic neutrophil activation is most prominent between 2 and 4 h after the induction of inflammation . the first measurement time point was therefore set at 3 h postinjury . the local ethics committee approved the study , and written informed consent was obtained from all patients or their legal representatives in accordance with the helsinki declaration . the following commercially available mouse antihuman monoclonal antibodies were purchased for analyzing neutrophil receptor expression by flowcytometry : fluorescein isothiocyanate ( fitc)-labeled igg1 isotype control ( clone mopc-21 , bd pharmingen , usa ) , alexa fluor 647-labeled igg1 isotype control ( clone mopc-21 , bd pharmingen , usa ) , r - phycoerythrin ( rpe)-labeled igg2a isotype control ( clone mrc ox-34 , serotec , germany ) , rpe - labeled igg1 anti-m ( cd11b ; clone 2lpm19c , dako , denmark ) , fitc - labeled igg1 anti - l - selectin ( cd62l ; clone dreg56 , bd pharmingen , usa ) , alexa fluor 647-labeled igg1 anti - fcriii ( cd16 ; clone 3g8 , bd pharmingen , usa ) , rpe - labeled igg2b anti - fcrii ( cd32 ; clone fli8.26 , bd pharmingen , usa ) , fitc - labeled igg2a anti - cxcr1 ( cd181a ; clone 42705 , r&d systems europe , uk ) , rpe - labeled igg2a anti - cxcr2 ( cd182b ; clone 48311 , r&d systems europe , uk ) , and fitc - labeled igg2a anti - c5ar ( cd88 ; clone p12/1 , serotec , germany ) . a fitc - labeled monoclonal phage antibody ( a27 ) , which recognizes the active configuration of fcrii ( cd32 ) , was manufactured at the department of respiratory medicine at the university medical centre utrecht ( mophab a27 , umc utrecht , the netherlands ) [ 21 , 27 ] . the functionality and configuration of fcrii ( cd32 ) on granulocytes is regulated by inside - out control . visualization of this process by the antibody a27 is a very sensitive means of monitoring the subtle activation of innate immune cells such as neutrophils in vivo . blood was collected in a vacutainer with sodium heparin as anticoagulant and cooled immediately on melting ice . after lysis , white blood cells were washed and resuspended in pbs2 + [ phosphate - buffered saline supplemented with sodium citrate ( 0.4% wt / vol ) and pasteurized plasma protein solution ( 10% vol / vol ) ] . resuspended cells were incubated for 45 min on ice with commercially obtained directly labeled antibodies against activation molecules : l - selectin , m , cxcr1 , cxcr2 , c5ar , fcrii and fcriii . after incubation and a final wash , expression was measured on a facscalibur flow cytometer ( becton , dickinson & co. , mountain view , ca , usa ) . the neutrophils were identified according to their specific side - scatter and forward - scatter signals . to measure fcrii * expression , whole blood was incubated with fitc - labeled monoclonal phage antibody a27 for 45 min on ice . active upregulation of fcrii * expression was measured after 5 min of stimulation of whole blood at 37c with n - formyl - methionyl - leucyl - phenylalanine ( fmlp 10 m ) to evaluate the responsiveness of the cells for bacterially derived protein products / peptides . after stimulation , the samples were put on ice again and stained with phage antibody a27 . after staining , red cells were lysed , and expression was measured on facscalibur , as described above . data from individual experiments are depicted as the median fluorescence intensity ( mfi ) of at least 10,000 neutrophils . plasma samples were obtained at 3 , 9 and 24 h after injury and stored at 80c until further analysis . il-6 levels were measured by an enzyme - linked immunosorbent assay ( elisa ) according to the manufacturer s protocol ( ebioscience , san diego , usa ) . all data are presented as means se , unless described otherwise . to compare differences in admission variables between patients or control values , the mann patients suffering from chest injury with an abbreviated injury score ( ais ) of 2 or more who were admitted to the trauma department of the university medical centre utrecht were included . patients with injuries with an ais > 2 in other regions than the thorax were excluded to reduce systemic inflammation caused by tissue damage outside the thorax . other exclusion criteria included age < 18 or > 70 years , death within 24 h after admission , and patients with altered immunological status ( e.g. , corticosteroid use or chemotherapy ) . at admission , injury severity score ( iss ) , new injury severity score ( niss ) , apache ii score , and leukocyte count the presence of ards was assessed according to their clinical criteria , as determined in the consensus conferences on ards . blood samples ware taken at approximately 3 ( 24 ) , 9 ( 810 ) and 24 ( 2226 ) h after the accident to investigate the relationship between chest injury and systemic neutrophil activation . in an in vivo human inflammation model , we saw previously that systemic neutrophil activation is most prominent between 2 and 4 h after the induction of inflammation . the first measurement time point was therefore set at 3 h postinjury . the local ethics committee approved the study , and written informed consent was obtained from all patients or their legal representatives in accordance with the helsinki declaration . antihuman monoclonal antibodies were purchased for analyzing neutrophil receptor expression by flowcytometry : fluorescein isothiocyanate ( fitc)-labeled igg1 isotype control ( clone mopc-21 , bd pharmingen , usa ) , alexa fluor 647-labeled igg1 isotype control ( clone mopc-21 , bd pharmingen , usa ) , r - phycoerythrin ( rpe)-labeled igg2a isotype control ( clone mrc ox-34 , serotec , germany ) , rpe - labeled igg1 anti-m ( cd11b ; clone 2lpm19c , dako , denmark ) , fitc - labeled igg1 anti - l - selectin ( cd62l ; clone dreg56 , bd pharmingen , usa ) , alexa fluor 647-labeled igg1 anti - fcriii ( cd16 ; clone 3g8 , bd pharmingen , usa ) , rpe - labeled igg2b anti - fcrii ( cd32 ; clone fli8.26 , bd pharmingen , usa ) , fitc - labeled igg2a anti - cxcr1 ( cd181a ; clone 42705 , r&d systems europe , uk ) , rpe - labeled igg2a anti - cxcr2 ( cd182b ; clone 48311 , r&d systems europe , uk ) , and fitc - labeled igg2a anti - c5ar ( cd88 ; clone p12/1 , serotec , germany ) . a fitc - labeled monoclonal phage antibody ( a27 ) , which recognizes the active configuration of fcrii ( cd32 ) , was manufactured at the department of respiratory medicine at the university medical centre utrecht ( mophab a27 , umc utrecht , the netherlands ) [ 21 , 27 ] . the functionality and configuration of fcrii ( cd32 ) on granulocytes is regulated by inside - out control . visualization of this process by the antibody a27 is a very sensitive means of monitoring the subtle activation of innate immune cells such as neutrophils in vivo . blood was collected in a vacutainer with sodium heparin as anticoagulant and cooled immediately on melting ice . after lysis , white blood cells were washed and resuspended in pbs2 + [ phosphate - buffered saline supplemented with sodium citrate ( 0.4% wt / vol ) and pasteurized plasma protein solution ( 10% vol / vol ) ] . resuspended cells were incubated for 45 min on ice with commercially obtained directly labeled antibodies against activation molecules : l - selectin , m , cxcr1 , cxcr2 , c5ar , fcrii and fcriii . after incubation and a final wash , expression was measured on a facscalibur flow cytometer ( becton , dickinson & co. , mountain view , ca , usa ) . the neutrophils were identified according to their specific side - scatter and forward - scatter signals . to measure fcrii * expression , whole blood was incubated with fitc - labeled monoclonal phage antibody a27 for 45 min on ice . active upregulation of fcrii * expression was measured after 5 min of stimulation of whole blood at 37c with n - formyl - methionyl - leucyl - phenylalanine ( fmlp 10 m ) to evaluate the responsiveness of the cells for bacterially derived protein products / peptides . after stimulation , the samples were put on ice again and stained with phage antibody a27 . after staining , red cells were lysed , and expression was measured on facscalibur , as described above . data from individual experiments are depicted as the median fluorescence intensity ( mfi ) of at least 10,000 neutrophils . plasma samples were obtained at 3 , 9 and 24 h after injury and stored at 80c until further analysis . il-6 levels were measured by an enzyme - linked immunosorbent assay ( elisa ) according to the manufacturer s protocol ( ebioscience , san diego , usa ) . all data are presented as means se , unless described otherwise . to compare differences in admission variables between patients or control values , the mann four patients were eventually excluded because considerable additional injury ( ais > 2 ) was diagnosed within 24 h after admission . all of the patients , nine of whom were male and four female , had blunt chest injuries . one of them was also hypothermic , with a body temperature of 35.4c.table 1patient characteristicsptgenderagemechanism of injurydiagnosisissnissthorax aisapache iileukocyte count ( 10)/l at admissioncomplications1m25mva8 unilateral rib fracturesbilateral pneumothorax16254312.0none2m62mva3 unilateral rib fracturesclavicular fractureorbital roof fracture17173158.0pneumonia3m51fall from height2 unilateral rib fracturesunilateral pneumothorax99369.0none4f47fall from horse10 unilateral rib fracturesflail thorax17174319.3none5m60mva5 unilateral rib fractures unilateral pneumothorax unilateral lung contusionpancreatic contusion21294917.6infected epidural catheter6m31mva3 unilateral rib fracturesunilateral pneumothoraxunilateral lung contusionhumeral luxationclavicular fracturediffuse axonal injury38433188.0none7f67mva5 unilateral rib fracturesflail thoraxunilateral pneumothorax bilateral lung contusionfracture of proc . transversus20364515.6none8m59fall from height6 unilateral rib fracturesunilateral pneumothoraxunilateral lung contusion162541815.9thorax empyema , sepsis9m69mva3 unilateral rib fractures55255.0infected epidural catheter10f62bicycle accident6 unilateral rib fracturesminor laceration of kidneyfacial hematoma1414378.5pleural effusion11f53fall from height4 unilateral rib fractrures1 contralateral rib fracturescapular fracturefracture of cervical vertebral body17173211.2pleural effusion12m62mva3 rib fracturesbilateral lung contusionminor liver laceration20294157.8pneumothorax13m59attacked by cowmultiple bilateral rib fracturesbilateral flail thoraxbilateral pneumothoraxbilateral lung contusionsternal fractureminor liver laceration29385618.9pneumonia patient characteristics the mean age was 54 4 years , the mean iss 18 2 , and the mean niss 23 3 . this patient suffered from diffuse axonal injury ( dai ) without signs of bleeding , edema or compression on ct scan ( ais of 5 ) . diagnosis was made several days after injury based on clinical presentation . since the high score assigned to dai is related to an increased risk of mortality caused by direct brain injury rather than to severity of inflammation , this patient was not excluded from further analysis . the mean apache ii score was 9 2 and the mean hospital stay amounted to 16 2 days . six patients needed mechanical ventilation during their hospital stays for a mean duration of 6 2 days . one patient developed sepsis due to thoracic empyema and underwent thoracotomy for debridement of the pleural cavity . l - selectin expression was decreased at 9 h postinjury ( p = 0.002 ) and remained decreased until 24 h ( p = 0.012 ) ( fig . m expression was significantly decreased at 9 h postinjury compared to control values ( fig . although neutrophil activation is typically characterized by m upregulation , expression of m declined at 9 h postinjury ( p = 0.020 ) to a minimum in this group of patients . this overall decrease in m expression is probably due to an increased amount of young neutrophils , which express m at low levels [ 29 , 30].fig . 1expression of l - selectin ( a ) , m ( cd11b ) ( b ) , cxcr1 ( c ) , cxcr2 ( d ) , and c5ar ( e ) on the neutrophil surface over time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) expression of l - selectin ( a ) , m ( cd11b ) ( b ) , cxcr1 ( c ) , cxcr2 ( d ) , and c5ar ( e ) on the neutrophil surface over time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) neutrophil activation is associated with reduced surface expression of cxcr1 , cxcr2 and c5ar [ 3133 ] . after chest injury , circulating neutrophils showed a temporary decline in the expression of chemokine receptors cxcr1 and cxcr2 and complement receptor c5ar ( fig . this decline was statistically significant for cxcr2 and c5ar at 3 h postinjury , but not for cxcr1 . it has been demonstrated that , upon activation , cxcr2 internalizes more rapidly than cxcr1 , which may explain the more pronounced decline of cxcr2 compared to cxcr1 in our results . the expression of cxcr2 and c5ar was gradually restored during the first 24 h after injury , indicating a transient activation of circulating neutrophils . cxcr1 expression increased above control values at 24 h after injury ( p = 0.039 ) . expression of fcrii was markedly decreased until 24 h after injury ( p < 0.010 ; fig . 2 ) . expression of the active form was slightly lower in trauma patients compared to control values , although this decline did not reach statistical significance . expression of fmlp - induced active fcrii , however , was significantly decreased until 9 h after injury ( p = 0.006 ) . expression of fcriii evidently dropped during the first 24 h after chest trauma ( p < 0.001 ) . fcriii is normally expressed at lower levels on young ( banded ) neutrophils compared with more mature forms . therefore , this decrease in overall fcriii suggests an influx of young neutrophils.fig . 2intrinsic expression of fcrii ( a ) , active fcrii ( b ) , fmlp - induced expression of active fcrii ( c ) , and expression of fcriii ( d ) on the neutrophil surface during time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) intrinsic expression of fcrii ( a ) , active fcrii ( b ) , fmlp - induced expression of active fcrii ( c ) , and expression of fcriii ( d ) on the neutrophil surface during time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . 0.05,**p < 0.01 ) il-6 levels were significantly enhanced at 3 h postinjury compared to control values [ mean concentration of 44 15 versus 0 pg / ml ( p < 0.001 ) ] . il-6 levels further increased to a maximum mean concentration of 86 31 pg / ml ( p < 0.001 compared to control values ) 24 h after blunt chest injury ( fig . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * * p < 0.01 ) levels of circulating il-6 . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * * p < 0.01 ) four patients were eventually excluded because considerable additional injury ( ais > 2 ) was diagnosed within 24 h after admission . all of the patients , nine of whom were male and four female , had blunt chest injuries . one of them was also hypothermic , with a body temperature of 35.4c.table 1patient characteristicsptgenderagemechanism of injurydiagnosisissnissthorax aisapache iileukocyte count ( 10)/l at admissioncomplications1m25mva8 unilateral rib fracturesbilateral pneumothorax16254312.0none2m62mva3 unilateral rib fracturesclavicular fractureorbital roof fracture17173158.0pneumonia3m51fall from height2 unilateral rib fracturesunilateral pneumothorax99369.0none4f47fall from horse10 unilateral rib fracturesflail thorax17174319.3none5m60mva5 unilateral rib fractures unilateral pneumothorax unilateral lung contusionpancreatic contusion21294917.6infected epidural catheter6m31mva3 unilateral rib fracturesunilateral pneumothoraxunilateral lung contusionhumeral luxationclavicular fracturediffuse axonal injury38433188.0none7f67mva5 unilateral rib fracturesflail thoraxunilateral pneumothorax bilateral lung contusionfracture of proc . transversus20364515.6none8m59fall from height6 unilateral rib fracturesunilateral pneumothoraxunilateral lung contusion162541815.9thorax empyema , sepsis9m69mva3 unilateral rib fractures55255.0infected epidural catheter10f62bicycle accident6 unilateral rib fracturesminor laceration of kidneyfacial hematoma1414378.5pleural effusion11f53fall from height4 unilateral rib fractrures1 contralateral rib fracturescapular fracturefracture of cervical vertebral body17173211.2pleural effusion12m62mva3 rib fracturesbilateral lung contusionminor liver laceration20294157.8pneumothorax13m59attacked by cowmultiple bilateral rib fracturesbilateral flail thoraxbilateral pneumothoraxbilateral lung contusionsternal fractureminor liver laceration29385618.9pneumonia patient characteristics the mean age was 54 4 years , the mean iss 18 2 , and the mean niss 23 3 . this patient suffered from diffuse axonal injury ( dai ) without signs of bleeding , edema or compression on ct scan ( ais of 5 ) . diagnosis was made several days after injury based on clinical presentation . since the high score assigned to dai is related to an increased risk of mortality caused by direct brain injury rather than to severity of inflammation , this patient was not excluded from further analysis . the mean apache ii score was 9 2 and the mean hospital stay amounted to 16 2 days . six patients needed mechanical ventilation during their hospital stays for a mean duration of 6 2 days . one patient developed sepsis due to thoracic empyema and underwent thoracotomy for debridement of the pleural cavity . l - selectin expression was decreased at 9 h postinjury ( p = 0.002 ) and remained decreased until 24 h ( p = 0.012 ) ( fig . m expression was significantly decreased at 9 h postinjury compared to control values ( fig . although neutrophil activation is typically characterized by m upregulation , expression of m declined at 9 h postinjury ( p = 0.020 ) to a minimum in this group of patients . this overall decrease in m expression is probably due to an increased amount of young neutrophils , which express m at low levels [ 29 , 30].fig . 1expression of l - selectin ( a ) , m ( cd11b ) ( b ) , cxcr1 ( c ) , cxcr2 ( d ) , and c5ar ( e ) on the neutrophil surface over time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) expression of l - selectin ( a ) , m ( cd11b ) ( b ) , cxcr1 ( c ) , cxcr2 ( d ) , and c5ar ( e ) on the neutrophil surface over time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) l - selectin expression was decreased at 9 h postinjury ( p = 0.002 ) and remained decreased until 24 h ( p = 0.012 ) ( fig . m expression was significantly decreased at 9 h postinjury compared to control values ( fig . although neutrophil activation is typically characterized by m upregulation , expression of m declined at 9 h postinjury ( p = 0.020 ) to a minimum in this group of patients . this overall decrease in m expression is probably due to an increased amount of young neutrophils , which express m at low levels [ 29 , 30].fig . 1expression of l - selectin ( a ) , m ( cd11b ) ( b ) , cxcr1 ( c ) , cxcr2 ( d ) , and c5ar ( e ) on the neutrophil surface over time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) expression of l - selectin ( a ) , m ( cd11b ) ( b ) , cxcr1 ( c ) , cxcr2 ( d ) , and c5ar ( e ) on the neutrophil surface over time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) neutrophil activation is associated with reduced surface expression of cxcr1 , cxcr2 and c5ar [ 3133 ] . after chest injury , circulating neutrophils showed a temporary decline in the expression of chemokine receptors cxcr1 and cxcr2 and complement receptor c5ar ( fig . this decline was statistically significant for cxcr2 and c5ar at 3 h postinjury , but not for cxcr1 . it has been demonstrated that , upon activation , cxcr2 internalizes more rapidly than cxcr1 , which may explain the more pronounced decline of cxcr2 compared to cxcr1 in our results . the expression of cxcr2 and c5ar was gradually restored during the first 24 h after injury , indicating a transient activation of circulating neutrophils . cxcr1 expression increased above control values at 24 h after injury ( p = 0.039 ) . expression of fcrii was markedly decreased until 24 h after injury ( p < 0.010 ; fig . 2 ) . expression of the active form was slightly lower in trauma patients compared to control values , although this decline did not reach statistical significance . expression of fmlp - induced active fcrii , however , was significantly decreased until 9 h after injury ( p = 0.006 ) . expression of fcriii evidently dropped during the first 24 h after chest trauma ( p < 0.001 ) . fcriii is normally expressed at lower levels on young ( banded ) neutrophils compared with more mature forms . 2intrinsic expression of fcrii ( a ) , active fcrii ( b ) , fmlp - induced expression of active fcrii ( c ) , and expression of fcriii ( d ) on the neutrophil surface during time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) intrinsic expression of fcrii ( a ) , active fcrii ( b ) , fmlp - induced expression of active fcrii ( c ) , and expression of fcriii ( d ) on the neutrophil surface during time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) il-6 levels were significantly enhanced at 3 h postinjury compared to control values [ mean concentration of 44 15 versus 0 pg / ml ( p < 0.001 ) ] . il-6 levels further increased to a maximum mean concentration of 86 31 pg / ml ( p < 0.001 compared to control values ) 24 h after blunt chest injury ( fig . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * * p < 0.01 ) levels of circulating il-6 . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * * p < 0.01 ) in this study we show that blunt chest injury leads to a systemic activation of circulating neutrophils , characterized by the shedding of l - selectin and the downregulation of cxcr2 and c5ar . it furthermore shows that blunt chest injury is associated with the mobilization of young ( fcriii - low ) neutrophils and with a reduced responsiveness of circulating neutrophils to an inflammatory stimulus . although seven patients had a chest ais of 4 , and two of these had a bilateral pulmonary contusion , none of these patients developed ards . lung injury results in endovascular changes , tissue barrier failure , and locally increased cytokine levels , enabling systemic activated neutrophils to infiltrate the parenchyma . despite vast local damage in the abovementioned cases , the innate immune response provoked was apparently not abundant enough to cause ards . this study showed that isolated lung contusion resulted in an increase in circulating il-6 , but it did not cause ards . these findings involved patients with minor as well as major lung contusions ( based on ct lung injury score ) . the same study also described an enhanced inflammatory response , with significantly increased levels of circulating il-6 and il-8 plus significantly elevated multiple organ failure ( mof ) scores in multi - trauma patients with major lung contusions compared to multi - trauma patients with minor or no lung contusions . however , the exact role of lung contusion in the immune response and the occurrence of organ dysfunction was not completely clear in these severely injured patients . the result was biased by an evidently higher iss in patients with major lung contusions compared to those with minor or no lung contusions . more severe chest injury is most often accompanied by more severe additional injury in at least one of the other regions , resulting in a higher iss . the majority of patients who presented at the emergency department with severe chest injury could not be included due to considerable injuries in regions other than the thorax . these challenging inclusion and exclusion criteria resulted in the inclusion of fewer patients , but also a more homogeneous group . earlier studies concerning multi - trauma patients demonstrated that fmlp - induced active - fcrii expression was decreased in patients compared to controls , and that the expression was negatively correlated with iss and adverse outcome [ 11 , 20 ] . a clear difference in the median fmlp - induced expression of active fcrii , measured during the first 24 h after injury , was seen between patients with ali / ards ( low expression ) and patients with an uneventful course ( high expression ) . the median values for the fmlp - induced expression of active fcrii measured in this study are comparable to multi - trauma patients with an uneventful clinical course . these findings support the hypothesis that isolated chest injury induces only a restricted activation of the systemic immune response . the increased number of circulating neutrophils after chest injury is most likely due to mobilization of young neutrophils , as displayed by a decrease in overall fcriii expression . however , delayed neutrophil apoptosis has been described after trauma [ 36 , 37 ] . although this phenomenon remains to be established in vivo , we can not exclude that the total number of neutrophils are biased by disturbed apoptosis , since apoptotic markers such as annexin v were not measured . the fact that isolated chest injury rarely leads to ards contrasts with other conditions in which the lung is locally affected and this complication is frequently seen , such as during pneumonia or after aspiration [ 25 , 38 , 39 ] . presumably , the systemic innate immune response evoked by isolated chest injury is less extensive than that during infection or after aspiration . in a rodent model , hoth et al . demonstrated that lung injury followed by exposure to e. coli lipopolysaccharide ( lps ) leads to massive neutrophil infiltration and lung damage , whereas tissue infiltration and damage was far less after lps or lung injury alone . in addition , serum il-6 levels were significantly increased compared to lps exposure or lung injury alone . they concluded that lung injury primes the systemic innate immune response , as was suggested by maier et al . in this study we show that the systemic innate immune response caused by isolated chest injury is transient and short . although il-6 levels remained elevated until 24 h after injury , activation of circulating neutrophils was partially restored . however , this mild systemic response may be sufficient to enhance the innate immune response caused by a second hit such as concomitant tissue damage , fat emboli , or infection . we therefore suggest that lung damage alone is not likely to result in an ards , but a synergism between inflammation caused by lung injury and an additional stimulus caused by a second hit results in a markedly increased risk of developing ards . in this study we demonstrated that isolated blunt chest injury caused transient systemic activation of neutrophils together with a mobilization of young neutrophils into the peripheral circulation . in addition , only severe chest injury ( ais > 4 ) results in an increased number of circulating neutrophils . however , it seems that chest injury alone is not sufficient to cause ards in these cases ; a second hit may be needed .

introductionthe acute respiratory distress syndrome ( ards ) is a severe and frequently seen complication in multi - trauma patients . ards is caused by an excessive innate immune response with a clear role for neutrophils . as ards is more frequently seen in trauma patients with chest injury , we investigated the influence of chest injury on the systemic neutrophil response and the development of ards.materials and methodsthirteen patients with isolated blunt chest injury [ abbreviated injury score ( ais ) 25 ] were included . to avoid systemic inflammation caused by tissue damage outside the thorax , injuries to other regions than the chest did not exceed an ais of 2 . at 3 , 9 and 24 h after injury , the expression of circulating activating molecules on neutrophils and levels of circulating interleukine ( il)-6 were determined . blood samples from eight healthy volunteers were used as control.resultsblunt chest injury resulted in the activation of circulating neutrophils , as characterized by a decreased expression of l - selectin ( cd62l ) , cxcr2 ( cd182b ) and c5ar ( cd88 ) compared to control ( p < 0.05 ) . expression of l - selectin , cxcr2 and c5ar was partially restored at 24 h after injury . in addition , the mean expression of fcriii ( cd16 ) dropped ( p < 0.001 ) , indicating the recruitment of young neutrophils into the circulation . il-6 levels increased to a maximum mean concentration of 86 31 pg / ml at 24 h postinjury . none of the patients developed ards.conclusionblunt chest trauma caused a systemic inflammatory reaction with transient activation of neutrophils and mobilization of young neutrophils into the circulation . isolated chest injury , however , was not abundant enough to cause ards , so a second hit appears crucial .

Introduction Methods Patients Expression of activation markers on neutrophils determined by flowcytometry IL-6 Statistics Results Patient demographics Receptor expression on the neutrophil surface None CXCR1, CXCR2 and C5aR FcRII, active FcRII and FcRIII IL-6 levels Discussion Conclusion

in multi - trauma patients , the acute respiratory distress syndrome ( ards ) is a commonly seen complication . important risk factors for the development of ards after trauma are severe injury [ injury severity score ( iss ) > 25 ] and pulmonary contusion [ 1 , 2 ] . although the occurrence of ards in multi - trauma patients increases considerably with severity of pulmonary contusion , the incidence is surprisingly low in patients with isolated chest injury [ 24 ] . due to the heterogeneity of injuries present in multi - trauma patients , the specific role of chest injury in the pathogenesis of ards is not well known . in this study we investigated the effect of isolated chest injury on the systemic innate immune response to test whether this increased risk of ards is caused by priming of the circulating neutrophils or we focused on differences in the phenotype of circulating neutrophils during the first 24 h after chest injury . systemic inflammation is characterized by the activation of circulating neutrophils [ 6 , 7 ] and the mobilization of young neutrophils from the bone marrow . systemic neutrophil activation is typically characterized by the shedding of l - selectin ( cd62l ) and the upregulation of expression of m integrin ( cd11b ) [ 1013 ] . decreased l - selectin and increased m expression have been shown to correlate with iss and to be associated with the development of posttraumatic complications such as ards [ 1420 ] . a previous study by our group showed a significant decreased responsiveness after in vitro stimulation of active fcrii ( cd32 ) , the main igg receptor on neutrophils , in multi - trauma patients . we investigated whether isolated chest injury leads to a systemic innate immune response quantified by the activation of circulating neutrophils . patients suffering from chest injury with an abbreviated injury score ( ais ) of 2 or more who were admitted to the trauma department of the university medical centre utrecht were included . patients with injuries with an ais > 2 in other regions than the thorax were excluded to reduce systemic inflammation caused by tissue damage outside the thorax . blood samples ware taken at approximately 3 ( 24 ) , 9 ( 810 ) and 24 ( 2226 ) h after the accident to investigate the relationship between chest injury and systemic neutrophil activation . the following commercially available mouse antihuman monoclonal antibodies were purchased for analyzing neutrophil receptor expression by flowcytometry : fluorescein isothiocyanate ( fitc)-labeled igg1 isotype control ( clone mopc-21 , bd pharmingen , usa ) , alexa fluor 647-labeled igg1 isotype control ( clone mopc-21 , bd pharmingen , usa ) , r - phycoerythrin ( rpe)-labeled igg2a isotype control ( clone mrc ox-34 , serotec , germany ) , rpe - labeled igg1 anti-m ( cd11b ; clone 2lpm19c , dako , denmark ) , fitc - labeled igg1 anti - l - selectin ( cd62l ; clone dreg56 , bd pharmingen , usa ) , alexa fluor 647-labeled igg1 anti - fcriii ( cd16 ; clone 3g8 , bd pharmingen , usa ) , rpe - labeled igg2b anti - fcrii ( cd32 ; clone fli8.26 , bd pharmingen , usa ) , fitc - labeled igg2a anti - cxcr1 ( cd181a ; clone 42705 , r&d systems europe , uk ) , rpe - labeled igg2a anti - cxcr2 ( cd182b ; clone 48311 , r&d systems europe , uk ) , and fitc - labeled igg2a anti - c5ar ( cd88 ; clone p12/1 , serotec , germany ) . plasma samples were obtained at 3 , 9 and 24 h after injury and stored at 80c until further analysis . to compare differences in admission variables between patients or control values , the mann patients suffering from chest injury with an abbreviated injury score ( ais ) of 2 or more who were admitted to the trauma department of the university medical centre utrecht were included . patients with injuries with an ais > 2 in other regions than the thorax were excluded to reduce systemic inflammation caused by tissue damage outside the thorax . at admission , injury severity score ( iss ) , new injury severity score ( niss ) , apache ii score , and leukocyte count the presence of ards was assessed according to their clinical criteria , as determined in the consensus conferences on ards . blood samples ware taken at approximately 3 ( 24 ) , 9 ( 810 ) and 24 ( 2226 ) h after the accident to investigate the relationship between chest injury and systemic neutrophil activation . antihuman monoclonal antibodies were purchased for analyzing neutrophil receptor expression by flowcytometry : fluorescein isothiocyanate ( fitc)-labeled igg1 isotype control ( clone mopc-21 , bd pharmingen , usa ) , alexa fluor 647-labeled igg1 isotype control ( clone mopc-21 , bd pharmingen , usa ) , r - phycoerythrin ( rpe)-labeled igg2a isotype control ( clone mrc ox-34 , serotec , germany ) , rpe - labeled igg1 anti-m ( cd11b ; clone 2lpm19c , dako , denmark ) , fitc - labeled igg1 anti - l - selectin ( cd62l ; clone dreg56 , bd pharmingen , usa ) , alexa fluor 647-labeled igg1 anti - fcriii ( cd16 ; clone 3g8 , bd pharmingen , usa ) , rpe - labeled igg2b anti - fcrii ( cd32 ; clone fli8.26 , bd pharmingen , usa ) , fitc - labeled igg2a anti - cxcr1 ( cd181a ; clone 42705 , r&d systems europe , uk ) , rpe - labeled igg2a anti - cxcr2 ( cd182b ; clone 48311 , r&d systems europe , uk ) , and fitc - labeled igg2a anti - c5ar ( cd88 ; clone p12/1 , serotec , germany ) . plasma samples were obtained at 3 , 9 and 24 h after injury and stored at 80c until further analysis . l - selectin expression was decreased at 9 h postinjury ( p = 0.002 ) and remained decreased until 24 h ( p = 0.012 ) ( fig . 1expression of l - selectin ( a ) , m ( cd11b ) ( b ) , cxcr1 ( c ) , cxcr2 ( d ) , and c5ar ( e ) on the neutrophil surface over time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) expression of l - selectin ( a ) , m ( cd11b ) ( b ) , cxcr1 ( c ) , cxcr2 ( d ) , and c5ar ( e ) on the neutrophil surface over time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) neutrophil activation is associated with reduced surface expression of cxcr1 , cxcr2 and c5ar [ 3133 ] . after chest injury , circulating neutrophils showed a temporary decline in the expression of chemokine receptors cxcr1 and cxcr2 and complement receptor c5ar ( fig . the expression of cxcr2 and c5ar was gradually restored during the first 24 h after injury , indicating a transient activation of circulating neutrophils . expression of fcrii was markedly decreased until 24 h after injury ( p < 0.010 ; fig . expression of the active form was slightly lower in trauma patients compared to control values , although this decline did not reach statistical significance . expression of fmlp - induced active fcrii , however , was significantly decreased until 9 h after injury ( p = 0.006 ) . expression of fcriii evidently dropped during the first 24 h after chest trauma ( p < 0.001 ) . 2intrinsic expression of fcrii ( a ) , active fcrii ( b ) , fmlp - induced expression of active fcrii ( c ) , and expression of fcriii ( d ) on the neutrophil surface during time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) intrinsic expression of fcrii ( a ) , active fcrii ( b ) , fmlp - induced expression of active fcrii ( c ) , and expression of fcriii ( d ) on the neutrophil surface during time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . 0.05,**p < 0.01 ) il-6 levels were significantly enhanced at 3 h postinjury compared to control values [ mean concentration of 44 15 versus 0 pg / ml ( p < 0.001 ) ] . il-6 levels further increased to a maximum mean concentration of 86 31 pg / ml ( p < 0.001 compared to control values ) 24 h after blunt chest injury ( fig . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . l - selectin expression was decreased at 9 h postinjury ( p = 0.002 ) and remained decreased until 24 h ( p = 0.012 ) ( fig . although neutrophil activation is typically characterized by m upregulation , expression of m declined at 9 h postinjury ( p = 0.020 ) to a minimum in this group of patients . 1expression of l - selectin ( a ) , m ( cd11b ) ( b ) , cxcr1 ( c ) , cxcr2 ( d ) , and c5ar ( e ) on the neutrophil surface over time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) expression of l - selectin ( a ) , m ( cd11b ) ( b ) , cxcr1 ( c ) , cxcr2 ( d ) , and c5ar ( e ) on the neutrophil surface over time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) l - selectin expression was decreased at 9 h postinjury ( p = 0.002 ) and remained decreased until 24 h ( p = 0.012 ) ( fig . although neutrophil activation is typically characterized by m upregulation , expression of m declined at 9 h postinjury ( p = 0.020 ) to a minimum in this group of patients . 1expression of l - selectin ( a ) , m ( cd11b ) ( b ) , cxcr1 ( c ) , cxcr2 ( d ) , and c5ar ( e ) on the neutrophil surface over time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) expression of l - selectin ( a ) , m ( cd11b ) ( b ) , cxcr1 ( c ) , cxcr2 ( d ) , and c5ar ( e ) on the neutrophil surface over time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) neutrophil activation is associated with reduced surface expression of cxcr1 , cxcr2 and c5ar [ 3133 ] . after chest injury , circulating neutrophils showed a temporary decline in the expression of chemokine receptors cxcr1 and cxcr2 and complement receptor c5ar ( fig . the expression of cxcr2 and c5ar was gradually restored during the first 24 h after injury , indicating a transient activation of circulating neutrophils . expression of fcrii was markedly decreased until 24 h after injury ( p < 0.010 ; fig . expression of the active form was slightly lower in trauma patients compared to control values , although this decline did not reach statistical significance . expression of fmlp - induced active fcrii , however , was significantly decreased until 9 h after injury ( p = 0.006 ) . expression of fcriii evidently dropped during the first 24 h after chest trauma ( p < 0.001 ) . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) intrinsic expression of fcrii ( a ) , active fcrii ( b ) , fmlp - induced expression of active fcrii ( c ) , and expression of fcriii ( d ) on the neutrophil surface during time . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * p < 0.05,**p < 0.01 ) il-6 levels were significantly enhanced at 3 h postinjury compared to control values [ mean concentration of 44 15 versus 0 pg / ml ( p < 0.001 ) ] . il-6 levels further increased to a maximum mean concentration of 86 31 pg / ml ( p < 0.001 compared to control values ) 24 h after blunt chest injury ( fig . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . open squares indicate control values from healthy controls ( n = 8) , whereas black squares represent patients ( n = 13 ) at 3 , 9 and 24 h postinjury . data are presented as mean se ( * * p < 0.01 ) in this study we show that blunt chest injury leads to a systemic activation of circulating neutrophils , characterized by the shedding of l - selectin and the downregulation of cxcr2 and c5ar . it furthermore shows that blunt chest injury is associated with the mobilization of young ( fcriii - low ) neutrophils and with a reduced responsiveness of circulating neutrophils to an inflammatory stimulus . despite vast local damage in the abovementioned cases , the innate immune response provoked was apparently not abundant enough to cause ards . the same study also described an enhanced inflammatory response , with significantly increased levels of circulating il-6 and il-8 plus significantly elevated multiple organ failure ( mof ) scores in multi - trauma patients with major lung contusions compared to multi - trauma patients with minor or no lung contusions . however , the exact role of lung contusion in the immune response and the occurrence of organ dysfunction was not completely clear in these severely injured patients . a clear difference in the median fmlp - induced expression of active fcrii , measured during the first 24 h after injury , was seen between patients with ali / ards ( low expression ) and patients with an uneventful course ( high expression ) . these findings support the hypothesis that isolated chest injury induces only a restricted activation of the systemic immune response . the increased number of circulating neutrophils after chest injury is most likely due to mobilization of young neutrophils , as displayed by a decrease in overall fcriii expression . presumably , the systemic innate immune response evoked by isolated chest injury is less extensive than that during infection or after aspiration . in this study we show that the systemic innate immune response caused by isolated chest injury is transient and short . although il-6 levels remained elevated until 24 h after injury , activation of circulating neutrophils was partially restored . however , this mild systemic response may be sufficient to enhance the innate immune response caused by a second hit such as concomitant tissue damage , fat emboli , or infection . we therefore suggest that lung damage alone is not likely to result in an ards , but a synergism between inflammation caused by lung injury and an additional stimulus caused by a second hit results in a markedly increased risk of developing ards . in this study we demonstrated that isolated blunt chest injury caused transient systemic activation of neutrophils together with a mobilization of young neutrophils into the peripheral circulation . in addition , only severe chest injury ( ais > 4 ) results in an increased number of circulating neutrophils . however , it seems that chest injury alone is not sufficient to cause ards in these cases ; a second hit may be needed .

the pig ( sus scrofa ) is the most relevant agricultural meat species as well as an important animal model for its numerous physiological and morphological similarities to the human . a parameter that is important for both aspects ( meat production and animal model ) is the level of fat deposition . for example , back fat thickness ( bft ) is a trait that affects ham and carcass values and , indirectly , correlates with production efficiency . for these reasons , breeding programs in most pig breeds and lines are designed to reduce bft and increase lean meat content . in a few pig lines , an excessive reduction of the level of bft could create problems to the meat processing industries as in the case of heavy pigs whose legs are cured for the production of dry - cured hams , and , for this reason , animals are selected to maintain an optimized fat thickness . this trait is also an interesting phenotype to consider the pig as a model for human obesity [ 4 , 5 ] that is one of the major health problems in both developed and developing countries . to understand the biological mechanisms affecting bft in pigs , we recently carried out several studies to elucidate the genetic factors involved in the definition of this trait and to obtain a systems biology comparative picture of human and pig obesity related traits . in a whole genome candidate gene approach , we reported that polymorphisms in genes already shown to affect fat deposition in humans and mice are associated with bft or correlated traits in commercial pigs and in the italian large white heavy pig breed [ 710 ] . in addition , a genome wide association ( gwa ) study which we carried out in the same breed using a selective genotyping approach and the illumina porcinesnp60 beadchip showed quite a large number of markers associated with bft ( each with a small effect that could not explain the whole genetic variability for this trait ) , with a limited overlap with other gwa studies that investigated the same or similar traits in other breeds and pig populations . this could be due to different experimental designs and incomplete power in the different studies as well as different linkage disequilibrium structures of the investigated populations that could not be captured completely by the genotyping tool ( illumina porcinesnp60 beadchip ) . taking advantage from the sequenced genome of the pig and its reference assembly ( sscrofa10.2 ) , it is now possible to use next generation sequencing ( ngs ) platforms to further investigate the level and extent of genetic variability in different breeds and populations ( i.e. , ) . the ion torrent technology is a cheap promising ngs platform that is based on a semiconductor detection of ph variation during the sequencing process that can be applied in different experimental approaches in which a medium - high throughput is needed . we already evaluated the ion torrent platform to analyse a mammalian genome by sequencing reduced representation libraries ( rrls ) obtained from rabbit genomic dna and identified thousands of new single nucleotide polymorphisms ( snps ) in this species . in this study , with the final aim to identify snps that could be useful to evaluate the peculiarities of the italian large white heavy pig breed and explain , at least in part , the missed genetic variability for the bft trait not completely captured by our previous association works , we tested the potential and limits of an experimental design in which we combined the ion torrent sequencing technology to sequence rrls . reduced representation libraries were obtained by enzymatically digest dna pools constructed from divergent italian large white pigs with extreme estimated breeding value ( ebv ) for bft . in addition , we used illumina porcinesnp60 beadchip genotyping data already generated from the same animals to obtain a comparative analysis and validation of the sequencing information . a subset of the italian large white pigs that were previously used in a gwa study , carried out to identify markers associated with bft ebv , were used to constitute two genomic dna pools . the selected animals were from two groups , each of 50 pigs , of two - generation unrelated gilts with extreme and divergent bft ebv ( 50 with the most negative bft ebv and 50 with the most positive bft ebv ) , selected among about 12,000 pigs individually performance - tested at the central test station of the national pig breeder association ( anas ) for the sib - testing evaluation of candidate boars within the national selection program of the italian large white breed [ 7 , 9 , 12 ] . average and standard deviation of bft ebv of the pigs in the negative and positive tails were 9.40 1.60 mm and + 8.00 5.95 mm , respectively . estimated breeding values for this trait were calculated by a blup - multiple trait animal model including the fixed factors of batch , age at the beginning of test , date of slaughtering , inbreeding coefficient , body weight at slaughter , and age at slaughter , besides the random factors of animal and litter . genomic dna was extracted from blood using the wizard genomic dna purification kit ( promega corporation , madison , wi , usa ) . extracted dna was quantified using a nanophotometer p-330 instrument ( implen gmbh , mnchen , germany ) and pooled at equimolar concentration to constitute two dna pools , one including dna from the 50 italian large white pigs with the lowest bft ebv and a second including dna from the 50 italian large white pigs with the highest bft ebv . the investigated animals were previously genotyped with the illumina porcinesnp60 beadchip ( illumina inc . , san diego , ca , usa ) , interrogating 62,163 snps . no filter was applied and all samples and genomic positions were retained for subsequent evaluation and comparison with sequencing data ( see below ) . ten micrograms of dna from each of the two pools were digested overnight with 50 u of haeiii restriction enzyme and the digested products were loaded in a 0.8% agarose gel . haeiii was selected as it did not produce visible patterns that could be ascribed to repetitive elements in the range of 500700 bp ( data not shown ) . dna fragments from this range obtained from haeiii digestion were purified from the agarose gel with the qiaquick gel extraction kit ( qiagen , hilden , germany ) according to the manufacturer instructions . obtained dna was used for library preparation and sequencing with the ion torrent pgm ( life technologies , carlsbad , ca , usa ) . sequencing of the two rrls was obtained using 200 ng of dna that was purified by agarose gel electrophoresis as described above , enzymatically sheared , end - repaired , and adapter - ligated using the ion xpress plus fragment library kit ( life technologies ) . obtained dna material was size - selected using the e - gel system ( invitrogen , carlsbad , ca , usa ) and bands corresponding to 100 bp of inserts were collected and quantified by qpcr using a steponeplus real - time pcr system ( life technologies ) . selected fragments were clonally amplified , purified , and sequenced using the ion one touch 100 template kit and the ion pgm sequencing kit with two ion 318 chips ( life technologies ) , for the two rrls . obtained sequencing reads were filtered and trimmed using the ion torrent suite v.2.2 ( life technologies ) which ( i ) eliminated polyclonal sequences and sequences of low quality and ( ii ) trimmed adapters and low quality 3-ends . then data were inspected with fastqc v.0.11.22 . sequenced reads were trimmed and filtered using prinseq lite v.0.20.4 as follows : ( i ) trimming at the 3-end up to 140 bp , ( ii ) trimming of the 5-end and 3-end for poly - a / t sequences > 5 , ( iii ) trimming the 5-end and 3-end up to reaching a base with a quality score > 20 , ( iv ) exclusion of reads having average quality < 20 , and ( v ) exclusion of reads shorter than 20 bp . after the pcr duplicates removing step , reads were merged , processed , and aligned on the sscrofa10.2 genome version using bwa v.0.7.7 . reads aligning in only one place of the genome and with mapping quality score ( qm ) > 20 were retained . snp calling was obtained using snape , setting divergence to 0.01 , prior nucleotide diversity ( ) of 0.001 , folded spectrum , and filtering by a posterior probability of segregation > 0.90 . snape input files ( pileup format ) were obtained using samtools v.0.1.4 [ 21 , 22 ] . snape filters were applied to consider only positions with minimum depth of 3x , to avoid indels ( as indel calling algorithm is not specific for pools ) . for each putative snp , we identified if it was already included in the dbsnp or if it was new using the ensembl biomart data mining tool , interrogating the ensembl variation 77 database ( october 2014 ) for sscrofa10.2 short variations and indels ( based on dbsnp build 140 ) . all the snps that did not match with those reported on dbsnps were also analyzed with the samtools mpileup function [ 21 , 22 ] . variant effect predictor ( vep ) tool ( http://www.ensembl.org/sus_scrofa/tools/vep ; ) was used to map gene positions and to predict the effect of each substitution . sift was used to evaluate if missense mutations could have deleterious effects on the translated proteins . in order to evaluate differences in allele frequency derived by the number of alternative reads between the two rrls , fisher 's exact test was computed for each alternative genomic position covered by a minimum depth of 3x . all the positions with pfisher < 0.05 were also visually inspected with igv ( integrative genomics viewer ) software . a total of 3,390,796 and 3,731,776 sequenced reads were obtained from the two rrls produced using the positive and negative bft ebv dna pools , respectively ( table 1 ) . after cleaning the datasets for duplicated reads , the number of unique reads was 2,692,605 and 2,885,815 , respectively ( table 1 ) . a total of 1,449,838 ( positive bft ebv rrl ) and 1,476,125 ( negative bft ebv rrl ) reads were mapped with high confidence to the sscrofa10.2 assembly of the pig genome . the merged dataset had an average read depth ( rd ) of 1.28x ( range from 1 to 426x ) . table s1 ( see supplementary material available online at http://dx.doi.org/10.1155/2015/950737 ) reports the number of reads and nucleotides mapped on the different pig chromosomes . sequence data obtained from the two rrls have been submitted to the european nucleotide archive database ( embl , http://www.ebi.ac.uk/ena/ ) and are indexed with the accession number erp009239 . using sequencing data , a total of 39,165 putative snps were called with high confidence by snape . of these snps 24,560 ( 62.5% ) were polymorphic carrying two alleles within the sequenced reads and 14,605 ( 37.58% ) were monomorphic for an alternative form than that of the reference genome . we detected 9,680 new putative snps not yet reported in dbsnp ( 24.72% of the called snps ) while the major part of identified variations ( 29,485 ; 75.28% ) was already present in dbsnp . the transition / transversion ratio considering all the detected snps is 2.08 , comparable to other mammalian genomes . in addition , 6,324 of the newly detected snps were also detected using samtools and 3,964 of these snps had score 20 . most of the snps were in intergenic ( 56.1% ) or in intronic ( 28.9% ) regions . the list of snps included in transcribed regions is reported in table s2 . among the putative snps predicted in coding regions , 217 were synonymous mutations , 159 were missense mutations , two were stop - gained mutations ( in the novel gene enssscg00000028324 and in the nut family member 2d gene , known as nutm2d ) , and one was a stop - lost variation ( in the putative pleckstrin and sec7 domain containing 2 gene ; ps2d ) . among the missense mutations , several genes with deleterious missense mutations ( e.g. , nadh dehydrogenase ( ubiquinone ) 1 , subcomplex unknown , 1 , 6kda ( ndufc1 ) ; parathyroid hormone 1 receptor ( pth1r ) ; glycerol-3-phosphate acyltransferase 2 , mitochondrial ( gpat2 ) ; and several olfactory receptor like genes ) play important roles in different biochemical and physiological cellular mechanisms . to validate some of the called snps we took advantage from the illumina porcinesnp60 beadchip genotyping data obtained on the same animals used to construct the two rrls . considering snp positions covered by a minimum of three reads , 661 out of 62,163 snps of the chip ( 1.1% ) were identified from the 13,596,939 sequenced positions ( 0.45% of the porcine genome ) . snape analysis over these positions reported that ( i ) 3 positions were discarded and 8 had read depth < 3 ( for further features of snape in addition to the general criteria adopted ) , ( ii ) 257 were identified as snps ( 152 polymorphic snps carrying two alleles while 105 snps were monomorphic for an alternative form from that of the reference genome ) , and ( iii ) 375 positions showed only the sequence of the reference genome . of the overlapping 653 positions ( 661 8 = 653 ) , ( i ) for 28 of them the chip genotype data of the individual pigs were not possible to retrieve ( probably due to problems in the design of the chip probes that could prevent the genotyping ) and ( ii ) for 63 dna positions having all individuals homozygous for only one genotype 59 of these base positions matched with the genotype inferred by ngs , whereas 2 were called as heterozygous and 2 were called as homozygous for a noncomplementary nucleotide by sequencing data ( table s3 ) . if we go into more details for the 28 snps that failed to report reliable genotyping data from the porcinesnp60 beadchip , for 12 out of 28 both alleles were present in the ngs reads ; 15 out of 28 showed only one allele and one was an erroneous snp . in addition to these overlaps between ngs sequencing and genotyping data , we wanted to evaluate if the estimated allele frequencies derived by ngs in rrls obtained from dna pools could match the true allele frequencies at the same positions obtained by using the porcinesnp60 beadchip . starting from 559 snps ( derived by the subsequent filtering steps of the 661 snps reported above ) , 262 ( 145 called snps by snape ) had the same type of substitution . excluding the transversions gc cg and at ta , for each one of the remaining 258 snps , we compared the minor allele frequency ( maf ) of the genotyping data against the frequency of the same allele derived by the sequencing . as expected , a low correlation from these two data was observed when considering all 258 snps due to the low coverage depth ( 3x ) that was not enough for a reliable allele frequency estimation from ngs data . this value increased up to 3 times setting a coverage depth equal to or higher than the double of the minimum coverage depth ( 6 ) . when adding data coming from monomorphic allele , correlation increased up to 0.70 . these data indicate that even using a coverage depth 6 the maf of these snps can be estimated with good approximation . for each of the two initial pileups we filtered out genomic positions having depth < 3x and then we used snape to extract the allele frequency of each genomic position taking the advantage of the filters implemented in it . polymorphic positions were compared among the 237,969 positions that were in common between the two rrls ( table 1 ) . among these nucleotides , 67 genomic positions ( filtered to 54 when tested by snape and inspected with igv ) showed a pfisher < 0.05 comparing alternative reads observed in the two rrls generated from dna of pigs with extreme and divergent bft ebv ( table s4 ) . these snps were located in several autosomal chromosomes ( ssc1 , ssc3 , ssc6 , ssc8 , ssc9 , ssc10 , ssc12 , ssc15 , ssc16 , ssc17 , and ssc18 ) . these variants ( only 12 of which already deposited in dbsnp ) were localized as follows : 63% were intergenic variants , 21% were in introns , 11% were upstream gene variants , and 5% were downstream gene variants . intronic variants were located in four genes of which only two were annotated with a known function : ( 1 ) dysbindin ( dystrobrevin binding protein 1 ) domain containing 1 ( dbndd1 ) ; ( 2 ) phosphatidic acid phosphatase type 2a ( ppap2a ) . in order to evaluate if the 54 snps that showed differences in number of alternative reads between the two rrls were located in chromosome regions associated with bft in italian large white pigs ( listed in table s4 ) , we compared their positions on the basis of our previous gwa study carried out in the same breed . we considered a window spanning 0.5 mbp from each marker having nominal p value < 0.05 in our previous study . the top pfisher for each of the identified regions is reported in table 4 ( the complete list is reported in table s5 ) . the most significant marker ( m1ga0008302 ; p = 1.65e 06 ) is located 72,572 bp downstream snp ssc6:859837 ( pfisher = 0.038 ) and 85,796 bp downstream the 6th top snp ssc6:873061 ( pfisher = 0.012 ) obtained from the list of the 54 snps . in this region there is the acyl - coa synthetase family member 3 ( acsf3 ) gene that belongs to a family of enzymes that activate fatty acids . in the same region we previously showed that other markers ( m1ga0008329 , ssc6:996248 , p = 9.35e 05 , and m1ga0008318 , ssc6:945991 , p = 4.41e 04 ) were associated with bft in the same breed . within table 4 , the second most significant marker as reported previously ( alga0000014 , p = 1.74e 05 ) is located close to the snp ssc1:68514 ( pfisher = 0.029 ) identified in the present study ( table 4 ) . in this region there is another marker associated with bft in the previous gwa study ( alga0000009 , p = 2.75e 03 ; ) . an interesting gene located in this part of the pig genome , delta - like 1 ( drosophila ) ( dll1 ) , seems associated to type 1 diabetes in humans . for marker drga009307 ( ssc9:17138159 , p = 8.66e 04 ) there is no annotated gene in a 500 kbp region . dias0000309 ( ssc12:48865200 , p = 9.96e 04 ) is near the active breakpoint cluster - related ( abr ) gene and enssscg00000017808 gene , orthologous of the acyl - coa - binding protein ( dbi ) gene . abr gene is annotated with two interesting gene ontology ( go ) terms : phospholipid binding and brain development . dbi gene functions as intracellular carrier of acyl - coa esters and it seems that it could act as a neuropeptide modulating the action of the gaba receptor . it is annotated with the go terms : long - chain fatty acyl - coa binding , transport , phosphatidylcholine acyl - chain remodeling , and triglyceride metabolic process that might suggest a potential role in fat metabolism and deposition . next generation sequencing is changing the way to identify markers associated with production traits in livestock species . several applications and strategies have been designed mainly using illumina platforms ( i.e. , ) . to our knowledge , this study applied for the first time the ion torrent technology to identify dna polymorphisms in the pig genome . the experimental design was quite simple as , at this stage , we wanted to test this ngs technology to identify markers that could be useful for subsequent association studies in the italian large white pig breed . the identification of polymorphisms was based on the construction and sequencing of two rrls generated from dna pools of pigs with extreme and divergent bft ebv . this approach was tested to set up a strategy for the identification of polymorphisms at a reduced fraction of the cost required for individual sequencing . in this way , we could also identify variants that might be enriched in one pool compared to the other one . to call snps we used snape that is a software package that implemented a bayesian approach for snp identification and maf estimation in sequenced pools . the validation of identified snps was obtained by comparing the genotyping data generated with the illumina porcinesnp60 beadchip on the same animals . as we sequenced dna in pools and genotyping data were obtained on individual animals , we evaluated how allele frequency correlated between the two approaches varying the depth of sequencing . this approach was able to define an interesting procedure to validate snps identified from dna pools . reduced representation libraries were generated as a simple strategy to reduce the complexity of mammalian genomes and to obtain information from a small part of it that can be sampled after restriction fragment digestion . several studies have already applied this strategy in farm animals for snp discovery [ 16 , 3032 ] . for example , in pigs , wiedmann et al . and ramos et al . sequenced rrls for the identification of snps that were used to construct the illumina porcinesnp60 beadchip genotyping platform . in our study , we identified about 40k snps in the pig genome . this is a quite large number of snps , considering the limited throughput of the benchtop ion torrent technology ( compared to illumina platforms ) and the stringent criteria that we used to call snps . as the technology is prone to errors in case of homopolymeric regions , indels were not considered in this study . that means that we could probably have discovered other short variants but we did not consider them to guarantee a high quality of the called polymorphisms . in addition , other bioinformatics tools should be developed to obtain a reliable maf estimation of indels from sequencing data generated from dna pools . among the 159 snps causing missense mutations , 37 these polymorphisms will be prioritized to evaluate their association with several production traits together with snps whose alleles were differentially enriched in the two rrls ( table 4 , table s4 , and table s5 ) . the identification of these latter snps was based on allele frequency generated by mapping alternative reads in the two extreme groups of pigs with divergent bft ebv . the low coverage of many snp positions in both rrls limited the possibility to identify markers associated with this trait . this problem is also due to the incomplete overlapping of read coverage between the two rrls . however , a comparative analysis of the nominally significant snps with our previous gwa study for bft obtained using the same animals analyzed in this study indirectly supported , to some extent , the identified association results . some of these markers were located close to genes already shown in humans and mouse to be involved in obesity related phenotypes and pathologies suggesting a potential effect of these polymorphisms on bft and fat deposition in italian large white pigs . these indications should be supported by association studies with fat deposition traits in the investigated breed or in other pig populations . several methodological approaches were tested in this study for the first time : ( i ) partial sequencing obtained with ion torrent technology of the pig genome from dna pools by using rrls ; ( ii ) the application of snp calling and maf estimation on ion torrent low coverage sequencing data from dna pools ; ( iii ) the validation of snp called in dna pools using individual genotyping data from the same animals of the pools ; ( iv ) the possibility to identify enriched alleles in the two sequenced rrls representing two extremes for important phenotypes ( bft ) . all these approaches were implemented in a case study that tried to identify additional markers associated with bft in the italian large white pig breed . the purpose was to set up a strategy that could reduce as much as possible the sequencing cost and that could produce data useful to identify novel markers for the targeted trait . association studies will be carried out to evaluate the effects of the 54 selected markers . ion torrent can be successfully applied for snp discovery even if its limited throughput reduced the possibilities to obtain reliable allele frequencies in the two dna pools . other reductionist approaches , like genotyping by sequencing or genotyping by genome reducing and sequencing [ 35 , 36 ] , might be used to identify and validate snps associated with bft .

the aim of this study was to identify single nucleotide polymorphisms ( snps ) that could be associated with back fat thickness ( bft ) in pigs . to achieve this goal , we evaluated the potential and limits of an experimental design that combined several methodologies . dna samples from two groups of italian large white pigs with divergent estimating breeding value ( ebv ) for bft were separately pooled and sequenced , after preparation of reduced representation libraries ( rrls ) , on the ion torrent technology . taking advantage from snape for snps calling in sequenced dna pools , 39,165 snps were identified ; 1/4 of them were novel variants not reported in dbsnp . combining sequencing data with illumina porcinesnp60 beadchip genotyping results on the same animals , 661 genomic positions overlapped with a good approximation of minor allele frequency estimation . a total of 54 snps showing enriched alleles in one or in the other rrls might be potential markers associated with bft . some of these snps were close to genes involved in obesity related phenotypes .

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

for example , back fat thickness ( bft ) is a trait that affects ham and carcass values and , indirectly , correlates with production efficiency . in a few pig lines , an excessive reduction of the level of bft could create problems to the meat processing industries as in the case of heavy pigs whose legs are cured for the production of dry - cured hams , and , for this reason , animals are selected to maintain an optimized fat thickness . to understand the biological mechanisms affecting bft in pigs , we recently carried out several studies to elucidate the genetic factors involved in the definition of this trait and to obtain a systems biology comparative picture of human and pig obesity related traits . in a whole genome candidate gene approach , we reported that polymorphisms in genes already shown to affect fat deposition in humans and mice are associated with bft or correlated traits in commercial pigs and in the italian large white heavy pig breed [ 710 ] . in addition , a genome wide association ( gwa ) study which we carried out in the same breed using a selective genotyping approach and the illumina porcinesnp60 beadchip showed quite a large number of markers associated with bft ( each with a small effect that could not explain the whole genetic variability for this trait ) , with a limited overlap with other gwa studies that investigated the same or similar traits in other breeds and pig populations . this could be due to different experimental designs and incomplete power in the different studies as well as different linkage disequilibrium structures of the investigated populations that could not be captured completely by the genotyping tool ( illumina porcinesnp60 beadchip ) . taking advantage from the sequenced genome of the pig and its reference assembly ( sscrofa10.2 ) , it is now possible to use next generation sequencing ( ngs ) platforms to further investigate the level and extent of genetic variability in different breeds and populations ( i.e. the ion torrent technology is a cheap promising ngs platform that is based on a semiconductor detection of ph variation during the sequencing process that can be applied in different experimental approaches in which a medium - high throughput is needed . we already evaluated the ion torrent platform to analyse a mammalian genome by sequencing reduced representation libraries ( rrls ) obtained from rabbit genomic dna and identified thousands of new single nucleotide polymorphisms ( snps ) in this species . in this study , with the final aim to identify snps that could be useful to evaluate the peculiarities of the italian large white heavy pig breed and explain , at least in part , the missed genetic variability for the bft trait not completely captured by our previous association works , we tested the potential and limits of an experimental design in which we combined the ion torrent sequencing technology to sequence rrls . reduced representation libraries were obtained by enzymatically digest dna pools constructed from divergent italian large white pigs with extreme estimated breeding value ( ebv ) for bft . in addition , we used illumina porcinesnp60 beadchip genotyping data already generated from the same animals to obtain a comparative analysis and validation of the sequencing information . a subset of the italian large white pigs that were previously used in a gwa study , carried out to identify markers associated with bft ebv , were used to constitute two genomic dna pools . the selected animals were from two groups , each of 50 pigs , of two - generation unrelated gilts with extreme and divergent bft ebv ( 50 with the most negative bft ebv and 50 with the most positive bft ebv ) , selected among about 12,000 pigs individually performance - tested at the central test station of the national pig breeder association ( anas ) for the sib - testing evaluation of candidate boars within the national selection program of the italian large white breed [ 7 , 9 , 12 ] . average and standard deviation of bft ebv of the pigs in the negative and positive tails were 9.40 1.60 mm and + 8.00 5.95 mm , respectively . extracted dna was quantified using a nanophotometer p-330 instrument ( implen gmbh , mnchen , germany ) and pooled at equimolar concentration to constitute two dna pools , one including dna from the 50 italian large white pigs with the lowest bft ebv and a second including dna from the 50 italian large white pigs with the highest bft ebv . the investigated animals were previously genotyped with the illumina porcinesnp60 beadchip ( illumina inc . , san diego , ca , usa ) , interrogating 62,163 snps . no filter was applied and all samples and genomic positions were retained for subsequent evaluation and comparison with sequencing data ( see below ) . haeiii was selected as it did not produce visible patterns that could be ascribed to repetitive elements in the range of 500700 bp ( data not shown ) . obtained dna was used for library preparation and sequencing with the ion torrent pgm ( life technologies , carlsbad , ca , usa ) . sequencing of the two rrls was obtained using 200 ng of dna that was purified by agarose gel electrophoresis as described above , enzymatically sheared , end - repaired , and adapter - ligated using the ion xpress plus fragment library kit ( life technologies ) . selected fragments were clonally amplified , purified , and sequenced using the ion one touch 100 template kit and the ion pgm sequencing kit with two ion 318 chips ( life technologies ) , for the two rrls . obtained sequencing reads were filtered and trimmed using the ion torrent suite v.2.2 ( life technologies ) which ( i ) eliminated polyclonal sequences and sequences of low quality and ( ii ) trimmed adapters and low quality 3-ends . sequenced reads were trimmed and filtered using prinseq lite v.0.20.4 as follows : ( i ) trimming at the 3-end up to 140 bp , ( ii ) trimming of the 5-end and 3-end for poly - a / t sequences > 5 , ( iii ) trimming the 5-end and 3-end up to reaching a base with a quality score > 20 , ( iv ) exclusion of reads having average quality < 20 , and ( v ) exclusion of reads shorter than 20 bp . after the pcr duplicates removing step , reads were merged , processed , and aligned on the sscrofa10.2 genome version using bwa v.0.7.7 . for each putative snp , we identified if it was already included in the dbsnp or if it was new using the ensembl biomart data mining tool , interrogating the ensembl variation 77 database ( october 2014 ) for sscrofa10.2 short variations and indels ( based on dbsnp build 140 ) . sift was used to evaluate if missense mutations could have deleterious effects on the translated proteins . in order to evaluate differences in allele frequency derived by the number of alternative reads between the two rrls , fisher 's exact test was computed for each alternative genomic position covered by a minimum depth of 3x . a total of 3,390,796 and 3,731,776 sequenced reads were obtained from the two rrls produced using the positive and negative bft ebv dna pools , respectively ( table 1 ) . a total of 1,449,838 ( positive bft ebv rrl ) and 1,476,125 ( negative bft ebv rrl ) reads were mapped with high confidence to the sscrofa10.2 assembly of the pig genome . table s1 ( see supplementary material available online at http://dx.doi.org/10.1155/2015/950737 ) reports the number of reads and nucleotides mapped on the different pig chromosomes . using sequencing data , a total of 39,165 putative snps were called with high confidence by snape . of these snps 24,560 ( 62.5% ) were polymorphic carrying two alleles within the sequenced reads and 14,605 ( 37.58% ) were monomorphic for an alternative form than that of the reference genome . we detected 9,680 new putative snps not yet reported in dbsnp ( 24.72% of the called snps ) while the major part of identified variations ( 29,485 ; 75.28% ) was already present in dbsnp . in addition , 6,324 of the newly detected snps were also detected using samtools and 3,964 of these snps had score 20 . most of the snps were in intergenic ( 56.1% ) or in intronic ( 28.9% ) regions . the list of snps included in transcribed regions is reported in table s2 . among the putative snps predicted in coding regions , 217 were synonymous mutations , 159 were missense mutations , two were stop - gained mutations ( in the novel gene enssscg00000028324 and in the nut family member 2d gene , known as nutm2d ) , and one was a stop - lost variation ( in the putative pleckstrin and sec7 domain containing 2 gene ; ps2d ) . to validate some of the called snps we took advantage from the illumina porcinesnp60 beadchip genotyping data obtained on the same animals used to construct the two rrls . considering snp positions covered by a minimum of three reads , 661 out of 62,163 snps of the chip ( 1.1% ) were identified from the 13,596,939 sequenced positions ( 0.45% of the porcine genome ) . snape analysis over these positions reported that ( i ) 3 positions were discarded and 8 had read depth < 3 ( for further features of snape in addition to the general criteria adopted ) , ( ii ) 257 were identified as snps ( 152 polymorphic snps carrying two alleles while 105 snps were monomorphic for an alternative form from that of the reference genome ) , and ( iii ) 375 positions showed only the sequence of the reference genome . of the overlapping 653 positions ( 661 8 = 653 ) , ( i ) for 28 of them the chip genotype data of the individual pigs were not possible to retrieve ( probably due to problems in the design of the chip probes that could prevent the genotyping ) and ( ii ) for 63 dna positions having all individuals homozygous for only one genotype 59 of these base positions matched with the genotype inferred by ngs , whereas 2 were called as heterozygous and 2 were called as homozygous for a noncomplementary nucleotide by sequencing data ( table s3 ) . if we go into more details for the 28 snps that failed to report reliable genotyping data from the porcinesnp60 beadchip , for 12 out of 28 both alleles were present in the ngs reads ; 15 out of 28 showed only one allele and one was an erroneous snp . in addition to these overlaps between ngs sequencing and genotyping data , we wanted to evaluate if the estimated allele frequencies derived by ngs in rrls obtained from dna pools could match the true allele frequencies at the same positions obtained by using the porcinesnp60 beadchip . starting from 559 snps ( derived by the subsequent filtering steps of the 661 snps reported above ) , 262 ( 145 called snps by snape ) had the same type of substitution . excluding the transversions gc cg and at ta , for each one of the remaining 258 snps , we compared the minor allele frequency ( maf ) of the genotyping data against the frequency of the same allele derived by the sequencing . as expected , a low correlation from these two data was observed when considering all 258 snps due to the low coverage depth ( 3x ) that was not enough for a reliable allele frequency estimation from ngs data . these data indicate that even using a coverage depth 6 the maf of these snps can be estimated with good approximation . for each of the two initial pileups we filtered out genomic positions having depth < 3x and then we used snape to extract the allele frequency of each genomic position taking the advantage of the filters implemented in it . among these nucleotides , 67 genomic positions ( filtered to 54 when tested by snape and inspected with igv ) showed a pfisher < 0.05 comparing alternative reads observed in the two rrls generated from dna of pigs with extreme and divergent bft ebv ( table s4 ) . these snps were located in several autosomal chromosomes ( ssc1 , ssc3 , ssc6 , ssc8 , ssc9 , ssc10 , ssc12 , ssc15 , ssc16 , ssc17 , and ssc18 ) . these variants ( only 12 of which already deposited in dbsnp ) were localized as follows : 63% were intergenic variants , 21% were in introns , 11% were upstream gene variants , and 5% were downstream gene variants . intronic variants were located in four genes of which only two were annotated with a known function : ( 1 ) dysbindin ( dystrobrevin binding protein 1 ) domain containing 1 ( dbndd1 ) ; ( 2 ) phosphatidic acid phosphatase type 2a ( ppap2a ) . in order to evaluate if the 54 snps that showed differences in number of alternative reads between the two rrls were located in chromosome regions associated with bft in italian large white pigs ( listed in table s4 ) , we compared their positions on the basis of our previous gwa study carried out in the same breed . the top pfisher for each of the identified regions is reported in table 4 ( the complete list is reported in table s5 ) . the most significant marker ( m1ga0008302 ; p = 1.65e 06 ) is located 72,572 bp downstream snp ssc6:859837 ( pfisher = 0.038 ) and 85,796 bp downstream the 6th top snp ssc6:873061 ( pfisher = 0.012 ) obtained from the list of the 54 snps . in the same region we previously showed that other markers ( m1ga0008329 , ssc6:996248 , p = 9.35e 05 , and m1ga0008318 , ssc6:945991 , p = 4.41e 04 ) were associated with bft in the same breed . within table 4 , the second most significant marker as reported previously ( alga0000014 , p = 1.74e 05 ) is located close to the snp ssc1:68514 ( pfisher = 0.029 ) identified in the present study ( table 4 ) . in this region there is another marker associated with bft in the previous gwa study ( alga0000009 , p = 2.75e 03 ; ) . an interesting gene located in this part of the pig genome , delta - like 1 ( drosophila ) ( dll1 ) , seems associated to type 1 diabetes in humans . next generation sequencing is changing the way to identify markers associated with production traits in livestock species . to our knowledge , this study applied for the first time the ion torrent technology to identify dna polymorphisms in the pig genome . the experimental design was quite simple as , at this stage , we wanted to test this ngs technology to identify markers that could be useful for subsequent association studies in the italian large white pig breed . the identification of polymorphisms was based on the construction and sequencing of two rrls generated from dna pools of pigs with extreme and divergent bft ebv . in this way , we could also identify variants that might be enriched in one pool compared to the other one . to call snps we used snape that is a software package that implemented a bayesian approach for snp identification and maf estimation in sequenced pools . the validation of identified snps was obtained by comparing the genotyping data generated with the illumina porcinesnp60 beadchip on the same animals . as we sequenced dna in pools and genotyping data were obtained on individual animals , we evaluated how allele frequency correlated between the two approaches varying the depth of sequencing . reduced representation libraries were generated as a simple strategy to reduce the complexity of mammalian genomes and to obtain information from a small part of it that can be sampled after restriction fragment digestion . for example , in pigs , wiedmann et al . sequenced rrls for the identification of snps that were used to construct the illumina porcinesnp60 beadchip genotyping platform . in our study , we identified about 40k snps in the pig genome . this is a quite large number of snps , considering the limited throughput of the benchtop ion torrent technology ( compared to illumina platforms ) and the stringent criteria that we used to call snps . as the technology is prone to errors in case of homopolymeric regions , indels were not considered in this study . in addition , other bioinformatics tools should be developed to obtain a reliable maf estimation of indels from sequencing data generated from dna pools . among the 159 snps causing missense mutations , 37 these polymorphisms will be prioritized to evaluate their association with several production traits together with snps whose alleles were differentially enriched in the two rrls ( table 4 , table s4 , and table s5 ) . the identification of these latter snps was based on allele frequency generated by mapping alternative reads in the two extreme groups of pigs with divergent bft ebv . the low coverage of many snp positions in both rrls limited the possibility to identify markers associated with this trait . however , a comparative analysis of the nominally significant snps with our previous gwa study for bft obtained using the same animals analyzed in this study indirectly supported , to some extent , the identified association results . some of these markers were located close to genes already shown in humans and mouse to be involved in obesity related phenotypes and pathologies suggesting a potential effect of these polymorphisms on bft and fat deposition in italian large white pigs . these indications should be supported by association studies with fat deposition traits in the investigated breed or in other pig populations . several methodological approaches were tested in this study for the first time : ( i ) partial sequencing obtained with ion torrent technology of the pig genome from dna pools by using rrls ; ( ii ) the application of snp calling and maf estimation on ion torrent low coverage sequencing data from dna pools ; ( iii ) the validation of snp called in dna pools using individual genotyping data from the same animals of the pools ; ( iv ) the possibility to identify enriched alleles in the two sequenced rrls representing two extremes for important phenotypes ( bft ) . all these approaches were implemented in a case study that tried to identify additional markers associated with bft in the italian large white pig breed . the purpose was to set up a strategy that could reduce as much as possible the sequencing cost and that could produce data useful to identify novel markers for the targeted trait . ion torrent can be successfully applied for snp discovery even if its limited throughput reduced the possibilities to obtain reliable allele frequencies in the two dna pools . other reductionist approaches , like genotyping by sequencing or genotyping by genome reducing and sequencing [ 35 , 36 ] , might be used to identify and validate snps associated with bft .

mass spectrometry ( ms ) is a powerful tool to assess the relative abundance of proteins among biological samples . numerous methodologies now support relative quantification measurements , providing a routine means to analyze protein expression patterns and post - translational modification states as a function of biological perturbation . one of the most popular methods for relative quantification through ms is stable isotope labeling of proteins in samples prior to analysis . labeling can be achieved by the application of combinatorial heavy isotopologues of c , h , n , and o and can be introduced in proteins either by metabolic means or through chemical derivatization processes . in vivo metabolic labeling approaches include techniques such as stable isotope labeling in mammals ( silam ) , stable isotope labeling by amino acids in cell culture ( silac ) , and neucode ( neutron encoding ) silac . the in vitro chemical derivatization processes include techniques such as isotope - coded affinity tags ( icat ) , dimethyl labeling , isobaric mass tags , and others . with the exception of isobaric mass tags , stable isotope derivatization methods introduce a small mass difference to identical peptides from two or more samples so that they can be distinguished in the ms1 spectrum . the relative - abundance ratio of peptides is experimentally measured by comparing heavy / light peptide pairs , and then protein levels are inferred from statistical evaluation of the peptide ratios . isobaric tags , on the other hand , use a different concept for peptide quantification . in isobaric labeling - based quantification , each sample is derivatized with a different isotopic variant of an isobaric mass tag from a set , and then the samples are pooled and analyzed simultaneously in ms . since the tags are isobaric , peptides labeled with isotopic variants of the tag appear as a single composite peak at the same m / z value in an ms1 scan with identical liquid chromatography ( lc ) retention time . the fragmentation of the modified precursor ion during ms / ms event generates two types of product ions : ( a ) reporter ion peaks and ( b ) peptide fragment ion peaks . the quantification is accomplished by directly correlating the relative intensity of reporter ions to that of the peptide selected for ms / ms fragmentation . the fragment ion peaks observed at higher m / z are specific for peptide amino acid sequence and are used for peptide identifications , which are eventually assigned to the proteins that they represent . since every tryptic peptide can be labeled in an isobaric labeling method , more than one peptide representing the same protein may be identified , thereby increasing the confidence in both the identification and quantification of the protein . this technology has proved to be successful in numerous experimental contexts for comparative analysis upon perturbation . the protocol involves extraction of proteins from cells or tissues followed by reduction , alkylation , and digestion . in the case of tmt 6-plex , up to six samples can be labeled with the six isobaric tags of the reagent . ( b ) in an ms1 scan , same - sequence peptides from the different samples appear as a single unresolved additive precursor ion . following fragmentation of the precursor ion during ms / ms , the six reporter ions appear as distinct masses between m / z 126131 , and the remainder of the sequence - informative b- and y - ions remains as additive isobaric signals . the reporter ion intensity indicates the relative amount of peptide in the mixture that was labeled with the corresponding reagent . isobaric mass tags include families of stable isotope chemicals that are used for labeling of peptides . they generate relative quantitative information in an isobaric labeling - based quantification strategy . isobaric mass tags have identical overall mass but vary in terms of the distribution of heavy isotopes around their structure . the most common isobaric tag is amine - reactive , but tags that react with cysteine residues and carbonyl groups in proteins are also available . the amine specificity of the amine - reactive isobaric mass tags makes most peptides in a sample amenable to this labeling strategy . the tags employ n - hydroxysuccinimide ( nhs ) chemistry , and the structure consists of three functional groups : an amine - reactive group and an isotopic reporter group ( n - methylpiperazine ) linked by an isotopic balancer group ( carbonyl ) for the normalization of the total mass of the tags . the amine - reactive , nhs - ester - activated group reacts with n - terminal amine groups and -amine groups of lysine residues to attach the tags to the peptides . the labeling is efficient for all peptides regardless of protein sequence or proteolytic enzyme specificity . the labeling does not occur , however , if the primary amino groups are modified , such as when n - terminal glutamine or glutamic acid forms a ring ( pyro - glutamic acid ) or if the group is acetylated . the nhs - based isobaric tags may lead to acylation of side chain hydroxyl group of serine , threonine , and tyrosine residues under reaction conditions normally employed for the acylation of primary amines . for successful quantification , labeling should be specific to the targeted residues ( n - terminal amine and lysyl -amine groups in a peptide ) and should proceed to completion . reversal of peptide o - acylation reactions can be achieved by treatment with hydroxylamine that has no disruptive effect on acyl modifications on primary amines . the mass normalization group balances the mass difference among the reporter ion groups so that different isotopic variants of the tag have the same mass . the overall mass of reporter and balance components of the molecule are kept constant using differential isotopic enrichment with c , n , and o atoms . the relative intensities of the reporter ion are used to derive quantitative information on the labeled peptides between the samples . figure 2 shows chemical structure of commercially available isobaric mass tags : tandem mass tag ( tmt ) and isobaric tags for relative and absolute quantification ( itraq ) . the complete molecule consists of a reporter group ( based on n - methylpiperazine ) , a mass balance group ( carbonyl ) , and a peptide - reactive group ( nhs ester ) . the overall mass of the reporter and balance components of the molecule are kept constant using differential isotopic enrichment with c , n , and o atoms . the reporter group ranges in mass from m / z 114117 , whereas the balance group ranges in mass from 28 to 31 da , such that the combined mass remains constant ( 145 da ) for each of the four reagents of the itraq 4-plex set . ( ii ) the tag reacts with peptide n - terminus or -amino group of lysine to form an amide linkage that fragments in a similar fashion to that of backbone peptide bonds when subjected to cid . following fragmentation of the tag amide bond , the balance ( carbonyl ) moiety is lost as neutral loss , whereas charge is retained by the reporter group . the number in parentheses in the table indicates the number of enriched centers in each section of the molecule . ( b ) chemical structure of a generic tmt reagent showing the three functional groups : an amine - reactive group that labels the n - terminus and -amine group of lysine in peptides , a mass normalization ( balance ) group that balances mass differences from individual reporter ions to ensure the same overall mass of the reagents , and a reporter group that provides the abundance of a peptide upon ms / ms in individual samples being mixed . the blue dashed lines indicate a cleavable linker that enables the release of the reporter ion from the whole tag upon ms / ms . the tmt reagent family consists of tmtzero , tmtduplex , tmt 6-plex , and tmt 10-plex sets , and each of them is based on the same chemical structure . the application of isobaric tags for simultaneous determination of both the identity and relative abundance of peptide pairs was first demonstrated by thompson et al . in 2003 . they synthesized peptides containing a tandem mass tag and showed that this strategy could be used to obtain relative quantification in ms / ms experiment . a year later , ross et al . published a similar approach using the itraq approach . in this study , they demonstrated for the first time the application of isobaric mass tags with 4-fold multiplexing to identify global protein expression trends in a set of isogenic yeast strains . an 8-plex series of itraq reagent performs similarly and increases throughput of analyses by a factor of 2 when compared to that of the 4-plex approach . a few year later , dayon et al . showed the increased multiplexing capability of tmt tags and demonstrated its application by using 6-plex tmt reagents in relative quantification of standard protein mixtures at various concentrations . in this study , tmt 6-plex was also used to assess the differential protein abundance in post - mortem cerebrospinal fluid samples after brain injury vs antemortem samples . isobaric reagents are commercially available through vendors such as ab sciex ( framingham , ma , usa ) and thermo scientific ( rockford , il , usa ) . the itraq reagents available from ab sciex are set of 4-plex and 8-plex mass tags that can be used to label and derive quantitative information on up to four and eight different biological samples simultaneously . the 4-plex itraq reagents have reporter ion masses at m / z 114117 and a corresponding balancer group added to accommodate the extra isotopes has masses of 2831 da such that they sum to about 145 da . the 8-plex reagents have reporter ion masses at m / z 113119 and 121 with a balance group ranging from 2431 da . mass 120 is omitted in itraq 8-plex to avoid contamination from phenylalanine immonium ion ( m / z 120.08 ) . thermo scientific tmt reagents , available as tmtzero , tmt duplex , tmt 6-plex , and tmt 10-plex , share an identical structure with each other but contain different numbers and combinations of c and n isotopes in the mass reporter region . the identical structure of tmt reagents facilitates efficient transition from method development using tmtzero or tmt duplex to multiplex quantification using tmt 6-plex or tmt 10-plex . the chemical structure of the tmt tag enables the introduction of five heavy isotopes ( c or n ) in the reporter group and five heavy isotopes ( c or n ) in the balancer group to provide six isobaric tags ( figure 3a ) . each of the six tags of tmt 6-plex has a specific reporter ion that appears at m / z 126 , 127 , 128 , 129 , 130 , and 131 . tmt 10-plex is an expansion of tmt 6-plex generated by combining current tmt 6-plex reagents with four isotope variants of the tag with 6.32 mda mass differences between n and c isotopes . even though the mass difference between these reporter ion isotopologues is incredibly small , current generation high - resolution and high mass accuracy analyzers can resolve these ions . the seemingly miniscule difference is sufficient to achieve baseline resolution between the reporter ions when high resolving power is employed ( 30 k at m / z 400 ) . figure 3b shows the substitution of n for c to generate new reporter ions that are lighter than the original forms used in tmt 6-plex . in cases where coalescence , fusion of the proximate reporter ion signals into a single measurable entity , phenomenon is observed , the artifact can be completely eliminated by lowering the maximum ion target for ms / ms spectra . this modified setting does not result in any losses in identification depth or quantification quality of proteins . the high - throughput tmt 10-plex reagent enables concurrent ms analysis and relative quantification of up to 10 different samples derived from cells , tissues , or biological fluids . the higher multiplexing potential also facilitates incorporation of replicates , providing additional statistical validation within any given isobaric labeling experiments . ( a ) chemical structure of tmt 6-plex reagents with c and n heavy isotope positions ( blue asterisks ) . the tags are isobaric , with a different distribution of isotopes between the reporter and mass normalization ( balance ) groups . ( b ) the substitution of n for c to generate new reporter ions that are 6.32 mda lighter than the original forms used in tmt 6-plex . the tmt 6-plex reagents in combination with four isotope variants of the tag with 6.32 mda mass differences were used to generate tmt 10-plex reagent . the numbers of identified peptides and proteins in shotgun proteomics experiments have been compared for the three commercially available isobaric mass tags : itraq 4-plex , tmt 6-plex , and itraq 8-plex . even though the number of identified proteins and peptides was largest with itraq 4-plex , followed by tmt 6-plex , and smallest with itraq 8-plex , the precision on the level of peptide the discrepancy in peptide identification observed with different n - plex isobaric mass tags was suggested to be due to combination of several factors , such as search algorithms and scoring functions , fragment ions derived from cleavage of the label itself or within the label from precursor ions , or disparate physiochemical properties conferred to the peptides depending on the type of isobaric mass tags used for their derivatization . however , in a study by pottiez et al . on comparison of quantitative measurements of proteins in human plasma samples by itraq 4-plex versus 8-plex reagents , 8-plex tagging provided more consistent ratios than that with 4-plex without compromising protein identification . the discrepancies in observations from pichler et al . and pottiez et al . could be due to different instruments ( ltq orbitrap versus mald - tof / tof 4800 platform ) and search algorithms ( mascot and proteome discoverer software versus proteinpilot 4.0 with paragon algorithm ) that were used for the data acquisition and analysis . nevertheless , the obvious advantage of 8-plex tagging is that it allows investigation of eight experimental conditions in one analytical experiment . for example , a study of one control and seven experimental conditions can be performed in one 8-plex experiment but would require at least three 4-plex experiments ( using the control and up to three experimental samples in each ) . the three 4-plex experiments would need more instrument time , likely introducing a source of variability , and would be more laborious . n , n - dimethyl leucine ( dileu ) is an isobaric tandem mass tagging reagent that uses isotope - encoded dimethylated leucine as reporters and serves as attractive alternative for itraq and tmt . labeling with dileu , however , requires activation of the reagents using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride ( dmtmm)/n - methylmorpholine ( nmm ) in n , n - dimethylformamide ( dmf ) . the general structure of dileu resembles that of other isobaric mass tags , with an amine - reactive group ( triazine ester ) targeting the n - terminus and -amino group of the lysine side chain of a peptide , a balance group , and a reporter group . a mass shift of 145.1 da is observed for each incorporated label . by using dileu isobaric tags , however , dileu - labeled peptides undergo better fragmentation and hence generate higher reporter ion intensities than itraq , thereby offering improved confidence for peptide identification and more reliable quantification . intense reporter ions ( dimethylated leucine a1 ion ) at m / z 115 , 116 , 117 , and 118 are observed for all pooled samples upon ms / ms . even though deuterium affects the retention time of small- to intermediate - sized peptides in reversed - phase chromatography , the increased polarity of the amine group offsets the small deuterium number difference in 4-plex dileu tags . ( a ) ( i ) general structure of dimethyl leucine isobaric ( dileu ) mass tag . ( ii ) illustration of formation of new peptide bond at n - terminus or -amino group of the lysine side chain and isotope combination of isobaric tags ( b ) chemical structure of diart isobaric reagents . positions containing heavy stable isotopes are illustrated as numbers in the structure , and the table lists the elemental composition of the corresponding numbers . during ms / ms , the diart - tagged peptides yield reporter ions ranging from m / z 114 to 119 . deuterium isobaric amine reactive tag ( diart ) is another alternative to itraq and tmt for isobaric tagging in quantitative proteomics . like itraq or tmt , diart reagents have three functional groups , an amine - reactive ( nhs ester ) group for coupling of peptides , a balancer , and a reporter ( n , n-dimethylleucine ) with a m / z range of 114119 ( figure 4b ) . up to six samples can be labeled with diart reagents and analyzed by ms . diart reagents have high isotope purity ; hence , unlike that for itraq , tmt , or dileu labeling , isotopic impurities correction is not required during data analysis of diart - labeled samples . the performances of diart and itraq , including their fragmentation mechanisms , the number of identified proteins , and the accuracy of quantification , have been compared . regardless of the peptide sequence , diart tags generate high - intensity reporter ions compared to those with itraq . since quantification accuracy is dependent on the intensity of reporter ions , as high - intensity reporter ions are less susceptible to underestimation effect , diart labeling quantifies more peptides , including low - abundance ones , and with reliable results . while dileu uses a nontraditional activation chemistry ( dmtmm / nmm in dmf ) to label peptides , diart uses the same labeling protocol ( nhs - ester - based peptide coupling chemistry ) as that of tmt and itraq , making it easy for users to switch between the techniques . however , unlike that for itraq or tmt , diart - labeled samples can not be analyzed by the hcd - only instrument method due to easy fragmentation of its reporter ions . nevertheless , diart and dileu serve as a cost - effective alternatives to tmt and itraq with comparable labeling efficiency . diart has been shown to be useful in labeling large quantities of proteins from cell lysates prior to tio2 enrichment in quantitative phosphoproteomics study . isobaric labeling - based quantification can also be used for differential quantification of various protein post - translational modifications . isobaric mass tags are available that are especially designed to measure relative abundance of modified cysteine residues or carbonylated residues in protein . carbonylation of proteins is caused by the reactive oxygen and carbonyl species generated as byproducts of lipid oxidation during oxidative stress . itraq hydrazide ( itraqh ) is a novel reagent for the selective labeling and relative quantitative analysis of carbonyl groups in proteins . itraqh was synthesized from itraq and an excess of hydrazine ( figure 5a ) . itraqh reacts with a carbonylated peptide , resulting in the formation of a hydrazone moiety . consistent with the isobaric labeling approach , peptides labeled with different isotopic variants of itraqh reagents are indistinguishable in ms scan . however , the itraqh reporter ions in the low m / z region of the ms / ms spectrum provide the relative abundance information on the carbonylated proteins in the samples . the itraqh reporter ions have been used as targets for precursor selection in precursor ion scan analysis , which allows selective acquisition of ms / ms spectra of only the carbonylated peptides . this eliminates the need for the step involving enrichment of modified peptides prior to lc ( a ) general structure of itraq hydrazide ( itraqh ) for relative quantitative analysis of carbonylation sites in proteins . ( b ) chemical structure of the carbonyl - reactive glyco - tmt compounds . red asterisks indicate c , and blue asterisks , n. the table below the compound structures shows isotope codes of the hydrazide- and aminoxy - functionalized glyco - tmt compounds . the carbonyl - reactive tags can be used to quantify a broad range of biologically important molecules including carbohydrates , steroids , or oxidized proteins . ( c ) chemical structure of the cysteine - reactive thermo scientific iodotmtzero isobaric mass tag . the iodotmt reagents are iodoacetyl - activated isobaric mass tags for covalent , irreversible labeling of sulfhydryl ( -sh ) groups . iodotmt 6-plex enable measurement of protein and peptide cysteine modifications ( s - nitrosylation , oxidation , and disulfide bridges ) by multiplex quantitative mass spectrometry . the workflow ( not shown in the image ) involves derivatization of modified peptides or proteins with the reagent , enrichment of tmt tagged peptide using anti - tmt antibody , and their subsequent elution . ms / ms to determine the sites of modification and to measure their relative abundance across samples . on the basis of similar chemistry as that of itraqh , the stable isotope - labeled carbonyl - reactive tandem mass tags ( glyco - tmts ) have been used for quantification of n - linked glycans . glyco - tmt reagents are derivatives of the original tmt compounds but are functionalized with carbonyl - reactive groups involving either hydrazide chemistry or aminoxy chemistry ( figure 5b ) . a study reported that aminooxy tmts outperformed their hydrazide counterparts in labeling efficiency and quantification . the glyco - tmt compounds are coded with stable isotopes and enable ( i ) isobaric quantification in ms / ms spectra and ( ii ) quantification in ms1 spectra using heavy / light pairs . isobaric quantification using glyco - tmt can be achieved by using the aminoxy tmt-128 and tmt-131 as well as the hydrazide tmt-126 and tmt-127 reagents ( figure 5b ) . the ms1 level quantification is accomplished by the mass difference of 5.0105 da between the light tmt and the heavy tmt reagents ( figure 5b ) that is sufficient to separate the isotopic patterns of all commonly existing n - glycans . glycan quantification using heavy and light glycol - tmts provided more accurate quantification in ms1 spectra over a broad dynamic range compared with that from quantification based on the reporter ions generated in ms / ms spectra . glyco - tmts with aminooxy - functionalized groups are available commercially from thermo scientific ( rockford , il , usa ) as aminoxytmtzero and aminoxytmt 6-plex reagents . labeling with aminoxytmt reagents involves treating intact proteins or proteolytic digests of proteins extracted from biological specimens with pngase f / a glycosidases to release n - linked glycans . the free glycans are subsequently purified from protein or peptide matrix and labeled at the reducing end with the aminoxytmt reagents . the derivatized glycans from individual samples are then combined and analyzed in ms to identify glycoforms in the sample and to quantify reporter ion relative abundance at ms / ms level . cysteine sulfhydryls in proteins are potential sites of reversible oxidative modification because of the unique redox chemistry of this amino acid . s - nitrosylation is a redox - based protein post - translational modification that occurs in response to nitric oxide signaling and is involved in a wide range of biological processes . it involves addition of a nitric oxide ( no ) group to a specific cysteine residue of a protein to form s - nitrosothiol . an analytical strategy to enrich and relatively quantify cysteine - containing peptides in complex mixtures has been reported . in this strategy , cysteine residues in proteins are first derivatized with n-{2-((2-acryloyl)amino)ethyl-1,3 thiazolidine-4-carboxamide ) ( atc ) followed by labeling with amine - reactive tmt tags for relative quantification of the targeted peptides after the covalent capture . the workflow involves reduction , derivatization of cysteine residue in protein samples with atc tag , digestion with trypsin , and differential labeling with tmt tags followed by pooling of the labeled samples . the atc - derivatized cysteinyl peptides are subsequently isolated on an aldehyde resin through the covalent capture technique and analyzed with lc the cysteine - reactive tmt reagents allow measurement of s - nitrosylation occupancy and determination of individual protein thiol reactivity . however , the disulfide linkage between the ( reversible ) cysteine - reactive tmt tag and protein thiol group can not survive the strong reducing conditions normally used during enzymatic digestion for subsequent shotgun proteomic analysis . an irreversible cysteine - reactive tmt reagent containing a sulfhydryl - reactive iodoacetyl reactive group called iodotmt has been developed . iodotmt reagents such as iodotmtzero and iodotmt 6-plex are commercially available from thermo scientific ( rockford , il , usa ) . each isobaric iodotmt 6-plex reagent within a set has the same nominal mass and consists of a thiol - reactive iodoacetyl functional group for covalent and irreversible labeling of cysteine , a balancer , and a reporter group . the quantification using iodotmt tags is achieved by inspection of the reporter ion region in ms / ms spectra . an iodotmt switch assay uses an isobaric set of thiol - reactive iodotmt 6-plex reagents to specifically detect and quantify protein s - nitrosylation . the iodotmt switch assay workflow includes irreversible labeling of s - nitrosylated cysteines followed by enrichment of s - nitrosylated peptides using high - affinity anti - tmt chromatography with competitive elution and finally multiplexed quantification of protein s - nitrosylation via six unique tmt reporter ions . isobaric labeling - based quantification has many advantages compared to other stable isotope labeling techniques , one of which is the ability to perform high - throughput quantification due to sample multiplexing . the ability to combine and analyze several samples within one experiment eliminates the need to compare multiple lc ms / ms data sets , thereby reducing overall analytical time and run - to - run variation . moreover , the information replication within lc ms / ms experimental regimes provides additional statistical validation within any given experiment . this is desirable in an analysis where conventional upregulation and downregulation measurements are not nearly as meaningful as obtaining temporal expression patterns of proteins throughout the experimental condition , such as in studies involving different stages of cell differentiation , comparisons of multiple drug treatments , identifications of protein drug interactions , measurement of inhibitor dose response , or time course comparisons . when each sample is run separately or with limited multiplexing , as required in label - free , metabolic - labeling and other ms1-based quantification methods , an ion selected for fragmentation on one lc ms / ms run may not be selected consistently in subsequent runs or spectra of suitable quality may not be acquired . the isobaric labeling strategy , however , is immune to the stochastic nature of data - dependent mass spectrometry because a common precursor ion is fragmented that corresponds to the same peptide species present in all of the labeled samples , yielding quantitative data across samples within an isobaric tagging experiment . isobaric labeling has been shown to surpass metabolic labeling in quantification precision and reproducibility . isobaric labeling exhibits a wide dynamic range in profiling both high- and low - abundance proteins and proteins with wide array of physiological properties . it can be used to identify and quantify proteins across diverse molecular weight and pi ranges , functional categories , and cellular locations . the isobaric mass tags do not interfere with peptide fragmentation , and the peptide length distribution profile and amino acid content of the isobarically derivatized peptides are similar to those obtained using other ms - based approaches . in fact , isobaric tags have been reported to improve the efficiency of ms / ms fragmentation and result in increased signal intensities of native peptides in samples of human parotid saliva that , in general , lack the uniform architecture of tryptic cleavage products , e.g. , a basic c - terminal amino acid residue . with an ms1-based quantification approach , the co - elution of light and heavy peptides can compromise sensitivity as the ion current is divided between multiple samples during ms analysis . occurrence of multiple precursor ion species in the ms1 level can also create redundancy in ms / ms scanning events of the same peptide bearing different labels . it is reported that up to 50% of ms / ms scans acquired during data acquisition can be redundant . by contrast , labeling of samples by isobaric mass tags does not increase the sample complexity during chromatographic separation and ms analysis because they are isotope - coded molecules with the same chemical structure and molecular weight , thus eluting at the same chromatographic time and with the same peptide mass . in fact , since differentially labeled but identical peptides from multiple samples are efficaciously merged , an improvement in overall signal - to - noise ratios occurs , allowing good - quality ms / ms data to be acquired from low - copy - number proteins . moreover , the sequence informative b- and y - ions in ms / ms spectra also show this summed intensity , which aids sensitivity . the in vitro labeling procedure used for isobaric labeling - based quantification strategy is highly efficient and enables this method to be applicable to wide variety of samples such as cultured cells , human tissues and biofluids , and tissues from model animals . this technique has been successfully applied to various biological studies , demonstrating its validity and robustness for quantitative ms - based proteomics . isobaric labeling , especially itraq has been used in identifying and distinguishing protease - generated neo - n termini from n - termini of mature proteins by performing terminal amine isotopic labeling of substrates ( tails ) . after tryptic digestion of itraq - labeled protein samples , n - terminal peptide separation is accomplished using a high - molecular - weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and c - terminal peptides that now have n - terminal alpha amines . the unbound itraq - labeled mature n - terminal and neo - n - terminal peptides and naturally blocked ( acetylated , cyclized , and methylated ) the neo - n - terminal peptides specific to the protease of interest appear only in the protease - treated sample and therefore show a high protease / untreated itraq reporter ion intensity ratio , thus differentiating them from trypsin cleavage products that are present in all samples in equal amounts and therefore have expected itraq ratios of 1 . the applications of the isobaric labeling strategy have also been extended to studies involving the characterization of post - translational modifications such as phosphorylation and other modifications ( discussed in the section above ) . initially , isobaric labeling experiments were carried out on maldi - tof / tof and quadrupole time - of - flight ( q - tof ) instruments . quadrupole and tof instruments are capable of detecting low m / z fragment ions in the region where reporter ions are observed . however , the large ion selection window of the tof / tof instrument can result in a relatively high background of chemical noise for the reporter groups , compressing the dynamic range of the ratios significantly . quadrupole ion trap geometries generally produce suboptimal results because the reporter ions often lie below the stability limit , as dictated by the precursor peptide mass - to - charge ratio and pseudopotential well parameters used for activation ( for example , activation q = 0.23 ) . the slow scanning q - tof instruments also have less sensitivity for complex mixtures compared to that of linear ion traps . isobaric quantification using standard collision - induced dissociation ( cid ) conditions is not feasible using ion traps . the /3 rule for ion - trap instruments restricts the analysis of product ions with m / z values less than 2530% of the precursor ion . this low mass cutoff limitation also applies to hybrid instruments containing an ion - trap for fragmentation , such as the ltq - ft and the ltq - orbitrap . this limitation can , in principle , be overcome by pulsed q dissociation ( pqd ) . pqd in the ion trap facilitates detection of low m / z reporter ions , bridging the gap between the linear ion trap with pqd and a quadrupole tof instrument . however , unlike conventional cid spectra , typical pqd spectra are dominated by the unfragmented precursor ion , indicating poor fragmentation efficiency and thus limiting its practical utility for quantification of peptides by itraq or tmt approaches . have shown that by carefully optimizing instrument parameters such as collision energy , activation q , delay time , ion isolation width , number of microscans , repeat count , and number of trapped ions , low m / z fragment ion intensities can be generated that enable accurate peptide quantification . a combined cid - pqd scan strategy exploits cid for efficient peptide identification and pqd for quantification . the development of higher energy collision - induced dissociation ( hcd ) in the ltq - orbitrap has also overcome the /3 rule limitation . in an ion trap cid is a resonance - based process , whereas hcd is a beam - type cid event that results in a different fragmentation pattern . during hcd , ions are accelerated as they leave the c - trap and then are fragmented in the nitrogen - filled collision cell . the resulting fragments are returned to the c - trap and detected in the orbitrap mass analyzer . this fragmentation technique allows analysis of the low m / z region of reporter ions in the orbitrap mass analyzer since there is no mass cutoff for the multipole . hcd enables efficient reporter ion generation with high mass accuracy detection , but , in general , it suffers from poor peptide sequence - ion recovery compared to that of the classical ion trap cid analysis . the combined use of cid and hcd for efficient identification and relative quantification of proteins with isobaric tags has been demonstrated . in this dual - fragmentation method , hcd is used to derive the accurate quantitative information from the reporter ions , whereas cid provides identification of the corresponding peptides . this method alternates ms / ms spectra generated by cid fragmentation with ms / ms spectra obtained from the same precursor ion by hcd fragmentation . since cid in the ion trap occurs in parallel to acquisition of hcd ms / ms spectra in the orbitrap , cid and hcd spectra are subsequently combined by merging the peptide sequence - ion m / z range from cid spectra and the reporter ion m / z range from hcd spectra . it should be noted that the extracted intensity values of the reporter ions from each hcd spectrum should be normalized to low ion counts when merging with the respective cid data , otherwise peptide scores can be significantly reduced . the cid - hcd method was shown to be superior to hcd alone in terms of sensitivity and ability to identify proteins in complex mixtures . however , a recent study has shown that with fine - tuning of the normalized collision energy values on orbitrap velos instruments , an hcd - only method can perform better than a cid - hcd dual - fragmentation method . this is due to the implementation of the new hcd cell with an axial electric field to push the fragment ions into the c - trap and mounted on orbitrap xl etd and orbitrap velos instruments that allows an improvement in the analytical precision of the acquired reporter ions . in addition , the redundancy in precursor selection in the dual cid - hcd method compared to that for the stand alone hcd method can result in a reduced number of total peptide and protein identifications . the use of a stepped hcd scheme in q exactive instruments has been shown to enhance the intensity of reporter ions without adversely affecting peptide identifications . another method for analyzing isobaric labeled samples is to use triple - stage mass spectrometry ( ms3 ) in a hybrid ion trap - orbitrap platform . in this approach , a peptide precursor ion is isolated and fragmented with cid - ms / ms to generate a plurality of first - generation product ion species comprising different respective m / z ratios . the most intense product ion in ms / ms scan is then selected for hcd - ms3 , yielding quantitative data . this method provides an experimental solution to remove interference , thus eliminating the ratio distortion problem ( discussed in the next section ) . a variant of this method referred to as multinotch ms3 involves selecting and co - isolating two or more of the first - generation product - ion species and fragmenting them to generate a plurality of second - generation fragment ion species including released isobaric tags ( figure 6 ) . the multinotch ms3 method significantly improves quantitative accuracy and increases the sensitivity of the ms experiment up to n - fold , where n is the number of ms fragments selected and simultaneously isolated . multinotch ms3 involves selecting and co - isolating multiple ms / ms product ion and fragmenting them to generate a plurality of second - generation fragment ion species including released isobaric tags . the method increases the sensitivity and quantitative accuracy achieved by isobaric labeling - based quantification approach . the ratios of the intensity of the reporter ions reflect the relative abundance of the peptides from which they are derived . the integration of the relative quantification data for the peptides allows elucidation of relative protein expression levels . this section discusses the various aspects of data analysis in isobaric labeling - based quantification . isobaric labeling is usually very efficient ; however , when primary amino groups are present elsewhere in the sample , they may interfere with the labeling reaction since they can react with the amine - reactive isobaric mass tags . hence , proper sample preparation is imperative for the success of an isobaric labeling - based quantification technique and includes either avoiding the use of primary amine - containing buffers such as tris and ammonium bicarbonate or performing sample cleanup prior to the isobaric labeling reaction . to improve detection limits and achieve a reliable estimate of quantification , it is recommended that the labeling efficiency be determined for each isobaric labeling experiment . the labeling efficiency can be ascertained by searching the data separately against protein database using tmt and itraq modifications as variable instead of fixed modifications . using these parameters , both labeled and unlabeled peptides can be identified and used to calculate labeling efficiency , which is defined as the percent of labeled peptides among all identified peptides . the labeling efficiency can be estimated aswhere nti and nki are the number of isobaric tag - labeled n - termini and lysine residues , respectively , and , ntt and nkt are the total number of peptide n - termini and lysine residues , respectively . additionally , due to isotopic contamination in isobaric mass tags , the peaks for each reporter ion will have some contribution from adjacent reporter ions . hence , prior to data analysis , each of the reporter ion peaks must be corrected to account for isotopic overlap ( values reported in the manufacturer s instruction sheet ) in order to achieve accurate quantification . the uncorrected data will appear distorted and confound the observed change in protein expression levels . a detailed procedure to calculate true peak areas that account for overlapping isotopic contributions using the reagent purity values provided by the manufacturer accurate ratios can be determined only when a single precursor ion is selected for fragmentation during an ms / ms scan event . it has been observed that the presence of co - eluting peptides within the isolation window used for the selection and subsequent fragmentation of individual peptide ions typically results in an underestimation or compression of actual protein abundance differences in the analyzed samples . this effect is ubiquitous and not dependent only on the instrument used to acquire the data . the compression in relative abundance is based on the assumption that the vast majority of proteins in biological studies do not change significantly ; therefore , when the peptides from these proteins co - fragment , the reporter ion intensity ratios generated will be less pronounced in terms of fold changes . precursor ions of similar intensities can produce reporter ions that span over 2 orders of magnitude in intensity . this means that very low intensity background ions can significantly contribute to reporter ion signals when they get co - fragmented with a selected precursor ion . additionally , if the coeluting peptides display a nonequimolar distribution of reporter ions , then the net effect of this co - selection is the unpredictable and context - specific distortion of reporter ion intensities . in addition to the distortion in quantification accuracy due to coselection phenomena , the source of quantification error can also be due to presence and interference from artifactual spectral peaks . the reporter ion region in orbitrap hcd ms / ms spectra contains many signals that are nearly isobaric with reporter ions generated from isobaric mass tags . these signals do not correspond to any plausible chemical compositions and may , in part , be attributed to artifacts related to amplifying and processing the transient signal of the orbitrap . depending on the mass tolerance used for picking the reporter ion signals , the presence of these nonreporter ion signals may distort the quantification results . peptide abundance ratios are calculated by combining data from multiple fractions across ms runs and then averaging across peptides to give an abundance ratio for each parent protein . the measured relative abundance can be influenced by the separation ( e.g. , scx ) stage in which the ms / ms was acquired , a phenomenon termed as fraction effect . fraction effect for a given peptide is defined as a significant dependence between the measured ratio and the fraction in which the reading was taken . the error within a fraction group for a peptide is smaller than the error between fraction groups and arises from the additional variance from the repeated scx separation stage . the observation of fraction effect could be due to differences in a peptide s concentration across fractions that contribute to variability in precursor ion intensity measures and subsequent reporter ion peak areas . in addition to fraction effect , the measured ratio is also dependent on the precursor ion ( i.e. , peptide ) used to characterize a protein . the measurement error within a peptide group for a protein the difference in quantitative value from one peptide to another , even though belonging to the same protein , might result from factors such as post - translational modifications and/or splice variants , tryptic digestion artifacts , peptide recovery , and stability . other factors of peptide effect include noise peaks with high signal - to - noise in the reporter ion region , sequence of the peptide used for quantification and the possibility of interference from the immonium ion signals in the reporter ion region , various charge states of the same peptide , and the number of isobaric tags per peptide . since interference due to coisolation is dependent on sample complexity and the number of co - eluting peptides , the ratio compression can be partly alleviated by better fractionation of complex biological samples at the protein or peptide level . ratio compression was observed to be smaller for enriched phosphoproteome samples compared to that for whole proteome samples due to their overall lower sample complexity . another approach involves using an optimized ( narrow ) ms / ms isolation width setting so that fewer contaminant ions are present during precursor ion activation . the high mass resolving power ( m/m > 15 000 ) in the reporter ion region also minimizes interference from potential contaminant species that may confound quantification data . delaying peptide selection and fragmentation until the apex of the chromatographic peak during lc ms / ms analysis has been shown to reduce co - fragmentation by 2-fold . with the delayed fragmentation approach , peptides were fragmented with 2.8-fold better signal - to - noise ratios , significantly improving the quantification . a targeted mass spectrometric data acquisition methodology with reporter ion - based quantification has been shown to be useful in applications where it is essential to reidentify and requantify a defined set of target proteins in a complex mixture . the gas - phase purification and ms3 methods also eliminate interfering ions in complex mixtures . in q - tof instruments , ion mobility ( i m ) separations have the potential to mitigate quantitative inaccuracies caused by isobaric interference since im - ms has the ability to separate ions based on charge , m / z , and collision cross section ( shape and size ) . the ratio correction can also be achieved by various computational approaches post data acquisition . one of the strategies is to use an algorithm that corrects experimental ratios on the basis of determined peptide interference levels . in this method , the measurement for spectrum purity in survey spectra ( signal - to - interference measure ) was used to improve the accuracy of protein quantification . signal - to - interference at the time of an ms / ms event is calculated by dividing precursor abundance by the sum of all ion signals observed within the isolation window . consequently , values close to one indicate little and values close to zero indicate a high degree of interference caused by co - eluting components . other informatics approaches include the intensity - based weighed average technique , variance - stabilizing normalization , and robust statistic - based metric called redescending m - estimator . the interference from non - tmt signals can be eliminated by mass difference processing in which tmt reporter ions in hcd spectra are identified via accurate mass differences between tmt reporter ions present within the same tandem mass spectrum instead of applying fixed mass error tolerances for all tandem mass spectra . this process leads to unambiguous reporter ion identification and eliminates all non - tmt ions from the spectra . zhang et al . developed an error model that relates the variance of measured ratios to observed reporter ion intensity and provides a p value , q value , and confidence interval for every peptide identified . the identification and exclusion of outlier data , with grubb s and rosner s tests , that alter or inappropriately skew the average observed expression ratios has shown to result in a more statistically robust estimation of relative protein abundance . the ability to consider outlier data , however , can occur only for proteins in which there are more than three ms / ms measurements of protein expression . in summary , even though all of the suggested strategies have merit , some techniques only partially remove the problem , and others come with decreased throughput or utilize specialized mass spectrometric instrumentation . isobaric labeling - based quantification accuracy is also influenced by reporter ion signal intensity and may result in either an underestimation or overestimation of quantification ratio if the signal intensity is outside the detector s saturation point . the reporter ions intensities will range between two extremes : the maximum intensity , which corresponds to saturation , and the minimum intensity , which corresponds to the lowest intensity detected . however , not all reporter ion intensity peaks will lead to accurate relative quantification . the peak intensities of high - abundance peptide ions may be underestimated by a saturation effect of the detector , which is instrument - dependent . nevertheless , high - intensity peptides convey more reliable quantitative information about the protein . larger variances of peptide ratios have been observed for reporter ions of lower intensity because the noise associated with low - intensity reporters constitutes a major handicap in determining the statistical significance of the differential expression of a protein . therefore , peptides with higher reporter ion intensities should be given higher weight when used to calculate a protein s abundance . reporter ion signal intensity can be increased by increasing the ms / ms acquisition duration ; however , this comes at the expense of decreased sampling , resulting in fewer protein identifications . it is therefore important to estimate the quantification limits of the instrument and the method used in order to assess the reliability of the obtained quantification measurement . this can be achieved by spiking samples with known quantities of reference proteins prior to analysis and confirming the expected protein ratio from the measured reporter ion intensity ratios . in isobaric labeling significant quantification errors arise if a quantified peptide is not unique to its corresponding protein . hence , relative quantification based on shared peptides ( i.e. , peptides that match multiple proteins or protein isoforms ) due to sequence homology should be interpreted with caution . for a distinct peptide , its relative abundance ratio is a direct measure of the abundance ratio of its corresponding protein . in contrast , the relative abundance ratio of a shared peptide is a weighted average of the abundance ratios of all its corresponding proteins , with the weighting factors being determined by the absolute abundance of those proteins in the samples . even though isobaric quantification is not dependent on the total number of spectra matching to each protein , a high number of relative abundance ratios obtained from multiple peptide / spectra increase the confidence in the observed protein ratios . both intact protein mass and abundance level influence the reliability of the quantification results since highly abundant proteins generate a larger number of peptides per protein . more data , whether from multiple observations per protein or from increasing replication , increases the detection of real signal and reduces false positives . the identification of so - called one - hit wonders should be filtered intelligently based on the goal of the study . these proteins deserve special attention if isobaric labeling is used as a screening tool since potentially important biological information or novel biomarkers may be discarded before they are even considered . fold change has been shown to be a function of protein mass and abundance , with small , low - abundance proteins showing the largest variance . a protein is considered to be differentially regulated if the measured fold change exceeds a certain threshold . the actual protein expression level is normally distorted by many factors , with biological variation being the most significant and which ultimately increases the cutoff point . the cutoff point that defines significant differential protein regulation upon perturbation can be estimated by including sample replicates in the experiment . the replicate samples can be technical , experimental , or biological . according to gan et al . , the definition of replicates in terms of isobaric labeling is as follows : a technical replicate will have two identical samples from the same biological source in an isobaric experiment set , whereas a biological replicate will have two distinct biological samples from same condition in an experiment set . the experimental replicate is the actual isobaric experiment replicate , the repetition of the same samples in two or more experimental sets , and they must have the same reference point or control . an illustration of the relationship among technical , experimental , and biological replicates in isobaric labeling experiments is depicted in figure 7 . an example defining the relation among technical , experimental , and biological replicates in isobaric labeling ( itraq 4-plex in this example ) experiments . a biological replicate has two distinct biological samples ( x1 and x2 ) from the same condition in an itraq set , whereas a technical replicate has two identical samples ( x1 and x1 ) from the same biological source in an itraq set . an experimental replicate is the repetitive analysis of the setup to assess the variation of the identical sample in two different itraq sets ( y1 and x1 in experiment 1 versus y1 and x1 in experiment 2 ) . r refers to a reference sample that can be an individual sample or a pooled sample and allows cross - set comparison . typically , a technical replicate assesses possible errors contributed from sample preparation , also commonly known as the sample variance . an experimental replicate compares the variation of an identical sample in two different isobaric experiment sets . during analysis of replicate samples , the theoretical relative quantification ratio should be 1:1 ; however , due to associated variations , the observed relative protein ratios might deviate from the theoretical value . the threshold should be chosen such that it encompasses the majority of technical and biological variation among the replicates . since in isobaric labeling multiple samples hence , the ratio cutoff applied for significant protein change via the isobaric labeling - based quantification approach is lower than the cutoff applied for the label - free quantification approach ; however , the researcher will need to assess whether such a change is biologically significant . for comparing biological replicates with isobaric labeling in multiple experimental designs , this can be accomplished by first comparing protein ratios of each sample against its reference within individual experiments and then extending the information among multiple experiments . the reference can be an individual sample or a pooled sample prepared by mixing small aliquots of equal amounts of protein from different individual samples . the composition of reference samples does not contribute to missing quantitative values , hence pooling to form a reference sample does not negatively impact the ability to quantitate peptides from comparative individual samples . the random biological variation in a pooled sample is generally lower , as the biological variation can be normalized by n samples before being introduced into the experiment . pooling provides a representative proteome of all of the samples that are detected in comparative samples and is needed for reliable quantification . , however , have shown that using a masterpool is counterproductive since the latter is also subjected to experimental noise and can result in highly variable estimates when ratios are calculated . according to their study , more precise estimates of protein relative abundance can be obtained by using the available biological data . when a reference sample is used , consistency of the reference is necessary throughout the entire experiment , otherwise even small changes to the reference sample are sufficient to alter the proteins that are reported as differentially expressed . regardless of using an individual sample or a pooled sample as a reference , before employing isobaric quantification results for follow - up studies , it is imperative to determine that the data was normalized adequately and the shortlisted protein targets hold merit . improper normalization might remove some of the biological effects , resulting in attenuated estimates of the protein fold change . like any other quantification technique , isobaric labeling - based quantification is also biased toward identifying and quantifying a larger percentage of the more abundant proteins , such as ribosomal proteins , heat shock proteins , cytoskeletal proteins , transcription factors , and many others , and often with multiple peptides . this is mainly due to the fact that their precursor ions have higher signal intensity . the greater signal intensity increases the likelihood that a given peptide will be selected for fragmentation during lc since , in most cases , the expression levels of these house - keeping proteins remain unperturbed in related cell types or growth conditions , they can be used as an effective means to determine the reliability of data normalization . normalizing by total intensity is not appropriate when the amount of protein is different in the different quantitative sample such as samples that are enriched for certain proteins by pull - down experiments . isobaric labeling experiments can be used for phosphopeptide quantification , and , in cases where the number of samples exceeds the number of isobaric mass tags available for labeling , the throughput can be increased by a hyperplexing method . amine - reactive isobaric mass tags have successfully been used in the quantification of post - translational modification such as phosphorylation . phosphopeptides exist in substoichiometric quantities , and because of the high background of nonphosphorylated peptides in a proteome digest , enrichment of phosphorylated peptides is necessary prior to introduction into the ms . phosphopeptide enrichment can be performed on isobaric - labeled peptides , or the phosphorylated peptide can be labeled upon enrichment . labeling before enrichment minimizes analytical variations caused by further sample manipulation of individual samples during enrichment , whereas labeling after enrichment might improve the yield of the labeling since the nonphosphorylated peptides would otherwise compete for isobaric reagent and interfere with the complete labeling of phosphopeptides . during the cid - hcd dual method for the quantification and identification of isobarically tagged phosphopeptides , cid with detection in the linear ion trap provides better sensitivity and can be an advantage for low - abundance precursors such as phosphopeptides . however , quantitative information from the low mass region of subsequent hcd scans may not be available for all such cid scans since hcd scans requires higher ion counts . wu et al . have examined the optimal fragmentation conditions using the cid - hcd method for itraq - labeled synthetic phosphopeptides in a complex phosphopeptide mix and phosphopeptides enriched from cells . during cid - ms / ms , the spectra derived from phosphoserine- and phosphothreonine - containing peptides show facile fragmentation of the phosphate group and dominance of neutral phosphate losses from the precursor ions . the neutral loss in hcd - ms / ms is much lower , and the sequence - specific fragments are significantly more abundant . with the increasing charge state of the precursor ions , the neutral loss in hcd - ms / however , the study of the effect of normalized collision energy on the hcd - ms / ms fragments and reporter ion abundances shows that the hcd identified phosphopeptides and the hcd spectra with reporter ion information are strongly dependent on precursor charge state . the 2 + charged precursors are more sensitive to the applied normalized collision energy values than the 3 + charged precursor ions in hcd experiments . have shown that derivatization with isobaric mass tags significantly increases the average ion charge state of phosphopeptides compared to that of nonlabeled peptides , resulting in a considerable reduction in the number of identified phosphopeptides . interestingly , it was demonstrated that adding a perpendicular flow of ammonia vapor between the needle and the ms orifice in lc ms / ms analyses reduced the average charge state of isobaric labeled peptides and resulted in an increase in peptide identification . thus , the application of isobaric labeling strategies for quantitative phosphopeptide analysis requires simultaneous monitoring of peptide backbone dissociation , loss of phosphoryl group , and the generation of reporter ions . with existing isobaric mass tags , the maximum number of samples that can be combined and analyzed in a single lc ms / ms experiment is eight in the case of itraq and 10 with tmt . an effort to increase the multiplexing capacity by the combined use of metabolic and isobaric labeling has been demonstrated . in this strategy , the mass separation of co - eluting intact peptides with the same sequence in an ms1 scan achieved by duplex ( heavy and light ) or triplex silac labeling was exploited to allow for the simultaneous quantification of multiple sets of tmt 6-plex isobaric labels in a single run . using a 3 6 hyperplexing experiment that enables simultaneous quantification of 18 samples , yeast response to the immunosuppressant drug rapamycin , which inhibits the kinase target of rapamycin ( tor ) , was monitored by measuring the changes in their protein abundance . in this study , three separate cultures of yeast cells grown in light , medium , or heavy silac culture medium were treated with 200 mm rapamycin , and samples were removed at 0 , 30 , 60 , 120 , and 180 min . equal amounts of peptides from each sample were labeled with 6-plex tmt reagents , mixed , and separated by scx before lc ms / ms analysis . the increased multiplexing capacity enabled analyses of multiple biological replicates of a time - course study in the same run , providing the statistical power required to identify significant trends . the hyperplexing technique with combined metabolic labeling and isobaric mass tags can also be extended to n - labeled samples . alternatively , the dimethyl chemical labeling technique can be combined with isobaric mass tags to increase the multiplexing capacity of quantitative proteomics . theoretically , the combination of itraq 8-plex or tmt 10-plex reagents and triplex silac would allow 24 or 30 channels to be monitored simultaneously . proteins that are identified and quantified as differentially expressed can be used for subsequent targeted studies using the isobaric labeling technique to assess reproducibility of the entire procedure and to validate the observed differences in protein expression levels between samples . during biomarker discovery experiments , targeted investigations are necessary to verify proteins with higher variance in additional patient samples or to obtain greater statistical power . for successful targeted analysis , peptides that allow clear protein quantification and isobaric mass tags are often used for discovery studies to reveal proteins being differentially expressed under any given conditions . however , stella et al . have shown that isobaric mass tags can be used in combination with multiple reaction monitoring ( mrm ) for targeted quantification . in this study , the instrument monitored the two reporter ions and three transitions for each peptide selected from the target membrane proteins . the relative quantification was achieved by comparing the intensities of the reporter ions generated from the labeled precursor peptide of two samples , wild - type ( reporter ion m / z 129 ) and prion protein(prp)-knockout ( reporter ion m / z 131 ) cerebellar granule neurons . byers et al . used isotopic versions of tmt reagents for targeted quantification to verify protein regulations observed in a discovery study . these isotopic sets of reagents are structurally identical to the isobaric ones but have different numbers of heavy isotopes incorporated and are referred to as light tmt and heavy tmt ( figure 8) . the labeling of peptides by these reagents results in an increase in mass of 224 and 229 da , respectively , per introduced tag . chemical structure of isotopic reagents , light tmt and heavy tmt , used for targeted quantification . the light reagent has no heavy isotope incorporated , whereas the heavy reagent has five heavy isotopes incorporated ( 4 c and 1 n ) . labeling with these reagents introduces mass differences into the peptides from different samples . in targeted experiments , quantification is obtained by structural b and/or y ions generated after collision - induced dissociation . the isobaric labeling - based quantification technique has developed as a powerful tool for obtaining the relative expression level of proteins in quantitative proteomics studies . moreover , the ability to multiplex with isobaric mass tags has expanded its applicability to a wide range of sample types . isobaric mass tags are isotope - coded molecules with the same chemical structure and molecular weight that are used to differentially label peptides without introducing mass difference and sample complexity . the isotopically derivatized peptides display a single peak on an ms spectrum and yield a series of low - mass reporter ions for quantification upon fragmentation in tandem mass spectrometry . however , since peptide quantification ratios are measured to determine protein relative abundance , the variance in peptide ratio measurements will contribute into the protein - level variance , affecting the accuracy of the quantification . herein , we have reviewed the studies of different aspects of an isobaric labeling - based quantification approach . this includes studies on different types of isobaric reagents and their applications , sources of variation that affect quantification , and the suggested combinations of experimental design and optimal data acquisition methods to increase the precision and accuracy of the measurements . we have also reviewed studies on challenges in data analysis and the proposed solutions for data processing to increase the confidence in the acquired data set .

mass spectrometry plays a key role in relative quantitative comparisons of proteins in order to understand their functional role in biological systems upon perturbation . in this review , we review studies that examine different aspects of isobaric labeling - based relative quantification for shotgun proteomic analysis . in particular , we focus on different types of isobaric reagents and their reaction chemistry ( e.g. , amine- , carbonyl- , and sulfhydryl - reactive ) . various factors , such as ratio compression , reporter ion dynamic range , and others , cause an underestimation of changes in relative abundance of proteins across samples , undermining the ability of the isobaric labeling approach to be truly quantitative . these factors that affect quantification and the suggested combinations of experimental design and optimal data acquisition methods to increase the precision and accuracy of the measurements will be discussed . finally , the extended application of isobaric labeling - based approach in hyperplexing strategy , targeted quantification , and phosphopeptide analysis are also examined .

Introduction Isobaric Mass Tags Benefits of Isobaric Labeling-Based Quantification Strategy Instrumentation and Data Acquisition Methods for Isobarically Labeled Samples Factors Affecting Quantification by Isobaric Labeling: Technical and Bioinformatics Issues Extended Applications of Isobaric Labeling-Based Quantification Strategy Summary

mass spectrometry ( ms ) is a powerful tool to assess the relative abundance of proteins among biological samples . one of the most popular methods for relative quantification through ms is stable isotope labeling of proteins in samples prior to analysis . the in vitro chemical derivatization processes include techniques such as isotope - coded affinity tags ( icat ) , dimethyl labeling , isobaric mass tags , and others . in isobaric labeling - based quantification , each sample is derivatized with a different isotopic variant of an isobaric mass tag from a set , and then the samples are pooled and analyzed simultaneously in ms . the fragmentation of the modified precursor ion during ms / ms event generates two types of product ions : ( a ) reporter ion peaks and ( b ) peptide fragment ion peaks . following fragmentation of the precursor ion during ms / ms , the six reporter ions appear as distinct masses between m / z 126131 , and the remainder of the sequence - informative b- and y - ions remains as additive isobaric signals . they generate relative quantitative information in an isobaric labeling - based quantification strategy . the most common isobaric tag is amine - reactive , but tags that react with cysteine residues and carbonyl groups in proteins are also available . the tags employ n - hydroxysuccinimide ( nhs ) chemistry , and the structure consists of three functional groups : an amine - reactive group and an isotopic reporter group ( n - methylpiperazine ) linked by an isotopic balancer group ( carbonyl ) for the normalization of the total mass of the tags . the overall mass of the reporter and balance components of the molecule are kept constant using differential isotopic enrichment with c , n , and o atoms . ( b ) chemical structure of a generic tmt reagent showing the three functional groups : an amine - reactive group that labels the n - terminus and -amine group of lysine in peptides , a mass normalization ( balance ) group that balances mass differences from individual reporter ions to ensure the same overall mass of the reagents , and a reporter group that provides the abundance of a peptide upon ms / ms in individual samples being mixed . the application of isobaric tags for simultaneous determination of both the identity and relative abundance of peptide pairs was first demonstrated by thompson et al . in this study , they demonstrated for the first time the application of isobaric mass tags with 4-fold multiplexing to identify global protein expression trends in a set of isogenic yeast strains . each of the six tags of tmt 6-plex has a specific reporter ion that appears at m / z 126 , 127 , 128 , 129 , 130 , and 131 . in cases where coalescence , fusion of the proximate reporter ion signals into a single measurable entity , phenomenon is observed , the artifact can be completely eliminated by lowering the maximum ion target for ms / ms spectra . even though the number of identified proteins and peptides was largest with itraq 4-plex , followed by tmt 6-plex , and smallest with itraq 8-plex , the precision on the level of peptide the discrepancy in peptide identification observed with different n - plex isobaric mass tags was suggested to be due to combination of several factors , such as search algorithms and scoring functions , fragment ions derived from cleavage of the label itself or within the label from precursor ions , or disparate physiochemical properties conferred to the peptides depending on the type of isobaric mass tags used for their derivatization . on comparison of quantitative measurements of proteins in human plasma samples by itraq 4-plex versus 8-plex reagents , 8-plex tagging provided more consistent ratios than that with 4-plex without compromising protein identification . the general structure of dileu resembles that of other isobaric mass tags , with an amine - reactive group ( triazine ester ) targeting the n - terminus and -amino group of the lysine side chain of a peptide , a balance group , and a reporter group . ( ii ) illustration of formation of new peptide bond at n - terminus or -amino group of the lysine side chain and isotope combination of isobaric tags ( b ) chemical structure of diart isobaric reagents . positions containing heavy stable isotopes are illustrated as numbers in the structure , and the table lists the elemental composition of the corresponding numbers . like itraq or tmt , diart reagents have three functional groups , an amine - reactive ( nhs ester ) group for coupling of peptides , a balancer , and a reporter ( n , n-dimethylleucine ) with a m / z range of 114119 ( figure 4b ) . the performances of diart and itraq , including their fragmentation mechanisms , the number of identified proteins , and the accuracy of quantification , have been compared . isobaric labeling - based quantification can also be used for differential quantification of various protein post - translational modifications . consistent with the isobaric labeling approach , peptides labeled with different isotopic variants of itraqh reagents are indistinguishable in ms scan . however , the itraqh reporter ions in the low m / z region of the ms / ms spectrum provide the relative abundance information on the carbonylated proteins in the samples . the workflow ( not shown in the image ) involves derivatization of modified peptides or proteins with the reagent , enrichment of tmt tagged peptide using anti - tmt antibody , and their subsequent elution . glyco - tmt reagents are derivatives of the original tmt compounds but are functionalized with carbonyl - reactive groups involving either hydrazide chemistry or aminoxy chemistry ( figure 5b ) . in this strategy , cysteine residues in proteins are first derivatized with n-{2-((2-acryloyl)amino)ethyl-1,3 thiazolidine-4-carboxamide ) ( atc ) followed by labeling with amine - reactive tmt tags for relative quantification of the targeted peptides after the covalent capture . however , the disulfide linkage between the ( reversible ) cysteine - reactive tmt tag and protein thiol group can not survive the strong reducing conditions normally used during enzymatic digestion for subsequent shotgun proteomic analysis . each isobaric iodotmt 6-plex reagent within a set has the same nominal mass and consists of a thiol - reactive iodoacetyl functional group for covalent and irreversible labeling of cysteine , a balancer , and a reporter group . the quantification using iodotmt tags is achieved by inspection of the reporter ion region in ms / ms spectra . isobaric labeling - based quantification has many advantages compared to other stable isotope labeling techniques , one of which is the ability to perform high - throughput quantification due to sample multiplexing . this is desirable in an analysis where conventional upregulation and downregulation measurements are not nearly as meaningful as obtaining temporal expression patterns of proteins throughout the experimental condition , such as in studies involving different stages of cell differentiation , comparisons of multiple drug treatments , identifications of protein drug interactions , measurement of inhibitor dose response , or time course comparisons . the isobaric labeling strategy , however , is immune to the stochastic nature of data - dependent mass spectrometry because a common precursor ion is fragmented that corresponds to the same peptide species present in all of the labeled samples , yielding quantitative data across samples within an isobaric tagging experiment . isobaric labeling exhibits a wide dynamic range in profiling both high- and low - abundance proteins and proteins with wide array of physiological properties . the isobaric mass tags do not interfere with peptide fragmentation , and the peptide length distribution profile and amino acid content of the isobarically derivatized peptides are similar to those obtained using other ms - based approaches . the in vitro labeling procedure used for isobaric labeling - based quantification strategy is highly efficient and enables this method to be applicable to wide variety of samples such as cultured cells , human tissues and biofluids , and tissues from model animals . the unbound itraq - labeled mature n - terminal and neo - n - terminal peptides and naturally blocked ( acetylated , cyclized , and methylated ) the neo - n - terminal peptides specific to the protease of interest appear only in the protease - treated sample and therefore show a high protease / untreated itraq reporter ion intensity ratio , thus differentiating them from trypsin cleavage products that are present in all samples in equal amounts and therefore have expected itraq ratios of 1 . the applications of the isobaric labeling strategy have also been extended to studies involving the characterization of post - translational modifications such as phosphorylation and other modifications ( discussed in the section above ) . however , the large ion selection window of the tof / tof instrument can result in a relatively high background of chemical noise for the reporter groups , compressing the dynamic range of the ratios significantly . this low mass cutoff limitation also applies to hybrid instruments containing an ion - trap for fragmentation , such as the ltq - ft and the ltq - orbitrap . have shown that by carefully optimizing instrument parameters such as collision energy , activation q , delay time , ion isolation width , number of microscans , repeat count , and number of trapped ions , low m / z fragment ion intensities can be generated that enable accurate peptide quantification . the method increases the sensitivity and quantitative accuracy achieved by isobaric labeling - based quantification approach . the ratios of the intensity of the reporter ions reflect the relative abundance of the peptides from which they are derived . this section discusses the various aspects of data analysis in isobaric labeling - based quantification . hence , proper sample preparation is imperative for the success of an isobaric labeling - based quantification technique and includes either avoiding the use of primary amine - containing buffers such as tris and ammonium bicarbonate or performing sample cleanup prior to the isobaric labeling reaction . additionally , due to isotopic contamination in isobaric mass tags , the peaks for each reporter ion will have some contribution from adjacent reporter ions . hence , prior to data analysis , each of the reporter ion peaks must be corrected to account for isotopic overlap ( values reported in the manufacturer s instruction sheet ) in order to achieve accurate quantification . the compression in relative abundance is based on the assumption that the vast majority of proteins in biological studies do not change significantly ; therefore , when the peptides from these proteins co - fragment , the reporter ion intensity ratios generated will be less pronounced in terms of fold changes . depending on the mass tolerance used for picking the reporter ion signals , the presence of these nonreporter ion signals may distort the quantification results . the measured relative abundance can be influenced by the separation ( e.g. other factors of peptide effect include noise peaks with high signal - to - noise in the reporter ion region , sequence of the peptide used for quantification and the possibility of interference from the immonium ion signals in the reporter ion region , various charge states of the same peptide , and the number of isobaric tags per peptide . since interference due to coisolation is dependent on sample complexity and the number of co - eluting peptides , the ratio compression can be partly alleviated by better fractionation of complex biological samples at the protein or peptide level . a targeted mass spectrometric data acquisition methodology with reporter ion - based quantification has been shown to be useful in applications where it is essential to reidentify and requantify a defined set of target proteins in a complex mixture . in q - tof instruments , ion mobility ( i m ) separations have the potential to mitigate quantitative inaccuracies caused by isobaric interference since im - ms has the ability to separate ions based on charge , m / z , and collision cross section ( shape and size ) . in this method , the measurement for spectrum purity in survey spectra ( signal - to - interference measure ) was used to improve the accuracy of protein quantification . other informatics approaches include the intensity - based weighed average technique , variance - stabilizing normalization , and robust statistic - based metric called redescending m - estimator . in summary , even though all of the suggested strategies have merit , some techniques only partially remove the problem , and others come with decreased throughput or utilize specialized mass spectrometric instrumentation . isobaric labeling - based quantification accuracy is also influenced by reporter ion signal intensity and may result in either an underestimation or overestimation of quantification ratio if the signal intensity is outside the detector s saturation point . it is therefore important to estimate the quantification limits of the instrument and the method used in order to assess the reliability of the obtained quantification measurement . in contrast , the relative abundance ratio of a shared peptide is a weighted average of the abundance ratios of all its corresponding proteins , with the weighting factors being determined by the absolute abundance of those proteins in the samples . , the definition of replicates in terms of isobaric labeling is as follows : a technical replicate will have two identical samples from the same biological source in an isobaric experiment set , whereas a biological replicate will have two distinct biological samples from same condition in an experiment set . the experimental replicate is the actual isobaric experiment replicate , the repetition of the same samples in two or more experimental sets , and they must have the same reference point or control . an illustration of the relationship among technical , experimental , and biological replicates in isobaric labeling experiments is depicted in figure 7 . an example defining the relation among technical , experimental , and biological replicates in isobaric labeling ( itraq 4-plex in this example ) experiments . during analysis of replicate samples , the theoretical relative quantification ratio should be 1:1 ; however , due to associated variations , the observed relative protein ratios might deviate from the theoretical value . since in isobaric labeling multiple samples hence , the ratio cutoff applied for significant protein change via the isobaric labeling - based quantification approach is lower than the cutoff applied for the label - free quantification approach ; however , the researcher will need to assess whether such a change is biologically significant . like any other quantification technique , isobaric labeling - based quantification is also biased toward identifying and quantifying a larger percentage of the more abundant proteins , such as ribosomal proteins , heat shock proteins , cytoskeletal proteins , transcription factors , and many others , and often with multiple peptides . isobaric labeling experiments can be used for phosphopeptide quantification , and , in cases where the number of samples exceeds the number of isobaric mass tags available for labeling , the throughput can be increased by a hyperplexing method . with the increasing charge state of the precursor ions , the neutral loss in hcd - ms / however , the study of the effect of normalized collision energy on the hcd - ms / ms fragments and reporter ion abundances shows that the hcd identified phosphopeptides and the hcd spectra with reporter ion information are strongly dependent on precursor charge state . thus , the application of isobaric labeling strategies for quantitative phosphopeptide analysis requires simultaneous monitoring of peptide backbone dissociation , loss of phosphoryl group , and the generation of reporter ions . an effort to increase the multiplexing capacity by the combined use of metabolic and isobaric labeling has been demonstrated . in this strategy , the mass separation of co - eluting intact peptides with the same sequence in an ms1 scan achieved by duplex ( heavy and light ) or triplex silac labeling was exploited to allow for the simultaneous quantification of multiple sets of tmt 6-plex isobaric labels in a single run . alternatively , the dimethyl chemical labeling technique can be combined with isobaric mass tags to increase the multiplexing capacity of quantitative proteomics . theoretically , the combination of itraq 8-plex or tmt 10-plex reagents and triplex silac would allow 24 or 30 channels to be monitored simultaneously . proteins that are identified and quantified as differentially expressed can be used for subsequent targeted studies using the isobaric labeling technique to assess reproducibility of the entire procedure and to validate the observed differences in protein expression levels between samples . the relative quantification was achieved by comparing the intensities of the reporter ions generated from the labeled precursor peptide of two samples , wild - type ( reporter ion m / z 129 ) and prion protein(prp)-knockout ( reporter ion m / z 131 ) cerebellar granule neurons . the isobaric labeling - based quantification technique has developed as a powerful tool for obtaining the relative expression level of proteins in quantitative proteomics studies . however , since peptide quantification ratios are measured to determine protein relative abundance , the variance in peptide ratio measurements will contribute into the protein - level variance , affecting the accuracy of the quantification . herein , we have reviewed the studies of different aspects of an isobaric labeling - based quantification approach . this includes studies on different types of isobaric reagents and their applications , sources of variation that affect quantification , and the suggested combinations of experimental design and optimal data acquisition methods to increase the precision and accuracy of the measurements . we have also reviewed studies on challenges in data analysis and the proposed solutions for data processing to increase the confidence in the acquired data set .

time perception is a critical element of both conscious and subconscious experience , providing synchrony and reliable representation of the surrounding environment . despite the significance of the temporal dimension , our knowledge of the neuropsychological mechanisms underlying timing processes and computations remains relatively poor [ 1 , 2 ] . modern theories about internal time representation share the view that the processing of temporal information is mediated by a distributed network with varying engagement of its individual components depending on the task requirements . however , there is substantial disagreement on the effects reported in various studies , providing no definitive anatomical or cognitive model [ 35 ] . several regions , including the basal ganglia , cerebellum , posterior parietal cortex , and frontal cortex , have been implicated as relevant to interval timing ; however , their precise role remains shrouded . the traditional model describing the processing of time passage in the brain is the pacemaker - accumulator model , a straightforward solution that is surprisingly effective in explaining behavioral data [ 6 , 7 ] . the pacemaker oscillates at a frequency subject to modulation by the temporal properties of sensory stimuli ; the pulse count is converted by the accumulator into a linear metric of time . however , recent advances point towards an alternative view offered by distributed timing models , deriving temporal information from the coincidental activation of different neural populations . although diverse , regarding the hypothesized specific neuropsychological mechanisms of time processing , this broad spectrum of models basically postulate the representation of time as ubiquitous in more networks that also encode other stimulus properties [ 4 , 9 ] . thus , temporal information may be encoded in the entire activity pattern of a neuronal mechanism , as suggested in the state - dependent network model [ 9 , 10 ] . at the same time , the majority of authors agree that , in the multisecond range , the linear integration of temporal signals is a plausible mechanism for temporal coding and considers it supplemental to the second class of models arising from the inherent temporal properties of the neural networks that provides automatic timing dealing with millisecond range durations [ 9 , 1113 ] . of particular relevance in this context is the observation that the processing of temporal information in the millisecond range involves a different network than multisecond operations [ 9 , 12 , 14 ] . following this distinction , short duration processes would be predominantly evaluated within the motor and sensory - motor network ( cerebellum , premotor cortex , and sensory cortex ) , whereas the timing analysis in longer intervals would be associated with the striatal - prefrontal circuit [ 15 , 16 ] . the suggestion that correct cerebellar function may be essential for successful temporal processing is not new ; its prominence in facilitating performance in time analysis has been further underscored by both clinical studies of patients with cerebellar pathology [ 1822 ] and neuroimaging studies [ 15 , 2325 ] , mostly based on paced finger tapping or temporal discrimination with the retrospective character of temporal computing ( for review see ) . however , surprisingly few studies have investigated the cerebellar role in the complex processes necessary for predictive timing the analysis of various environmental factors in the subsecond spectrum leading to a delineation of the probable future state of the surroundings which enables preemptive action in simple , routine activities and even in life - threatening situations . possible variations in the composition of the timing network related to gender might be of significant interest as well , as reports are available , albeit scarce , of both structural and functional dissimilarities between men and women in the infratentorial area . behavioral performance differences between males and females have been well known for decades , with the field of numeric operations , spatial processing , and probability and data analysis dominated by men and language and social skills being the realm of women . interestingly , a similar disparity has been repeatedly confirmed in complex analyses of reaction time to various stimuli revealing a significant disadvantage for women [ 3134 ] and a differential pattern of reaction times as a function of stimuli location and character that might reflect different information processing strategies . hence , understanding the underlying neural mechanisms associated with contingent performance and functional differences in specific processes in the context of gender may offer an important insight into neurological and psychiatric diseases with strong sex biases . we wished to establish the neuroanatomical signature of complex predictive motor timing in a well - characterized sample of healthy individuals using the fmri blood oxygen level dependent ( bold ) signal and to contrast the performance of men to that of women . forty healthy volunteers , 24 men ( mean age 40.15 ; sd 15.20 ) and 16 women ( mean age 43.85 ; sd 14.76 ) , with no symptoms of neurologic diseases participated in the study . all of the subjects underwent a screening to ensure a safe mri procedure , and their anatomical mri scans did not show any structural pathology . all participants provided their written informed consent prior to the experiment in accordance with the guidelines of the local research ethics committee . the participants performed the same motor - timing computer task as employed in our earlier studies [ 20 , 22 , 36 , 37 ] . the subjects were required to press a key with the dominant hand to launch a projectile that was intended to intercept a green circular target moving from the left side of the screen toward the upper right corner ( figure 1(a ) ) . the target ( diameter 1 cm ) moved to the fixed interception zone at three possible speeds ( slow , medium , and fast ) , at three possible angles relative to the horizontal plane ( 0 , 15 , 30 ) , and in three different manners ( constant speed , deceleration , and acceleration ) , reaching 27 distinct combinations of movement parameters with the intention of moderating the expected learning effect and providing varying levels of difficulty for further fmri models and analyses . the projectile ( diameter 0.3 cm ) was launched from the lower right corner of the screen at a constant speed of 20.0 cm / s on a vertical , unchanging trajectory . the subjects were instructed to press the key only once at the optimal time for the projectile to intercept the moving circular target in the upper right corner of the screen . a successful interception was followed by a small explosion animation as feedback for the subject ( figure 1(b ) ) . the participants were specifically asked to visually follow the target at all times and encouraged to use the same strategy throughout the experiment . due to the varying , complex movement parameters of the target , simplified approaches such as fixating the gaze on a launching area consciously estimated based on the previous trials and waiting for the target to reach the area or mentally counting the time from the target 's appearance to a preselected point would prove useless . the experiment was organized into 6 blocks , each formed by 54 trials , with pseudorandomized combinations of the target movement parameters to provide 12 repetitions of each combination for a total of 324 trials . we employed a counterbalanced presentation of various stimuli types within each block to minimize the repetition of the same movement parameter combinations in consecutive trials . the duration of one trial was on average 3.5 s. the entire paradigm consisted of a training session ( performed while scanning , but not subject to analysis ) and the main task . the duration of the whole session ( including the acquisition of anatomical scans , the training session , and the main session with short breaks between the blocks to prevent motor or cognitive fatigue ) was about 50 minutes . the task was presented using the labview 6.1 ( national instruments , austin , tx , usa ) interface . anne 's university hospital , brno , on a 1.5 t siemens symphony scanner equipped with numaris 4 system ( mrease ) . high - resolution anatomical t1-weighted images were acquired using the magnetization - prepared rapid gradient - echo ( mprage ) sequence ( tr = 1.7 s , te = 3.93 ms , ti = 1100 ms , in - plane voxel size 0.96 0.96 mm , 160 sagittal slices , slice thickness 1.17 mm , matrix 256 256 160 ) . a total of 850 volumes of functional images were acquired during the entire run ( tr = 2.3 s , te = 35 ms , fa = 90 , fov = 220 180 mm , in - plane voxel size = 3.44 3.44 mm , 28 axial slices , slice thickness 4.40 mm ) , but only 490 volumes corresponding to the main task were used for further analyses ( the remaining volumes scanned during the training part were discarded ) . two dummy scans , acquired before each fmri run in order for the fmri signal to reach a steady state , were discarded from the analysis as well . the main variable of interest was the success rate ( i.e. , the hit ratio ) across the different types of movements ( stable speed , acceleration , and deceleration ) and speed types . the angle was excluded from the analyses , since it was proven to have no effect on the hit ratio [ 20 , 38 ] . we computed the average success rate in each subject for every movement type in order to be able to use parametric statistical analysis . the effects of the movement parameters on the main variable were assessed using anova analysis , with the hit ratio as a dependent variable and the speed and the movement type as independent factors . the fmri data were preprocessed and analyzed using spm8 ( wellcome department of cognitive neurology , london , uk ) implemented in matlab r2013 ( mathworks inc . , no subject 's movements exceeded 3 mm in each spatial direction or 3 of rotation . afterwards , the coregistration of functional and anatomical images , interpolation in time to correct for volume acquisition phase progression , spatial normalization into the stereotactic montreal neurological institute ( mni ) space , and spatial smoothing using an isotropic gaussian kernel of 8 mm full - width at half - maximum were performed . to minimize low frequency artifacts possibly associated with the block length , the data were high - pass filtered with a gaussian kernel filter of 128 s. at the first level , the general linear model of bold activations consisted of bidirectional comparisons of successful hits and errors in each subject separately . the individual design matrix for every participant also included the speed , as interpreting the results of this parameter was more straightforward than with the complicated scheme of the movement type ( acceleration , deceleration , and stable speed ) . the onset was modelled as the moment the green circular target appeared on the left side of the screen and the end was the moment when the subject pressed the key to launch the projectile . in the analysis of the main hypothesis , six contrast maps were created at the first level ( two possible results [ hit or miss ] three speeds ) and a second level factorial design ( 2 3 ) was implemented with the following factors : success ( two levels : hit and miss ) and speed ( three levels : slow , medium , and high ) . the analysis of our secondary aim , the gender distinctions , was based on a first level model ( 3 speeds 3 movement types ( stable , acceleration , deceleration ) ) and the successive second level model with gender , speed , and the movement type as factors ( i.e. , 2 3 3 ) and the respective hit ratios for the individual target movement parameters as a nuisance variable to control for the effect associated primarily with the individual success rate differences . the results were considered significant at p < 0.05 ; family - wise error ( fwe ) corrected for multiple comparisons at the whole brain level with a cluster threshold of 35 contiguous voxels in all the comparisons . the analysis of pooled data provided a mean hit ratio of 0.4281 ( sd = 0.1534 ) , with a significant effect of movement type ( f2 , 354 = 24.75 ; p < 0.001 ) ( figure 2(a ) ) and a borderline trend in the effect of speed ( f2 , 354 = 2.054 ; p = 0.11 ) ( figure 2(b ) ) . overall , acceleration was associated with a lower success rate than either deceleration or stable speed . furthermore , the character and timing of errors were put to test , distinguishing between early errors ( i.e. , errors when the subject pressed the button too early ) and late errors ( the subject pressed the button too late ) . there was no difference between the occurrence of early and late errors ; early errors constituted 51.6% and the late errors , 48.4% of all the errors ( p > 0.05 ) . due to this even distribution of errors , the average response time ( period between the onset of the stimulus and the motor response key press ) did not differ between successful trials , hits ( 1.301 s ) , and unsuccessful trials , misses ( 1.265 s ) ( p > 0.05 ) . interestingly , significant gender differences were found , with a mean hit ratio of 0.4589 ( sd = 0.1552 ) in men and 0.3803 ( sd = 0.1383 ) in women ( p < 0.001 ) . sex did not interact significantly with acceleration ( f2 , 354 = 0.2864 ; p = 0.75 ) ( figure 2(a ) ) or speed ( only a borderline trend of performance decrease in women with higher speeds than men f2 , 354 = 2.235 ; p = 0.11 ) ( figure 2(b ) ) . the results of the 2 3 anova ( hit / miss speed ) were as follows:(1)main effect of hit versus miss : the areas significantly more activated in successful trials included the basal ganglia ( specifically the putamen and caudate bilaterally ) and a larger cluster comprising the posterior cerebellum ( lobule vi and crus 1 on the right side ) and the inferior part of the occipital lobe ( figure 3(a ) , table 1 ) . the reverse contrast ( miss > hit ) did not provide any results surviving the threshold of p < 0.05 , fwe - corrected.(2)the effect of speed : speed changes of the target were closely associated with cerebellar activity in the posterior cerebellum ( specifically vermis 6 and vermis 7 , bilateral lobules vi , and crura 1 , with the predominance of the right cerebellar hemisphere ) and , furthermore , with the right middle temporal lobe , the middle occipital lobe on the left side , and smaller clusters of activity in the putamen bilaterally and in the left precentral gyrus ( figure 3(b ) , table 1).(3)interaction of hit versus miss and speed : no clusters survived at the level of p < 0.05 with fwe - correction at the whole brain level.the analysis of the effect of gender , with the hit ratio as a nuisance variable due to performance differences ( see the behavioral results ) , yielded the following activation maps:(1)women > men : figure 3(c ) demonstrates the patterns of hyperactivation in women when compared to men , comprising primarily of the posterior cerebellum ( specifically the left lobules iv - v , viii , ix , and the right viii , vii , and vi with left - sided predominance ) . further regions found in this contrast included two clusters in the left temporal gyrus ( brodmann area 39 ) ( figure 3(c ) , table 1).(2)men > women : the reverse contrast revealed differences of lesser magnitude than the ones reported above , including the cingulum and brodmann areas 45 and 21.(3)the interaction between gender and speed or movement type did not yield any results surviving the threshold of p > 0.0 , fwe - corrected at the level of whole brain . main effect of hit versus miss : the areas significantly more activated in successful trials included the basal ganglia ( specifically the putamen and caudate bilaterally ) and a larger cluster comprising the posterior cerebellum ( lobule vi and crus 1 on the right side ) and the inferior part of the occipital lobe ( figure 3(a ) , table 1 ) . the reverse contrast ( miss > hit ) did not provide any results surviving the threshold of p < 0.05 , fwe - corrected . the effect of speed : speed changes of the target were closely associated with cerebellar activity in the posterior cerebellum ( specifically vermis 6 and vermis 7 , bilateral lobules vi , and crura 1 , with the predominance of the right cerebellar hemisphere ) and , furthermore , with the right middle temporal lobe , the middle occipital lobe on the left side , and smaller clusters of activity in the putamen bilaterally and in the left precentral gyrus ( figure 3(b ) , table 1 ) . interaction of hit versus miss and speed : no clusters survived at the level of p < 0.05 with fwe - correction at the whole brain level . women > men : figure 3(c ) demonstrates the patterns of hyperactivation in women when compared to men , comprising primarily of the posterior cerebellum ( specifically the left lobules iv - v , viii , ix , and the right viii , vii , and vi with left - sided predominance ) . further regions found in this contrast included two clusters in the left temporal gyrus ( brodmann area 39 ) ( figure 3(c ) , table 1 ) . men > women : the reverse contrast revealed differences of lesser magnitude than the ones reported above , including the cingulum and brodmann areas 45 and 21 . the interaction between gender and speed or movement type did not yield any results surviving the threshold of p > 0.0 , fwe - corrected at the level of whole brain . research efforts have been increasingly directed towards understanding the neuroanatomical substrates of time perception by studying the characteristic patterns of neuronal activity and behavioral correlates in both healthy individuals and specific patient populations . although a large corpus of research has implicated complex , overlapping networks bringing together information from multiple modalities , including the basal ganglia , cerebellum , posterior parietal cortex , and frontal cortex , substantial disagreement persists regarding the relevance of these structures and the specific computing models used to delineate temporal parameters from various inputs [ 1 , 6 , 7 , 9 , 10 ] . functional neuroimaging studies have consistently used tasks based on paced finger tapping and temporal discrimination . building upon the previous research , but with a substantial step forwards , the current study is not concerned with the simple ability to maintain certain intervals or to assess and compare the length of various stimuli , that is , with retrospective temporal processing . it is designed to analyze the neural networks involved in predictive timing processes in the subsecond spectrum that enable preemptive action on the basis of the estimated future state of our environment , an essential ability whose absence would virtually preclude even simply catching a ball . our results highlight the specific functional role of several areas , including the putamen and caudate , the temporal cortex , and primarily the cerebellum , an area previously commonly associated with temporal tasks , although probably of an underspecified level of contribution . our finding of high activation in the cerebellum associated with increasing speed , and hence also with the difficulty of the task , is well in accordance with previous studies of subsecond spectrum retrospective temporal processing utilizing transcranial magnetic stimulation of the cerebellum [ 3941 ] and imaging methods [ 25 , 4246 ] , and also with the hypothesis that this neural node acts as a timing system for brief intervals . even if reported as the most commonly engaged structure in retrospective subsecond temporal computing , the extent of the volume of activated cerebellar tissue and the dominance of the cerebellar cluster in the current study far exceeds the volumes and the rank among the other neural nodes reported in retrospective timing tasks . in contrast to the prevailing retrospective tasks , only a few studies have focused primarily on temporal predictions ; the anterior - inferior parietal cortex and ventral premotor cortex in the posner paradigm and the anterior - inferior parietal cortex in trajectory prediction have been suggested . the findings presented here are also very much in keeping with the analysis of perceptual prediction change and trajectory extrapolation associated with the notable engagement of the posterior cerebellum . turning to the other regions implied in the current study , the putamen , clearly revealed here in both the speed and success versus failure analysis , is extensively documented in temporal processing in the subsecond and multisecond range in both animals and humans ( for review see ) . it was highlighted in tasks requiring the comparison of durations with a probe stimulus [ 51 , 52 ] and even proposed as a core timer across the whole temporal spectrum , responsible for the early stages of temporal computation , temporary storage , or as a functional center in stimulus encoding , integrating the temporal signals and generating a subjective perception . our findings of a significant relation of putaminal activity to the hit ratio in predictive temporal computing not only resonate with the previous hypotheses , but also go beyond these and underscore its function as a neural node engaged in the evaluation of success and precision of the undergoing prospective temporal analysis . furthermore , although often not commented upon , the activity in the middle temporal lobe during timing tasks has also been implicated before [ 15 , 5153 ] . this supramodal involvement of the auditory cortex was demonstrated both in auditory and visual timing . our secondary aim , delineating gender differences in predictive timing , was already partly addressed in the analysis of behavioral data revealing a significantly lower success rate in women . the emergence of the cerebellum as the main hyperactivity cluster in women in the neuroimaging analysis , even when corrected for the hit ratio difference , may seem rather surprising , as the cerebellum is traditionally considered to be a fairly monomorphic structure with minimal sex dissimilarities . neuroimaging studies seem to provide contradictory results , and a higher volume of cerebellar hemispheres in men [ 26 , 56 ] , analogous to observations of the cerebral cortex , was reported , although other analyses , volumetric assessments based on mri and examination of fixed human tissue , failed to reveal a significant relationship between gender and cerebellar structure . pet confusingly revealed increased metabolism in women in the cerebellum , corresponding to the findings of our study , decreased metabolism in women in the cerebellum , or no gender differences at all in the infratentorial area . the lack of association with gender , though not completely surprising given the character of the stimulation task , was also reported in fmri studies [ 62 , 63 ] . on the other hand , strong cerebellar hyperactivity in women during simple silent counting , markedly similar to our results , provided a foundation for the hypothesis of distinct cognitive and executive strategies in response to various stimuli between the genders . moreover , behavioral performance differences between the genders have been well known for decades , for example , in language , dominated by women , and visuospatial tasks , where men show superior results . due to the versatile nature of cerebellar functions , this neural node has been implicated in many of those areas [ 6567 ] . hence , even the extent and character of cerebellar engagement may be of different magnitude in men and women . future studies incorporating complex analyses will be helpful in expanding upon our current results and confirming the disputable dissimilarities in activity patterns in the infratentorial area . the mechanisms underlying the increased cerebellar activity in women may have the character of a compensatory hyperactivation of one node of a complex neural network commonly responsible for temporal analysis , but without further support from other studies using various methods , this hypothesis is far from elucidating this poorly understood issue . first , given the temporal constraints of fmri , the plausibility of disambiguation of individual components and their specific functional signature in the process of prospective temporal timing in intervals of short duration used in our task may be considered borderline at best . ergo , no further hypotheses about the probable temporal processing model utilized in predictive timing may be provided , except for the obvious statement of multiple neural nodes engaged , with cerebellar dominance . second , the emergence of occipital cortical areas in the analysis of hit versus miss and , to a lesser extent , even in the analysis of the speed effect may not be surprising given the increased demands for both primary and secondary image processing necessary for the successful interception of the moving target . however , in the light of this distinction , the interpretation of other significant clusters as core structures for predictive temporal processing might be challenged . moreover , the notion of variable analyzed time interval with the offset of the regressor corresponding to the motor response may point to possible contamination with the signal associated with motor processing due to the temporal proximity of early motor responses ( early errors ) . however , due to the even distribution of errors between too early and too late responses , this convolution seems unlikely . nonetheless , our results are well in accord with the previous body of research on retrospective timing , thus allowing us confidence in our findings . the final issue worth reiterating in the context of gender distinctions is the complexity of the task ; it consists of series of component processes , and the neural activity in males and females may show differences for only specific segments , unfortunately outside the temporal resolution of fmri . further work will be necessary to tease apart the extent to which cerebellar activation varies between genders and to develop experimental tasks optimized to probe specific hypotheses about the gender differences . taken together , our findings underscore the pivotal role of the cerebellum in predictive temporal processing , with considerably higher engagement than in retrospective timing tasks , and provide novel insights for understanding different neural mechanisms in men and women . moreover , our results offer a more nuanced perspective on the contribution of the putamen in analyzing predictive temporal processing precision and success as a possible node in a feedback loop activated more in successful trials than in errors . despite the increasing amount of research it will be important for future studies to expand upon the current results in other domains of complex prospective timing .

time perception is an essential part of our everyday lives , in both the prospective and the retrospective domains . however , our knowledge of temporal processing is mainly limited to the networks responsible for comparing or maintaining specific intervals or frequencies . in the presented fmri study , we sought to characterize the neural nodes engaged specifically in predictive temporal analysis , the estimation of the future position of an object with varying movement parameters , and the contingent neuroanatomical signature of differences in behavioral performance between genders . the established dominant cerebellar engagement offers novel evidence in favor of a pivotal role of this structure in predictive short - term timing , overshadowing the basal ganglia reported together with the frontal cortex as dominant in retrospective temporal processing in the subsecond spectrum . furthermore , we discovered lower performance in this task and massively increased cerebellar activity in women compared to men , indicative of strategy differences between the genders . this promotes the view that predictive temporal computing utilizes comparable structures in the retrospective timing processes , but with a definite dominance of the cerebellum .

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

time perception is a critical element of both conscious and subconscious experience , providing synchrony and reliable representation of the surrounding environment . despite the significance of the temporal dimension , our knowledge of the neuropsychological mechanisms underlying timing processes and computations remains relatively poor [ 1 , 2 ] . modern theories about internal time representation share the view that the processing of temporal information is mediated by a distributed network with varying engagement of its individual components depending on the task requirements . several regions , including the basal ganglia , cerebellum , posterior parietal cortex , and frontal cortex , have been implicated as relevant to interval timing ; however , their precise role remains shrouded . however , recent advances point towards an alternative view offered by distributed timing models , deriving temporal information from the coincidental activation of different neural populations . thus , temporal information may be encoded in the entire activity pattern of a neuronal mechanism , as suggested in the state - dependent network model [ 9 , 10 ] . at the same time , the majority of authors agree that , in the multisecond range , the linear integration of temporal signals is a plausible mechanism for temporal coding and considers it supplemental to the second class of models arising from the inherent temporal properties of the neural networks that provides automatic timing dealing with millisecond range durations [ 9 , 1113 ] . of particular relevance in this context is the observation that the processing of temporal information in the millisecond range involves a different network than multisecond operations [ 9 , 12 , 14 ] . following this distinction , short duration processes would be predominantly evaluated within the motor and sensory - motor network ( cerebellum , premotor cortex , and sensory cortex ) , whereas the timing analysis in longer intervals would be associated with the striatal - prefrontal circuit [ 15 , 16 ] . the suggestion that correct cerebellar function may be essential for successful temporal processing is not new ; its prominence in facilitating performance in time analysis has been further underscored by both clinical studies of patients with cerebellar pathology [ 1822 ] and neuroimaging studies [ 15 , 2325 ] , mostly based on paced finger tapping or temporal discrimination with the retrospective character of temporal computing ( for review see ) . however , surprisingly few studies have investigated the cerebellar role in the complex processes necessary for predictive timing the analysis of various environmental factors in the subsecond spectrum leading to a delineation of the probable future state of the surroundings which enables preemptive action in simple , routine activities and even in life - threatening situations . possible variations in the composition of the timing network related to gender might be of significant interest as well , as reports are available , albeit scarce , of both structural and functional dissimilarities between men and women in the infratentorial area . behavioral performance differences between males and females have been well known for decades , with the field of numeric operations , spatial processing , and probability and data analysis dominated by men and language and social skills being the realm of women . hence , understanding the underlying neural mechanisms associated with contingent performance and functional differences in specific processes in the context of gender may offer an important insight into neurological and psychiatric diseases with strong sex biases . we wished to establish the neuroanatomical signature of complex predictive motor timing in a well - characterized sample of healthy individuals using the fmri blood oxygen level dependent ( bold ) signal and to contrast the performance of men to that of women . all of the subjects underwent a screening to ensure a safe mri procedure , and their anatomical mri scans did not show any structural pathology . all participants provided their written informed consent prior to the experiment in accordance with the guidelines of the local research ethics committee . the subjects were required to press a key with the dominant hand to launch a projectile that was intended to intercept a green circular target moving from the left side of the screen toward the upper right corner ( figure 1(a ) ) . the target ( diameter 1 cm ) moved to the fixed interception zone at three possible speeds ( slow , medium , and fast ) , at three possible angles relative to the horizontal plane ( 0 , 15 , 30 ) , and in three different manners ( constant speed , deceleration , and acceleration ) , reaching 27 distinct combinations of movement parameters with the intention of moderating the expected learning effect and providing varying levels of difficulty for further fmri models and analyses . the subjects were instructed to press the key only once at the optimal time for the projectile to intercept the moving circular target in the upper right corner of the screen . due to the varying , complex movement parameters of the target , simplified approaches such as fixating the gaze on a launching area consciously estimated based on the previous trials and waiting for the target to reach the area or mentally counting the time from the target 's appearance to a preselected point would prove useless . the experiment was organized into 6 blocks , each formed by 54 trials , with pseudorandomized combinations of the target movement parameters to provide 12 repetitions of each combination for a total of 324 trials . the duration of one trial was on average 3.5 s. the entire paradigm consisted of a training session ( performed while scanning , but not subject to analysis ) and the main task . the duration of the whole session ( including the acquisition of anatomical scans , the training session , and the main session with short breaks between the blocks to prevent motor or cognitive fatigue ) was about 50 minutes . a total of 850 volumes of functional images were acquired during the entire run ( tr = 2.3 s , te = 35 ms , fa = 90 , fov = 220 180 mm , in - plane voxel size = 3.44 3.44 mm , 28 axial slices , slice thickness 4.40 mm ) , but only 490 volumes corresponding to the main task were used for further analyses ( the remaining volumes scanned during the training part were discarded ) . , the hit ratio ) across the different types of movements ( stable speed , acceleration , and deceleration ) and speed types . the effects of the movement parameters on the main variable were assessed using anova analysis , with the hit ratio as a dependent variable and the speed and the movement type as independent factors . afterwards , the coregistration of functional and anatomical images , interpolation in time to correct for volume acquisition phase progression , spatial normalization into the stereotactic montreal neurological institute ( mni ) space , and spatial smoothing using an isotropic gaussian kernel of 8 mm full - width at half - maximum were performed . to minimize low frequency artifacts possibly associated with the block length , the data were high - pass filtered with a gaussian kernel filter of 128 s. at the first level , the general linear model of bold activations consisted of bidirectional comparisons of successful hits and errors in each subject separately . the individual design matrix for every participant also included the speed , as interpreting the results of this parameter was more straightforward than with the complicated scheme of the movement type ( acceleration , deceleration , and stable speed ) . the onset was modelled as the moment the green circular target appeared on the left side of the screen and the end was the moment when the subject pressed the key to launch the projectile . in the analysis of the main hypothesis , six contrast maps were created at the first level ( two possible results [ hit or miss ] three speeds ) and a second level factorial design ( 2 3 ) was implemented with the following factors : success ( two levels : hit and miss ) and speed ( three levels : slow , medium , and high ) . the analysis of our secondary aim , the gender distinctions , was based on a first level model ( 3 speeds 3 movement types ( stable , acceleration , deceleration ) ) and the successive second level model with gender , speed , and the movement type as factors ( i.e. , 2 3 3 ) and the respective hit ratios for the individual target movement parameters as a nuisance variable to control for the effect associated primarily with the individual success rate differences . the results were considered significant at p < 0.05 ; family - wise error ( fwe ) corrected for multiple comparisons at the whole brain level with a cluster threshold of 35 contiguous voxels in all the comparisons . the analysis of pooled data provided a mean hit ratio of 0.4281 ( sd = 0.1534 ) , with a significant effect of movement type ( f2 , 354 = 24.75 ; p < 0.001 ) ( figure 2(a ) ) and a borderline trend in the effect of speed ( f2 , 354 = 2.054 ; p = 0.11 ) ( figure 2(b ) ) . furthermore , the character and timing of errors were put to test , distinguishing between early errors ( i.e. there was no difference between the occurrence of early and late errors ; early errors constituted 51.6% and the late errors , 48.4% of all the errors ( p > 0.05 ) . due to this even distribution of errors , the average response time ( period between the onset of the stimulus and the motor response key press ) did not differ between successful trials , hits ( 1.301 s ) , and unsuccessful trials , misses ( 1.265 s ) ( p > 0.05 ) . interestingly , significant gender differences were found , with a mean hit ratio of 0.4589 ( sd = 0.1552 ) in men and 0.3803 ( sd = 0.1383 ) in women ( p < 0.001 ) . the results of the 2 3 anova ( hit / miss speed ) were as follows:(1)main effect of hit versus miss : the areas significantly more activated in successful trials included the basal ganglia ( specifically the putamen and caudate bilaterally ) and a larger cluster comprising the posterior cerebellum ( lobule vi and crus 1 on the right side ) and the inferior part of the occipital lobe ( figure 3(a ) , table 1 ) . (2)the effect of speed : speed changes of the target were closely associated with cerebellar activity in the posterior cerebellum ( specifically vermis 6 and vermis 7 , bilateral lobules vi , and crura 1 , with the predominance of the right cerebellar hemisphere ) and , furthermore , with the right middle temporal lobe , the middle occipital lobe on the left side , and smaller clusters of activity in the putamen bilaterally and in the left precentral gyrus ( figure 3(b ) , table 1). (3)interaction of hit versus miss and speed : no clusters survived at the level of p < 0.05 with fwe - correction at the whole brain level.the analysis of the effect of gender , with the hit ratio as a nuisance variable due to performance differences ( see the behavioral results ) , yielded the following activation maps:(1)women > men : figure 3(c ) demonstrates the patterns of hyperactivation in women when compared to men , comprising primarily of the posterior cerebellum ( specifically the left lobules iv - v , viii , ix , and the right viii , vii , and vi with left - sided predominance ) . further regions found in this contrast included two clusters in the left temporal gyrus ( brodmann area 39 ) ( figure 3(c ) , table 1). main effect of hit versus miss : the areas significantly more activated in successful trials included the basal ganglia ( specifically the putamen and caudate bilaterally ) and a larger cluster comprising the posterior cerebellum ( lobule vi and crus 1 on the right side ) and the inferior part of the occipital lobe ( figure 3(a ) , table 1 ) . the effect of speed : speed changes of the target were closely associated with cerebellar activity in the posterior cerebellum ( specifically vermis 6 and vermis 7 , bilateral lobules vi , and crura 1 , with the predominance of the right cerebellar hemisphere ) and , furthermore , with the right middle temporal lobe , the middle occipital lobe on the left side , and smaller clusters of activity in the putamen bilaterally and in the left precentral gyrus ( figure 3(b ) , table 1 ) . women > men : figure 3(c ) demonstrates the patterns of hyperactivation in women when compared to men , comprising primarily of the posterior cerebellum ( specifically the left lobules iv - v , viii , ix , and the right viii , vii , and vi with left - sided predominance ) . further regions found in this contrast included two clusters in the left temporal gyrus ( brodmann area 39 ) ( figure 3(c ) , table 1 ) . research efforts have been increasingly directed towards understanding the neuroanatomical substrates of time perception by studying the characteristic patterns of neuronal activity and behavioral correlates in both healthy individuals and specific patient populations . although a large corpus of research has implicated complex , overlapping networks bringing together information from multiple modalities , including the basal ganglia , cerebellum , posterior parietal cortex , and frontal cortex , substantial disagreement persists regarding the relevance of these structures and the specific computing models used to delineate temporal parameters from various inputs [ 1 , 6 , 7 , 9 , 10 ] . building upon the previous research , but with a substantial step forwards , the current study is not concerned with the simple ability to maintain certain intervals or to assess and compare the length of various stimuli , that is , with retrospective temporal processing . it is designed to analyze the neural networks involved in predictive timing processes in the subsecond spectrum that enable preemptive action on the basis of the estimated future state of our environment , an essential ability whose absence would virtually preclude even simply catching a ball . our results highlight the specific functional role of several areas , including the putamen and caudate , the temporal cortex , and primarily the cerebellum , an area previously commonly associated with temporal tasks , although probably of an underspecified level of contribution . our finding of high activation in the cerebellum associated with increasing speed , and hence also with the difficulty of the task , is well in accordance with previous studies of subsecond spectrum retrospective temporal processing utilizing transcranial magnetic stimulation of the cerebellum [ 3941 ] and imaging methods [ 25 , 4246 ] , and also with the hypothesis that this neural node acts as a timing system for brief intervals . even if reported as the most commonly engaged structure in retrospective subsecond temporal computing , the extent of the volume of activated cerebellar tissue and the dominance of the cerebellar cluster in the current study far exceeds the volumes and the rank among the other neural nodes reported in retrospective timing tasks . in contrast to the prevailing retrospective tasks , only a few studies have focused primarily on temporal predictions ; the anterior - inferior parietal cortex and ventral premotor cortex in the posner paradigm and the anterior - inferior parietal cortex in trajectory prediction have been suggested . the findings presented here are also very much in keeping with the analysis of perceptual prediction change and trajectory extrapolation associated with the notable engagement of the posterior cerebellum . turning to the other regions implied in the current study , the putamen , clearly revealed here in both the speed and success versus failure analysis , is extensively documented in temporal processing in the subsecond and multisecond range in both animals and humans ( for review see ) . it was highlighted in tasks requiring the comparison of durations with a probe stimulus [ 51 , 52 ] and even proposed as a core timer across the whole temporal spectrum , responsible for the early stages of temporal computation , temporary storage , or as a functional center in stimulus encoding , integrating the temporal signals and generating a subjective perception . our findings of a significant relation of putaminal activity to the hit ratio in predictive temporal computing not only resonate with the previous hypotheses , but also go beyond these and underscore its function as a neural node engaged in the evaluation of success and precision of the undergoing prospective temporal analysis . furthermore , although often not commented upon , the activity in the middle temporal lobe during timing tasks has also been implicated before [ 15 , 5153 ] . this supramodal involvement of the auditory cortex was demonstrated both in auditory and visual timing . our secondary aim , delineating gender differences in predictive timing , was already partly addressed in the analysis of behavioral data revealing a significantly lower success rate in women . the emergence of the cerebellum as the main hyperactivity cluster in women in the neuroimaging analysis , even when corrected for the hit ratio difference , may seem rather surprising , as the cerebellum is traditionally considered to be a fairly monomorphic structure with minimal sex dissimilarities . neuroimaging studies seem to provide contradictory results , and a higher volume of cerebellar hemispheres in men [ 26 , 56 ] , analogous to observations of the cerebral cortex , was reported , although other analyses , volumetric assessments based on mri and examination of fixed human tissue , failed to reveal a significant relationship between gender and cerebellar structure . pet confusingly revealed increased metabolism in women in the cerebellum , corresponding to the findings of our study , decreased metabolism in women in the cerebellum , or no gender differences at all in the infratentorial area . on the other hand , strong cerebellar hyperactivity in women during simple silent counting , markedly similar to our results , provided a foundation for the hypothesis of distinct cognitive and executive strategies in response to various stimuli between the genders . moreover , behavioral performance differences between the genders have been well known for decades , for example , in language , dominated by women , and visuospatial tasks , where men show superior results . hence , even the extent and character of cerebellar engagement may be of different magnitude in men and women . the mechanisms underlying the increased cerebellar activity in women may have the character of a compensatory hyperactivation of one node of a complex neural network commonly responsible for temporal analysis , but without further support from other studies using various methods , this hypothesis is far from elucidating this poorly understood issue . first , given the temporal constraints of fmri , the plausibility of disambiguation of individual components and their specific functional signature in the process of prospective temporal timing in intervals of short duration used in our task may be considered borderline at best . ergo , no further hypotheses about the probable temporal processing model utilized in predictive timing may be provided , except for the obvious statement of multiple neural nodes engaged , with cerebellar dominance . second , the emergence of occipital cortical areas in the analysis of hit versus miss and , to a lesser extent , even in the analysis of the speed effect may not be surprising given the increased demands for both primary and secondary image processing necessary for the successful interception of the moving target . however , in the light of this distinction , the interpretation of other significant clusters as core structures for predictive temporal processing might be challenged . moreover , the notion of variable analyzed time interval with the offset of the regressor corresponding to the motor response may point to possible contamination with the signal associated with motor processing due to the temporal proximity of early motor responses ( early errors ) . however , due to the even distribution of errors between too early and too late responses , this convolution seems unlikely . nonetheless , our results are well in accord with the previous body of research on retrospective timing , thus allowing us confidence in our findings . the final issue worth reiterating in the context of gender distinctions is the complexity of the task ; it consists of series of component processes , and the neural activity in males and females may show differences for only specific segments , unfortunately outside the temporal resolution of fmri . taken together , our findings underscore the pivotal role of the cerebellum in predictive temporal processing , with considerably higher engagement than in retrospective timing tasks , and provide novel insights for understanding different neural mechanisms in men and women . moreover , our results offer a more nuanced perspective on the contribution of the putamen in analyzing predictive temporal processing precision and success as a possible node in a feedback loop activated more in successful trials than in errors .

excitatory amino acid transporters ( eaats ) are membrane proteins that remove glutamate from the synaptic environment to terminate neurotransmission and help prevent neurotoxicity caused by high concentrations of the neurotransmitter . in the central nervous system , these proteins perform the glutamate reuptake into the presynaptic cells or astrocytes by coupling the transport of one glutamate substrate to the cotransport of three sodium ions and carry out antiport of one proton and one potassium ion while using this transport for a flux of chloride ions . eaats are important drug targets because their dysfunction is related to a variety of neurological and other conditions , including depression , schizophrenia , stroke , alzheimer s disease , or human dicarboxylic aminoaciduria . a major breakthrough toward a molecular mechanistic understanding of substrate transport in eaats has been the elucidation of crystal structures of gltph , a bacterial homologue that shares about 37% sequence identity with eaats . gltph transports one aspartic acid substrate molecule in symport with three sodium ions while also enabling chloride conductance . like the mammalian eaats , the bacterial gltph forms trimers , which in the crystals were shown to have a c3 symmetry that produces a bowl - shaped , membrane - traversing structure that presumably reaches deep into the lipid bilayer ( figure 1 ) . this symmetry is remarkable given the fact that the monomers in gltph and eaat have been suggested to transport substrate independently and are expected to move stochastically and independently the neurotransmitter transport mechanism has long been considered to involve a sequential opening of the transporter molecule toward the extracellular environment ( outward , to receive the substrate ) and an opening toward the intracellular environment ( inward , to release the substrate ) . the conformations of the gltph observed in the two available crystal structures are considered to represent , respectively , an outward - facing closed conformation ( ofcc ) ( figure 1a and 1c ) and an inward - facing closed conformation ( ifcc ) ( figure 1b and 1d ) . while the ofcc and ifcc structures present the substrate binding site in the respective directions , the site is occluded in both of them ( hence closed ) by two structural hairpin motifs that face the extracellular environment and intracellular cytoplasm , respectively . on the basis of these configurations , the structural information suggest that the outward- and inward - facing structures are the end - point conformations of gltph s substrate translocation path and thereby provide invaluable information about the structural relation between protein , substrate , and presumably sodium ions . outward - facing closed conformation ( ofcc ) ( a and c ) and inward - facing closed conformation ( ifcc ) ( b and d ) , viewed from the side , parallel to the membrane ( top ) and from the top ( bottom ) . sequence stretches in the n- and c - terminal loops that were unresolved in the crystal structures have been modeled using the program fugue , and parts of the tm34 loop , unresolved in the ofcc , were modeled with archpred , as described in methods . a comparison of the two crystal structures suggests that monomers undergo a rigid - body conformational rearrangement . however , the mechanism of substrate translocation across the membrane through the transporter molecule remains unknown despite the breakthrough crystallographic data and other observations from experimental and computational studies with regard to ion and substrate binding , extracellular capture , and intracellular release . to gain insight about the substrate translocation pathway ( stp ) at a detailed molecular level , we modeled structural intermediates along a putative transition path between the two crystal structures . these intermediates were constructed with the motion planning ( mp ) approach and analyzed with molecular dynamics ( md ) simulations and mixed elastic network models ( menm ) . together , they provide an all - atom model of the translocation path within each monomer in the context of the gltph trimer and the surrounding lipid membrane . we find that the modeled translocation path involves both the transport domain and the trimerization domain that were identified earlier in the crystal structures , moving in opposite directions ( and to different extents ) along the membrane normal . we also observe a tilt of the transport domain with respect to the membrane normal axis , and a global conformational change of the tm34 loop with respect to the transport domain . the md simulations of intermediate trimers constructed from initial identical monomers preserved their tertiary structure and the quaternary frame of the trimer , while one exhibited an asymmetric structure in which the monomers adopted different conformations . a striking change observed from the comparison of md simulation results from all systems ( including both the crystal structures and the modeled intermediates ) is the change in character of the interfaces between the transport and the trimerization domains ( transport trimerization domain interface , ttdi ) along the modeled stp . the pattern of changes in size and water accessibility of the ttdi indicates how the domain dynamics is facilitated along the translocation path and suggests the identity of specific residues that can be used to test experimentally the proposed stp modeled by the intermediates we calculated . to model conformations representing intermediates between the outward- and the inward - facing conformations observed crystallographically ( pdb codes 2nwx and 3kbc , respectively , figure 1 ) , we first used the motion planning module pathrover , implemented in the rosetta docking distribution . in pathrover , a protein structure is represented by the dihedral angles of its heavy - atom backbone and the c atoms . starting from this source representation , the conformational space of the protein is explored with a rapidly exploring random tree ( rrt ) algorithm , in our case using the rmsd minimize predicate ( which applies a transformation from dihedral to cartesian coordinates ) to bias the search toward a target conformation . here , the source and target are the monomers from an averaged , md - equilibrated state of the gltph trimer obtained from either the ofcc or the ifcc ( see molecular dynamics , md1 ) and they serve alternately . the mp paths are produced for single monomers , based on the experimental evidence that ( 1 ) individual monomers do transport substrate independently , ( 2 ) the ofcc and ifcc crystal structures preserve the trimerization interfaces in gltph , as indicated by superposition results ( c-rmsd = 0.5 of residues 6064 , 139161 , 183190 of all three monomers ) , and ( 3 ) residue pairs in these interfaces may be cross - linked without affecting substrate transport . the degrees of freedom for the conformational search included all the backbone dihedrals of residue i { 13 , ... , 414 } with i or i 20 , where i or i are the two average backbone dihedral differences between the ofcc and the ifcc for any residue i. the complete set of pathrover parameters we used is presented in table si12 , supporting information , and is default , except for max_tree_size and extend_max_step_size , which are , respectively , larger and smaller than in default , and energy_function , which is set to the centroid score4 function . the path - searching strategy was to use the path conformation with the lowest possible target rmsd from each run and to restart the search from this conformation . twelve such subsequent replicated restarts were used to construct an ensemble of mp conformations . predicate runs , which did not produce any paths with a target rmsd below 7.9 ( from an initial 9.7 ) for 25 initial replicated runs . to choose representative intermediates along this path ensemble , we grouped the mp conformations in ranges of 0.5 ( rmsdifcc.a ) and identified for each group the monomer with the lowest mean pairwise rmsd . the representative intermediates from five of these groups are denoted as pri.2 , pri.4 , pri.5 , pri.11 , and pri.12 . the starting structures for the md simulations of the ofcc and ifcc , respectively md1 and md2 , were taken from the corresponding pdb entries 2nwx and 3kbc . calculation of pka values for all titratable residues with the program mm_scp showed that each titratable residue in both the ofcc and the ifcc is likely in a protonation state corresponding to standard aqueous conditions at ph = 7.0 . to account for the recently reported transport stoichiometry of three sodium ions per substrate , we placed a third sodium ion ( na3 ) into the ofcc and the ifcc structures at a site near residues n310 , d312 , and t92 as identified in ref ( 19 ) . for the ofcc , residues missing from the tm34 loop were modeled with the template - based loop structure prediction software archpred . for the md2 simulations we constructed trimer intermediates , termed mdi.x ( x = 2 , 4 , 5 , 11 ) , from each selected monomer pri.x and added the substrate ( aspartate ) and sodium ions from the ofcc monomer to positions identified by superimposing the c atoms of hp1 and hp2 ( which interact with the substrate ) onto pri.x ( i.e. , of residues 262282 , 342367 , rmsds = 0.61.5 ) . finally , each pri.x monomer was triplicated and superimposed on each of the monomers of the ofcc s crystal structure by matching only the residues in the central inner cavity of gltph ( i.e. , c atoms of residues 6064 , 139161 , and 183190 ) . this procedure to construct trimer intermediates is made possible by the conserved structural frame of the trimerization domains observed in the crystal structures ( figure 1 ) and the symmetric monomer conformations in the ofcc and ifcc crystal structures . after this construction of a symmetric starting point , symmetry was not imposed on the monomers in the course of the md simulations , which nevertheless remained substantially symmetrical except for a special case of asymmetry discussed below ( section 3.3 ) . the n- and c - terminal end residues ( including residues 111 in the ofcc and mdi.x intermediates , 15 in the ifcc , 417422 in all conformations ) that were not resolved in the crystal structures were modeled using the homology server fugue and available crystal data from the ifcc . for md2 , internal water molecules were calculated for each starting conformation with the programs dowser and mmc ( these waters left the ttdi in the end conformation during the simulations ) . after adding missing protein hydrogen atoms with the vmd plugin psfgen , each starting conformation for the md1 and md2 protocol ( see below ) was embedded in a popc membrane bilayer model using guided positional information obtained from the program tip3p waters was added above and below each popc protein complex . with nacl concentrations at 150 mm ( vmd plugins solvate , autoionize ) , the systems are composed of 250 000300 000 atoms , including 50 00070 000 waters and 700900 lipid molecules in simulation boxes of 200 200 100 . all md simulations were performed with the all - atom charmm22 protein / water / ion force field ( using cmap corrections ) with the program namd . the popc molecules of the membrane were simulated with the charmm27 lipid force model in md1 ( input for motion planning ) and with charmm36 in md2 ( used to analyze the mdi.x intermediates and the end conformations ) ; charmm36 was not yet available for the md1 simulations . each system was equilibrated with md until all rmsds of the protein with respect to the trimer and monomers of the two crystal structures and the protein s starting conformation had converged ( not shown ) . as a result , the ofcc was equilibrated for 105 ns , the intermediate mdi.2 for 107 ns , mdi.4 for 164 ns , mdi.5 for 178 ns , mdi.11 for 173 ns , mdi.12 for 109 ns , and the ifcc for 115 ns ( the production phases were at least 40 ns long for each run ) , indicating that some of the modeled intermediates needed longer equilibration times than the crystal structures of the end conformations . we performed md simulations with an integration step of 1 fs for the first 5 ns of equilibration and 2 fs thereafter , a temperature of 310 k , a langevin damping coefficient of 5/ps , a nonbonded cutoff of 12 , switching distance of 10 , the particle - mesh - ewald algorithm to treat electrostatic interactions , and the shakeh algorithm to fix bonded interactions between hydrogens and heavy atoms ( rigidbonds all ) . the first 0.5 ns of each md equilibration was treated in the nvt and all subsequent phases of our md simulation were in the semi - isotropic npt ensemble ( p = 1 atm with a 200 fs nos . the transport domain and trimerization domain are defined based on the crystal structures ( i.e. , residues 76129 , 226422 and residues 175 , 130225 , respectively ) . helices are defined with the program stride in vmd applied to the last 16 ns of both the ofcc and the ifcc md - equilibrated trajectories , i.e. , residues 1430 , 4455 , 5965 , 83106 , 143146 , 151167 , 175199 , 208214 , 227243 , 248252 , 259262 , 266274 , 281290 , 298300 , 302303 , 305306 , 313328 , 339349 , 363364 , 378385 , and 396415 . the angle between tm6 and the membrane normal was measured using the anglebetweenhelices script in pymol . all rmsds in md were calculated with vmd considering the c atoms of the helical residues only . since the rotational orientation of our lipid protein system was well maintained throughout the simulations , we first translated the latest snapshot of each md - equilibrated simulation along the membrane normal , such that its membrane midplane would match the latest snapshot of the ofcc . each snapshot was then translated perpendicular to the membrane normal , so that the center of mass of the central cavity of the trimer ( c atoms of residues 6064 , 139161 , 183190 ) , projected on the midplane , matched the projected center of mass in the ofcc . distance matrices were computed with the vmd plugin itrajcomp from average cc distances over the last 16 ns of each md simulation . these matrices were then converted to pairwise distances between structural elements by averaging over all cc distances between each pair of these elements . relative solvent - accessible surface areas ( sasarel ) were computed with the program naccess on the last 16 ns of each simulation , with the default probe radius of 1.4 . on the basis of single - residue - accessibility studies , we defined a residue to be solvent accessible if its average relative solvent - accessibility surface area ( plus standard deviation ) was larger than the threshold sasarel , thres . of 11% and to be solvent inaccessible otherwise . a residue i is considered to be part of the ttdi if sasarel , i , isolated sasarel , i , isolated is the sasarel of residue i in the context of an isolated domain ( either transport or trimerization ) to which it belongs ; sasarel , i , complex is the sasarel of residue i in the context of the full trimerization / transport domain complex . this particular choice of sasattdi yields a fairly robust interface definition , as the number of ttdi residues in any of our monomers changed by < 16% when different values of sasattdi were used for the definition ( 13% , 14% , 16% , 17% ) . represent the sasas of the isolated trimerization and transport domains , respectively , and sasatrim.+trans . menms were constructed between minimized , md - equilibrated trimer conformations of the ofcc and ifcc . for these elastic networks , the uniform force constant was set to 0.03 kcal / mol / to normalize the calculated enm fluctuations of the end conformations ( at t = 310 k ) to their corresponding experimental b factors . the distance cutoff of 15 for the models was chosen to maximize the correlations between calculated fluctuations and b factors . the menm energy barriers were computed as described in ref ( 49 ) modifying an in - house enm python script . in the absence of experimental data about the relative distributions of the end conformations , the reduction of ttdi contacts was modeled as a reduction in 0.5 for all elastic bonds between the transport and the trimerization domains ( denoted as substrate translocation in gltph is a rare event , occurring on the time scale of micro- to milliseconds . therefore , in order to characterize a putative accessible path connecting the two end structures of the stp , we modeled conformations with the motion planning ( mp ) approach to obtain reasonable intermediate structures that can serve as input for md simulations . when applied starting from the outward- and the inward - facing conformations as described in methods , the mp computations resulted in an ensemble of 23 000 monomer conformations . figure 2 illustrates the progression of the structural intermediates obtained with this algorithm as expressed by their rmsd from the corresponding crystal structures of the monomers with chain identity a ( see pdb entries 2nwx and 3kbc ) . the plot of rmsdofcc.a against rmsdifcc.a ( figure 2 , black and gray points ) is calculated for the c atoms of the helices in the compared structures . it shows that the intermediate conformations determined with mp span the entire rmsd range of the rmsd between the monomers in the ofcc and ifcc end structures , rmsdo , i = 9.1 . rmsd landscape of all computed conformations along the modeled translocation path of gltph . monomer c-rmsds with respect to the a monomer in the ofcc crystal structure ( rmsdofcc.a ) are plotted against the ones calculated with respect to monomer a of the ifcc crystal structure ( rmsdifcc.a ) for all modeled mp monomers ( mpo2i and mpi2o , black and gray filled circles , respectively ) ; values of representative pri.x ( x = 2 , 4 , 5 , 11 , 12 ) intermediates are included . all monomers of the starting mdi.x ( x = 2 , 4 , 5 , 11 , 12 ) intermediates and crystal monomer conformations are included ( color - filled diamonds : ofcc in yellow , pri.2/starting mdi.2 in magenta , pri.4/starting mdi.4 in cyan , pri.5/starting mdi.5 in white , pri.11/starting mdi.11 , orange , pri.12/starting mdi.12 , purple , and ifcc , brown ) . red , blue , and green arrows point to the values for the corresponding monomers ( monomers a , b , and c , respectively ) after the md simulations done in the context of the trimer . the linear interpolation between the two crystal structures is drawn as a black dashed line . the representative intermediates pri.x ( x = 2 , 4 , 5 , 11 , 12 ) defined in methods are depicted in figure 3 in which their deviations from the crystal structures in figure 2 are marked by filled diamonds . the rearrangements are detectable in figure 3 in terms of a change in positioning of the transport domain ( orange ) relative to the trimerization domain ( green ) . in this modeled path the transport domain moves toward the intracellular cytoplasm , while the tertiary structure of both domains is largely preserved . the tm34 loop ( blue ) , which is expected to be highly flexible , appears fairly rigid along this path , although it is defined in the set of the degrees of freedom used by the motion planning algorithm ( see methods ) . participation of this loop in the transition is revisited in the md simulations discussed below . sequential series of structural intermediates in the substrate translocation path ( stp ) modeled with motion planning , superimposed on the ofcc and ifcc crystal structures . transport and trimerization domains are shown as orange and green ribbons , respectively ; the tm34 loop is colored in blue . to evaluate the feasibility of the pri.x intermediates calculated with the mp approach , each of the intermediates was used in turn to construct a symmetrical trimer mdi.x ( x = 2 , 4 , 5 , 11 , 12 ) , as described in methods . each one of these trimers and the two crystal trimers were simulated with all - atom md for at least 100 ns in the context of a solvated popc lipid membrane model . as seen from the positioning of the monomers in these trimers ( rmsd plot in figure 2 ) , the intermediate structures evolve into md - equilibrated conformations with rmsd values marked by the tip of the corresponding arrows ( figure 2 ) . in mdi.2 and mdi.11 , these equilibrated monomers have lower rmsdofcc.a ( higher rmsdifcc.a ) values compared to their starting values , whereas in mdi.5 and mdi.12 this trend is reversed . this indicates that the nearest feasible crystal structures are attraction points in the equilibration of the intermediates , with the exception of mdi.11 , which appears to be caught near an intermediate state . a special feature of asymmetry developed in mdi.4 , as individual monomers in this trimer were found to evolve toward either the ofcc ( monomers b and c ) or the ifcc ( monomer a ) , thus generating an asymmetric structure ; this is discussed further below ( section 3.3 ) . it is noteworthy that overall the converged rmsd values obtained from md fall near the ones generated with mp , indicating that the structures are consistent in the two approaches . comparison of the end - point crystal structures reveals large rearrangements of the transport domains , presumably outlining the changes required for substrate translocation . the corresponding conformational changes are evidenced in the md - equilibrated conformations changing from the outward - facing structure , via the five mdi.x intermediates , to the inward - facing structure ; this is illustrated in figure 4 with respect to the surrounding lipid membrane . along this modeled translocation path in the trimer , the three transport domains ( orange ribbons ) are seen to transition from the ofcc , along the membrane s normal axis , toward the intracellular cytoplasm , displaced by as much as 11 1 . this displacement is indicated by the change in position of the red bead marking their center of mass in all panels of figure 4 . these rearrangements of the transport domains are opposite in direction and much larger than those observed for the trimerization domains ( green ribbons in figure 4 ) . by following the change in position of the trimerization domain s center of mass ( blue bead ) in traversing the panels from the ofcc to the ifcc the repositioning of the domain is seen to be toward the extracellular cytoplasm by as much as 4 1 along the same axis . notably , there is also a tilt of the transport domain , measured by changes in the angle between tm6 and the membrane s normal axis . as shown in figure 4 , the value of ( averaged over all three monomers ) changes along the modeled stp from 27 3 in the ofcc to 44 3 in the ifcc , which corresponds to a tilt of the transport domain by 17 4. md - equilibrated trimers of the modeled substrate translocation path ( stp ) for gltph shown in the reference frame of the lipid membrane ( phosphor atoms as gray beads , lipid tail carbon atoms as cyan lines ) for ( a ) ofcc , ( b ) ifcc , ( c ) mdi.4 , and ( d ) mdi.5 . the center of mass of the three transport domains ( orange ) is indicated by a red sphere ; the center of mass for the trimerization domains ( green ) is marked by a blue sphere ; residues 130168 ( tm4a c ) in monomer b were omitted for clarity . the angle between the membrane normal ( black bar ) and tm6 of monomer a ( purple helix ) is inscribed in black . the structural integrity of the simulated systems that exhibit these large rearrangements is evident in the distance difference matrices ( ddms ) . as described in methods , these matrices were constructed from distances between structural segments ( i.e. , tm16 , hp1 , tm7 , hp2 , and tm8 of each monomer ) with respect to the outward - facing crystal structure ( figure si2 , supporting information ) . the positions of the regions where the absolute differences are small , <3 , show that the tertiary structure of each monomer as well as the trimeric frame of the quaternary structure are largely preserved along the modeled translocation path . because the monomers in gltph and eaats have been suggested to transport substrate independently , we investigated whether monomers can adopt different conformations in our md - equilibrated stp trimers . table si3 , supporting information , shows that mdi.4 has the highest average rmsd between monomers ( 4.2 0.1 ) , whereas the simulated ofcc and ifcc structures have the lowest ones of 1.0 0.1 and 1.2 0.1 , respectively . this observation suggests that the mdi.4 trimer is more asymmetric than other trimers along our modeled translocation path . the structural context of this asymmetry is evident from the comparison of two mdi.4 monomers in figure 5 . clearly , the transport domain is positioned differently in monomer b ( figure 5a ) than in monomer a ( figure 5b ) , i.e. , more toward the extracellular region ( into the figure plane ) in monomer b than in monomer a. this observation is recorded by smaller values of rmsdofcc.a ( and larger rmsdifcc.a ) for monomers b and c than for monomer a ( figure 2 ) . two monomers illustrate the conformational asymmetry of the mdi.4 trimer intermediate : the transport domain in monomer a of mdi.4 ( mdi.4.a ) ( b ) has moved deeper toward the intracellular region than in monomer mdi.4.b ( a ) ( the md starting conformations of all monomers in a trimer are identical ) . changes in the tm34 loop conformation ( in green ) along the modeled translocation path are shown with respect to every other residue of the monomer : each such residue is colored by an average correlation coefficient ( see text ) , i.e. , in red if the residue tends to get closer to the tm34 loop along the path from the outward- to the inward - facing conformation and in blue if it tends to move farther away from the tm34 loop ; residues colored in white are not correlated , on average , with the tm34 loop motion ( tm34 is necessary for substrate transport and undergoes substrate - dependent conformational changes ) . a recent study on gltph suggested that the tm34 loop is essential for substrate transport and undergoes substrate - induced conformational changes . before investigating these changes along our modeled translocation path , we identified a conformational difference of this loop between monomer a and monomer b in mdi.4 , which coincides with the different positioning of the transport domain , described above ( figure 5 ) : with respect to the extracellular part of the transport domain , the tm34 loop ( shown in green ) is shifted inward in monomer b ( figure 5a ) compared to the one in monomer a ( figure 5b ) . thus , in monomer b this loop is closer to the tip of hp2 ( average cc distance between residues 108127 and 352357 : d108127,352357 = 20.7 0.7 ) than to the loop between tm7b and hp2a ( d108127,330337 = 23.8 0.3 ) . for monomer a , which is closer to the intracellular end , we observe the opposite trend for the position of the tm34 loop ( d108127,352357 = 32.1 0.5 , d108127,330337 = 13.1 0.2 ) . to study the dynamics of loop tm34 along the modeled transition , we constructed a translocation path series from all 21 monomers of the six averaged , md - equilibrated trimers ordered by the transition parameter stp = ( + rmsdo , i)/(2 rmsdo , i ) , where rmsdofcc.a rmsdifcc.a and rmsdo , i 9.1 is the rmsd between the ofcc and ifcc crystal monomers . to capture the loop s different conformations along the path , we measured all possible values of dij , defined as the average ( over the last 16 ns of each simulation ) of cc distance between a tm34 loop residue i , and any non - tm34 loop residue j of the same monomer . the pearson correlation coefficients cij between each dij and stp quantify the trends of changes of the loop s conformation along the modeled translocation path series . figure 5 presents average values of the cij over all residues i ( shown in green ) mapped onto each residue j. negative correlation coefficients indicate that the distance to the tm34 loop tends to decrease along the transition from the outward - facing toward the inward - facing conformation ; positive correlations indicate residues for which the distance increases along the transition path . consequently , figure 5 indicates the correlation between the movement of the transport domain and that of the tm34 loop is such that as the transport domain moves toward ( or away from ) the intracellular end , the tm34 loop moves outward ( or inward ) , respectively , relative to the extracellular part of the transport domain . in movies available in si4 , supporting information , we present a molecular illustration of the rearrangements along stp , in an individual monomer , including the conformational changes of loop tm34 . the rearrangement of the spatial relationship between the transport and the trimerization domains is interpreted from a comparison of the crystallographic data as a change in their interface . similar comparisons among the mdi.x intermediates and the end states reveal gradual changes in this interface ( ttdi ) along the modeled transition path . to characterize these changes quantitatively we used differences in relative solvent - accessible surface areas ( sasas ) calculated as described in methods . figure 6 illustrates side views of the superimposed transport and trimerization domain of monomer a in the ofcc ( a ) , mdi.4 ( b ) , and ifcc ( c ) together with the corresponding ttdi residues . side of the ttdi is seen to be largely preserved among these conformations and contains residues of transmembrane segments ( tm ) tm1 and 2 , tm4a / c , and tm5 . side of the ttdi are greater in all three monomers , with the contacting residues coming from tm6 , hairpin motif hp2a , and tm8a . additional interface contacts are observed in some but not all monomers : residue contacts with hp1b and tm7a are formed only in the ofcc , residue contacts with hp1a are formed only in the ofcc and mdi.4 , and contacts with hp2b are formed only in mdi.4 and the ifcc . in figure 6 , the ttdi in monomer a of mdi.4 ( i.e. , mdi.4.a ) is seen to have a smaller area than the end states . in fact , the ttdi areas in the end conformations reach as high as 4000 , whereas in the intermediates they may be as low as 2900 . this intermediate reduction may affect greatly the energies of interactions and thus the dynamics of the interface along the translocation path . to estimate these effects in the form of changes in transition energy barriers between the outward- and the inward - facing conformations , we used mixed elastic network models ( menms ) in which interactions are modeled as elastic bonds . in figure si5 , supporting information , we plot the transition energy profiles of the occifcc menms ( w/ , red ) and the ones with reduced ttdi contacts ( see methods ) in the ofcc ( w/<_f0 , blue ) or the ifcc ( w/<_f1 , green ) . for this menm , a reduction of ttdi contacts in either conformation lowers the transition energy barrier , indicating increased flexibility in the menm transition between the two end conformations . trimerization domain interface ( ttdi ) along the modeled translocation path : ( a ) monomer a of the ofcc , ( b ) mdi.4 , and ( c ) ifcc . residues at the interface are rendered as white surfaces on the structural cartoon of the transport domain ( left , orange ribbons ) and trimerization domain ( right , green ribbons ) , respectively . concomitant with the reduction in the ttdi surface area , water molecules are observed to penetrate the ttdi region . this is illustrated by a snapshot of mdi.4.a in which a pore ( semitransparent colored surfaces ) of 20 water molecules is seen in the ttdi region ( figure 7b ) . note that this water pore does not form in either the outward- ( figure 7a ) or the inward - facing conformation ( figure 7c ) . on average , i.e. , over the last 16 ns of each equilibrated trajectory ( table si6 , supporting information ) , the space at the ttdi contains as much as nwater = 15 3 water molecules in the intermediates , significantly more than in the end conformations , which have no more than nwater = 5 1 . the interface between the transport and the trimerization domain ( ttdi ) shows water penetrating in the intermediates but much less so in the end - point conformations . the ttdi is shown for monomer a of the simulated ofcc ( a ) , mdi.4 ( b ) , and ifcc ( c ) from the structures shown in figure 4 . explicit water molecules within 8 of residues 1530 ( tm1 ) and 207216 ( tm5 ) are visualized as red white sticks ; computed water pores are rendered as surfaces colored from purple via cyan to blue to represent small to large pore radii . residues 3645 ( tm2 ) and 190222 ( tm5 ) are omitted for clarity . the results indicating the existence of a water pore at the ttdi observed in the intermediates but much less so in the end states can be compared to results from experimental measurements of solvent accessibilities in the literature . to this end , we first determined the particular set x of ttdi residues that are solvent inaccessible in any monomer of the end conformations ( see methods ) but are solvent accessible in the intermediates ( i.e. , for at least one intermediate monomer ) . we then classified any residue in the set x based on three criteria related to available experimental data ( table 1 and figure si7 , supporting information ) as successful scam prediction if this position is known ( e.g. , from published scams ) to be solvent accessible ( in either gltph or its corresponding site in an eaat homologue ) and unsuccessful scam prediction if this position has been tested , e.g. , with scam , but not found to be solvent accessible . those residues that should be accessible but to the best of our knowledge have not been reported out from any solvent - accessibility measurements . the three successful scam positions identified in this way among the nine positions in the ttdi support the validity of our model and the identified water pores at the ttdi . we predict that residue v209 , the only apparently unsuccessful scam prediction residue , would become accessible with scam using a smaller reagent than mts , such as hgcl , as was used in ref ( 5 ) . we note the remaining five positions identified here as new scam prediction positions that can be tested experimentally to further validate and understand the stp model . solvent - accessible residues predicted from the simulations are listed as successful scam prediction if the position is known ( e.g. , from published scams ) to be solvent accessible in gltph or its corresponding site of an eaat homologue ; it is identified as unsuccessful scam prediction if this position has been tested , e.g. , with scam but not found to be solvent accessible so far ; it is listed in new scam predictions if to date , to the best of our knowledge , no solvent - accessibility measurements have been performed on this residue in gltph or its eaat counterpart . residues marked as also showed some solvent accessibilty in the end conformations , measuring one or more average water molecules within 3 of the residue s side chain ( see figure si7 , supporting information ) . in the max sasarel column , for each residue in the set x we list the maximum sasarel values observed in the ofcc and ifcc . the availability of crystal structures for the outward- and inward - facing states of gltph has given a strong structural context to the studies of gltph and eaats that identify mechanistic elements of binding , extracellular capture , and intracellular release of both substrate and ions . since these two structures are considered to represent the end states of gltph s substrate translocation mechanism , it became clear from their differences that significant rearrangements of key functional domains are central to this mechanism . in spite of the very attractive hypothesis presented on the basis of these two end - point structures , the path leading from the outward- to the inward - facing state remained unexplored . in the present study , we obtained and evaluated dynamic molecular models of structural intermediates along gltph s substrate translocation path obtained from application of a combination of computational methods ( motion planning , molecular dynamics , mixed elastic network models ) to seek out salient conformational changes and mechanistic elements . motion planning , with which we calculated intermediate conformations of gltph monomers , is well known in computer science and robotics and was recently adapted to study proteins as large as the kcsa potassium channel . it was selected here as a relatively inexpensive computational tool for modeling the transition paths in terms of clash - free conformations as input for md simulations exploring the dynamic properties of these states . from the md simulations of the intermediate structures identified with pathrover , our results specify the rearrangements in the reference frame of the surrounding lipid membrane in terms of relative movements between the transport and the trimerization domains . these movements are in opposite directions along the membrane normal , and the computational results confirm as well a tilt of the transport domain with respect to this axis ( cf . . the dynamic role of the tm34 loop in the mechanistic substrate translocation pathway model resulting from our study agrees with the suggestion that this loop is involved in gltph s substrate transport , undergoing substrate - induced conformational changes . indeed , a comparison between the outward- and the inward - facing crystal structures suggests a repositioning of this loop with respect to the transport domain . however , since parts of the 60 loop are unresolved in the outward - facing crystal structure , it remained unclear whether this conformational difference distinguishes the ofcc from the ifcc and how this conformational change is accomplished . our study proposes a path for this conformational change of the loop with respect to the transport domain , which correlates well with the larger transitions along the translocation path ( see movies in si4 , supporting information ) . comparing the conformations of the tm34 loop in our modeled pri.x ( figure 3 ) and mdi.x ( figures 4 and 5 ) intermediates , we observed that after motion planning alone ( figure 3 ) this loop appears intrinsically less flexible than is expected from the data for residue - specific , substrate - dependent solvent - accessibility changes in this loop . much longer md simulations will be required to cover the likely complex spectrum of dynamics of this 60 long loop . we observed in our results a specific dynamic context for available experimental data indicating that gltph and eaat monomers function independently and move stochastically and independently . this appeared in one of the md simulations where a trimer intermediate was shown capable of adopting structural asymmetry , despite the fact that all the starting structures had identical monomer conformations ( figure 5 ) . the mixed elastic network model approach , which had been used successfully to reproduce protein fluctuations from crystallographic data to predict large collective conformational changes and to identify minimum energy conformations therein , served here to examine further the dynamics of the transition between the outward- and the inward - facing conformation . combined with the structural pathway indicated by our mp and md simulations , the findings provide details of the pathway that agrees substantially with the inferences from the crystal structures of gltph regarding a transport mechanism in which the transport domain undergoes a large vertical repositioning with respect to the trimerization domain in a rearrangement inferred to involve a rigid - bundle movement that preserves gltph s tertiary structure and its quaternary frame . however , accessibility studies using the scam method suggested that the intermediates between the two crystal structures representing the ofcc and the ifcc must involve intricate rearrangements . in particular , these studies identified as accessible some residues positioned in the interface between the transport and the trimerization domains ( the ttdi ) , although this interface is compact in both crystal structures , and not suggestive of solvent ( or reagent ) accessibility to the sites identified as reactive with the scam approach . while remaining in agreement with the inferences from crystallographic data , our model of the translocation path resolves this apparent conflict as it describes significant changes in contact area of the ttdi in transition intermediates and shows significant water penetration in the interface region in the intermediate conformations equilibrated computationally . thus , the contact area of the ttdi is reduced along our modeled translocation path by nearly 20% compared to the end states . this reduction is consistent with facilitated dynamics along gltph s substrate translocation path ( figure si5 , supporting information ) and exposes some of the residues near the ttdi to solvent ( figure 7 and table si6 , supporting information ) . our model is supported on a residue - specific level by experimental accessibility ( scam ) results . in contrast to the interface observed in either the outward- or the inward - facing structure of gltph , which would not allow reagent accessibility to residues i61 , i213 , and m395 , we show these to be accessible in the intermediates . moreover , we predict residues l54 , v62 , l66 , v198 , and a205 to become solvent accessible in the intermediates but not in the end conformations of gltph s substrate translocation path , thus suggesting direct modes of experimental validation of the modeled stp , e.g. , via scams . with our current approach , we were unable to distinguish whether the water pore along the modeled stp is initiated from the extra- or intracellular region . during the final preparation of this manuscript , a new crystal structure of an asymmetric intermediate of gltph was reported featuring two monomers in the inward - facing conformation ( rmsdifcc.a = 0.7 ) and one monomer in an intermediate conformation ( rmsdofcc.a = 3.0 , rmsdifcc.a = 6.8 ) . this structure ( pdb accession code 3v8 g ) exhibits remarkable similarity to the first intermediates in our modeled stp . , our mp conformations have a minimum rmsdiofs.c of 1.9 ( not shown ) . the pri.2 conformation has the lowest rmsdiofs.c ( 2.3 ) among all pri.x intermediates ( table si8 , supporting information ) . notably , the mdi.2 monomers conformations exhibit even lower minimum rmsdiofs.c values ( 1.7 ) ( table si9 , supporting information ) , with the rmsdiofs.c for their average structure being 1.8 ( figure si10 , supporting information ) . all these rmsd values are smaller than the crystallographic resolution of 3.5 for the gltph - s . taken together , these structural comparisons support the validity of the intermediates modeled from our computations , which have remarkable resemblance to available crystallographic data of gltph .

excitatory amino acid transporters ( eaats ) are membrane proteins responsible for reuptake of glutamate from the synaptic cleft to terminate neurotransmission and help prevent neurotoxically high , extracellular glutamate concentrations . important structural information about these proteins emerged from crystal structures of gltph , a bacterial homologue of eaats , in conformations facing outward and inward . these remarkably different conformations are considered to be end points of the substrate translocation path ( stp ) , suggesting that the transport mechanism involves major conformational rearrangements that remain uncharted . to investigate possible steps in the structural transitions of the stp between the two end - point conformations , we applied a combination of computational modeling methods ( motion planning , molecular dynamics simulations , and mixed elastic network models ) . we found that the conformational changes in the transition involve mainly the repositioning the transport domain and the trimerization domain identified previously in the crystal structures . the two domains move in opposite directions along the membrane normal , and the transport domain also tilts by 17 with respect to this axis . moreover , the tm34 loop undergoes a flexible , restraining bar-like conformational change with respect to the transport domain . as a consequence of these conformational rearrangements along the transition path we calculated a significant decrease of nearly 20% in the area of the transport - to - trimerization domain interface ( ttdi ) . water penetrates parts of the ttdi in the modeled intermediates but very much less in the end - point conformations . we show that these characteristics of the modeled intermediate states agree with experimental results from residue - accessibility studies in individual monomers and identify specific residues that can be used to test the proposed stp . moreover , md simulations of complete gltph trimers constructed from initially identical monomer intermediates suggest that asymmetry can appear in the trimer , consonant with available experimental data showing independent transport kinetics by individual monomers in the trimers .

Introduction Methods Results Discussion

excitatory amino acid transporters ( eaats ) are membrane proteins that remove glutamate from the synaptic environment to terminate neurotransmission and help prevent neurotoxicity caused by high concentrations of the neurotransmitter . a major breakthrough toward a molecular mechanistic understanding of substrate transport in eaats has been the elucidation of crystal structures of gltph , a bacterial homologue that shares about 37% sequence identity with eaats . this symmetry is remarkable given the fact that the monomers in gltph and eaat have been suggested to transport substrate independently and are expected to move stochastically and independently the neurotransmitter transport mechanism has long been considered to involve a sequential opening of the transporter molecule toward the extracellular environment ( outward , to receive the substrate ) and an opening toward the intracellular environment ( inward , to release the substrate ) . on the basis of these configurations , the structural information suggest that the outward- and inward - facing structures are the end - point conformations of gltph s substrate translocation path and thereby provide invaluable information about the structural relation between protein , substrate , and presumably sodium ions . sequence stretches in the n- and c - terminal loops that were unresolved in the crystal structures have been modeled using the program fugue , and parts of the tm34 loop , unresolved in the ofcc , were modeled with archpred , as described in methods . to gain insight about the substrate translocation pathway ( stp ) at a detailed molecular level , we modeled structural intermediates along a putative transition path between the two crystal structures . these intermediates were constructed with the motion planning ( mp ) approach and analyzed with molecular dynamics ( md ) simulations and mixed elastic network models ( menm ) . we find that the modeled translocation path involves both the transport domain and the trimerization domain that were identified earlier in the crystal structures , moving in opposite directions ( and to different extents ) along the membrane normal . we also observe a tilt of the transport domain with respect to the membrane normal axis , and a global conformational change of the tm34 loop with respect to the transport domain . the md simulations of intermediate trimers constructed from initial identical monomers preserved their tertiary structure and the quaternary frame of the trimer , while one exhibited an asymmetric structure in which the monomers adopted different conformations . a striking change observed from the comparison of md simulation results from all systems ( including both the crystal structures and the modeled intermediates ) is the change in character of the interfaces between the transport and the trimerization domains ( transport trimerization domain interface , ttdi ) along the modeled stp . the pattern of changes in size and water accessibility of the ttdi indicates how the domain dynamics is facilitated along the translocation path and suggests the identity of specific residues that can be used to test experimentally the proposed stp modeled by the intermediates we calculated . to model conformations representing intermediates between the outward- and the inward - facing conformations observed crystallographically ( pdb codes 2nwx and 3kbc , respectively , figure 1 ) , we first used the motion planning module pathrover , implemented in the rosetta docking distribution . the mp paths are produced for single monomers , based on the experimental evidence that ( 1 ) individual monomers do transport substrate independently , ( 2 ) the ofcc and ifcc crystal structures preserve the trimerization interfaces in gltph , as indicated by superposition results ( c-rmsd = 0.5 of residues 6064 , 139161 , 183190 of all three monomers ) , and ( 3 ) residue pairs in these interfaces may be cross - linked without affecting substrate transport . the degrees of freedom for the conformational search included all the backbone dihedrals of residue i { 13 , ... , 414 } with i or i 20 , where i or i are the two average backbone dihedral differences between the ofcc and the ifcc for any residue i. the complete set of pathrover parameters we used is presented in table si12 , supporting information , and is default , except for max_tree_size and extend_max_step_size , which are , respectively , larger and smaller than in default , and energy_function , which is set to the centroid score4 function . this procedure to construct trimer intermediates is made possible by the conserved structural frame of the trimerization domains observed in the crystal structures ( figure 1 ) and the symmetric monomer conformations in the ofcc and ifcc crystal structures . each system was equilibrated with md until all rmsds of the protein with respect to the trimer and monomers of the two crystal structures and the protein s starting conformation had converged ( not shown ) . as a result , the ofcc was equilibrated for 105 ns , the intermediate mdi.2 for 107 ns , mdi.4 for 164 ns , mdi.5 for 178 ns , mdi.11 for 173 ns , mdi.12 for 109 ns , and the ifcc for 115 ns ( the production phases were at least 40 ns long for each run ) , indicating that some of the modeled intermediates needed longer equilibration times than the crystal structures of the end conformations . since the rotational orientation of our lipid protein system was well maintained throughout the simulations , we first translated the latest snapshot of each md - equilibrated simulation along the membrane normal , such that its membrane midplane would match the latest snapshot of the ofcc . each snapshot was then translated perpendicular to the membrane normal , so that the center of mass of the central cavity of the trimer ( c atoms of residues 6064 , 139161 , 183190 ) , projected on the midplane , matched the projected center of mass in the ofcc . a residue i is considered to be part of the ttdi if sasarel , i , isolated sasarel , i , isolated is the sasarel of residue i in the context of an isolated domain ( either transport or trimerization ) to which it belongs ; sasarel , i , complex is the sasarel of residue i in the context of the full trimerization / transport domain complex . in the absence of experimental data about the relative distributions of the end conformations , the reduction of ttdi contacts was modeled as a reduction in 0.5 for all elastic bonds between the transport and the trimerization domains ( denoted as substrate translocation in gltph is a rare event , occurring on the time scale of micro- to milliseconds . therefore , in order to characterize a putative accessible path connecting the two end structures of the stp , we modeled conformations with the motion planning ( mp ) approach to obtain reasonable intermediate structures that can serve as input for md simulations . red , blue , and green arrows point to the values for the corresponding monomers ( monomers a , b , and c , respectively ) after the md simulations done in the context of the trimer . the rearrangements are detectable in figure 3 in terms of a change in positioning of the transport domain ( orange ) relative to the trimerization domain ( green ) . the tm34 loop ( blue ) , which is expected to be highly flexible , appears fairly rigid along this path , although it is defined in the set of the degrees of freedom used by the motion planning algorithm ( see methods ) . sequential series of structural intermediates in the substrate translocation path ( stp ) modeled with motion planning , superimposed on the ofcc and ifcc crystal structures . comparison of the end - point crystal structures reveals large rearrangements of the transport domains , presumably outlining the changes required for substrate translocation . the corresponding conformational changes are evidenced in the md - equilibrated conformations changing from the outward - facing structure , via the five mdi.x intermediates , to the inward - facing structure ; this is illustrated in figure 4 with respect to the surrounding lipid membrane . along this modeled translocation path in the trimer , the three transport domains ( orange ribbons ) are seen to transition from the ofcc , along the membrane s normal axis , toward the intracellular cytoplasm , displaced by as much as 11 1 . by following the change in position of the trimerization domain s center of mass ( blue bead ) in traversing the panels from the ofcc to the ifcc the repositioning of the domain is seen to be toward the extracellular cytoplasm by as much as 4 1 along the same axis . notably , there is also a tilt of the transport domain , measured by changes in the angle between tm6 and the membrane s normal axis . as shown in figure 4 , the value of ( averaged over all three monomers ) changes along the modeled stp from 27 3 in the ofcc to 44 3 in the ifcc , which corresponds to a tilt of the transport domain by 17 4. md - equilibrated trimers of the modeled substrate translocation path ( stp ) for gltph shown in the reference frame of the lipid membrane ( phosphor atoms as gray beads , lipid tail carbon atoms as cyan lines ) for ( a ) ofcc , ( b ) ifcc , ( c ) mdi.4 , and ( d ) mdi.5 . the positions of the regions where the absolute differences are small , <3 , show that the tertiary structure of each monomer as well as the trimeric frame of the quaternary structure are largely preserved along the modeled translocation path . changes in the tm34 loop conformation ( in green ) along the modeled translocation path are shown with respect to every other residue of the monomer : each such residue is colored by an average correlation coefficient ( see text ) , i.e. , in red if the residue tends to get closer to the tm34 loop along the path from the outward- to the inward - facing conformation and in blue if it tends to move farther away from the tm34 loop ; residues colored in white are not correlated , on average , with the tm34 loop motion ( tm34 is necessary for substrate transport and undergoes substrate - dependent conformational changes ) . before investigating these changes along our modeled translocation path , we identified a conformational difference of this loop between monomer a and monomer b in mdi.4 , which coincides with the different positioning of the transport domain , described above ( figure 5 ) : with respect to the extracellular part of the transport domain , the tm34 loop ( shown in green ) is shifted inward in monomer b ( figure 5a ) compared to the one in monomer a ( figure 5b ) . to study the dynamics of loop tm34 along the modeled transition , we constructed a translocation path series from all 21 monomers of the six averaged , md - equilibrated trimers ordered by the transition parameter stp = ( + rmsdo , i)/(2 rmsdo , i ) , where rmsdofcc.a rmsdifcc.a and rmsdo , i 9.1 is the rmsd between the ofcc and ifcc crystal monomers . to capture the loop s different conformations along the path , we measured all possible values of dij , defined as the average ( over the last 16 ns of each simulation ) of cc distance between a tm34 loop residue i , and any non - tm34 loop residue j of the same monomer . figure 5 presents average values of the cij over all residues i ( shown in green ) mapped onto each residue j. negative correlation coefficients indicate that the distance to the tm34 loop tends to decrease along the transition from the outward - facing toward the inward - facing conformation ; positive correlations indicate residues for which the distance increases along the transition path . consequently , figure 5 indicates the correlation between the movement of the transport domain and that of the tm34 loop is such that as the transport domain moves toward ( or away from ) the intracellular end , the tm34 loop moves outward ( or inward ) , respectively , relative to the extracellular part of the transport domain . the rearrangement of the spatial relationship between the transport and the trimerization domains is interpreted from a comparison of the crystallographic data as a change in their interface . similar comparisons among the mdi.x intermediates and the end states reveal gradual changes in this interface ( ttdi ) along the modeled transition path . to estimate these effects in the form of changes in transition energy barriers between the outward- and the inward - facing conformations , we used mixed elastic network models ( menms ) in which interactions are modeled as elastic bonds . for this menm , a reduction of ttdi contacts in either conformation lowers the transition energy barrier , indicating increased flexibility in the menm transition between the two end conformations . trimerization domain interface ( ttdi ) along the modeled translocation path : ( a ) monomer a of the ofcc , ( b ) mdi.4 , and ( c ) ifcc . the interface between the transport and the trimerization domain ( ttdi ) shows water penetrating in the intermediates but much less so in the end - point conformations . the results indicating the existence of a water pore at the ttdi observed in the intermediates but much less so in the end states can be compared to results from experimental measurements of solvent accessibilities in the literature . to this end , we first determined the particular set x of ttdi residues that are solvent inaccessible in any monomer of the end conformations ( see methods ) but are solvent accessible in the intermediates ( i.e. in spite of the very attractive hypothesis presented on the basis of these two end - point structures , the path leading from the outward- to the inward - facing state remained unexplored . in the present study , we obtained and evaluated dynamic molecular models of structural intermediates along gltph s substrate translocation path obtained from application of a combination of computational methods ( motion planning , molecular dynamics , mixed elastic network models ) to seek out salient conformational changes and mechanistic elements . from the md simulations of the intermediate structures identified with pathrover , our results specify the rearrangements in the reference frame of the surrounding lipid membrane in terms of relative movements between the transport and the trimerization domains . these movements are in opposite directions along the membrane normal , and the computational results confirm as well a tilt of the transport domain with respect to this axis ( cf . indeed , a comparison between the outward- and the inward - facing crystal structures suggests a repositioning of this loop with respect to the transport domain . our study proposes a path for this conformational change of the loop with respect to the transport domain , which correlates well with the larger transitions along the translocation path ( see movies in si4 , supporting information ) . comparing the conformations of the tm34 loop in our modeled pri.x ( figure 3 ) and mdi.x ( figures 4 and 5 ) intermediates , we observed that after motion planning alone ( figure 3 ) this loop appears intrinsically less flexible than is expected from the data for residue - specific , substrate - dependent solvent - accessibility changes in this loop . the mixed elastic network model approach , which had been used successfully to reproduce protein fluctuations from crystallographic data to predict large collective conformational changes and to identify minimum energy conformations therein , served here to examine further the dynamics of the transition between the outward- and the inward - facing conformation . combined with the structural pathway indicated by our mp and md simulations , the findings provide details of the pathway that agrees substantially with the inferences from the crystal structures of gltph regarding a transport mechanism in which the transport domain undergoes a large vertical repositioning with respect to the trimerization domain in a rearrangement inferred to involve a rigid - bundle movement that preserves gltph s tertiary structure and its quaternary frame . however , accessibility studies using the scam method suggested that the intermediates between the two crystal structures representing the ofcc and the ifcc must involve intricate rearrangements . in particular , these studies identified as accessible some residues positioned in the interface between the transport and the trimerization domains ( the ttdi ) , although this interface is compact in both crystal structures , and not suggestive of solvent ( or reagent ) accessibility to the sites identified as reactive with the scam approach . while remaining in agreement with the inferences from crystallographic data , our model of the translocation path resolves this apparent conflict as it describes significant changes in contact area of the ttdi in transition intermediates and shows significant water penetration in the interface region in the intermediate conformations equilibrated computationally . thus , the contact area of the ttdi is reduced along our modeled translocation path by nearly 20% compared to the end states . in contrast to the interface observed in either the outward- or the inward - facing structure of gltph , which would not allow reagent accessibility to residues i61 , i213 , and m395 , we show these to be accessible in the intermediates . moreover , we predict residues l54 , v62 , l66 , v198 , and a205 to become solvent accessible in the intermediates but not in the end conformations of gltph s substrate translocation path , thus suggesting direct modes of experimental validation of the modeled stp , e.g.

the plasma membrane serves as a highly dynamic platform for binding of peripheral membrane proteins and their complexes , a phenomenon that is involved in the regulation of a wide range of cellular and physiological processes , e.g. , intercellular signaling , membrane trafficking , and coagulation . as the association of peripheral membrane proteins is often intended to be transient , these proteins have evolved specialized domains , termed membrane anchoring domains , which are responsible for reversibly binding to the membrane . these membrane - anchoring domains take advantage of different molecular strategies ( various types of specific lipid protein interactions ) in achieving membrane affinities tuned to the function of the proteins , although the binding can be broadly viewed as a combination of electrostatic interactions with ( often anionic ) lipids and hydrophobic interactions with the apolar core of the membrane . in many cases , membrane binding constitutes a step in the activation of the protein , a process which is chiefly controlled through modulation of the lipid composition of the membrane in specific areas . developing a molecular view of how membrane - anchoring domains interact with the membrane is therefore crucial to our understanding of peripheral proteins function . despite the high relevance of the phenomenon , due to technical challenges involved in obtaining high - resolution information on the process of membrane insertion / binding , there have been relatively few studies achieving atomistic details on the membrane - bound forms of peripheral proteins.(9 ) furthermore , different experimental approaches have often resulted in different membrane - bound models for the same anchoring domain , adding to the uncertainty of the problem . for instance , a variety of experimental techniques , including epr , fluorescent labeling,(12 ) and nmr,(14 ) have been utilized to characterize the membrane - bound forms of the c2 domain , a common anchoring domain in signal transduction and membrane trafficking of proteins . these experiments , however , have resulted in membrane - bound models ranging from 5.2 insertion and making an angle of 77 with the membrane normal to the domain being inserted 10 and making an angle of 52 with the membrane normal . the roles of the three ca - binding loops ( cbl ) and the -groove in membrane binding are also disputed.(16 ) similar discrepancies are found in the studies of the gla domains , the anchoring domain of vitamin k - dependent coagulation factors , where the relative orientation and depth of penetration into the membrane are largely disputed , and the fyve domain , where the membrane binding motif has not even been characterized . ( clarification : gla refers to -carboxyglutamic acid ; gla refers to a gla - rich anchoring domain . ) computational studies on membrane anchoring domains have proven equally difficult . in contrast to integral membrane proteins , whose membrane binding mode is fairly well understood , and for which even semiautomated insertion protocols exist , there are currently no robust methods to determine how deep and in what orientation a peripheral membrane protein will insert into a biological membrane , or even in some cases which residues comprise the membrane anchor . these anchors usually comprise a small portion of the anchoring domain and could be as small as a few amino acids , making the prediction of the anchors rather difficult . compounded further by the need of describing the protein s structural changes that are induced / accompanied by insertion into the membrane , especially with regard to the side chains in and around the anchor region , and the slow diffusion of lipid molecules around the insertion point , describing the process of membrane insertion has proven extremely challenging in simulation studies . of the small number of computational studies conducted on peripheral proteins , a majority have focused on only a handful of anchoring domains , namely , the bar , c2,(31 ) gla,(21 ) fyve,(32 ) px , and membrane binding domain of prostaglandin h2 synthase . naturally , most of these studies had to rely on initial embedding ( partially or fully ) of the anchoring domain into the lipid bilayer . while these studies have provided important information on how the anchoring domains might be stabilized within the membrane , they give little detail on the extremely dynamic process of insertion . modeling the membrane - bound form in these studies also requires a priori knowledge of the anchoring region ( the anchor ) . in addition , manually embedding the protein in the membrane introduces biased sampling and might restrict the states available to the protein . however , in order to observe membrane insertion within the time scales accessible to molecular dynamics ( md ) simulations , these studies had to make use of methods such as implicit membranes , steered molecular dynamics ( smd)(21 ) or relaxing the anchoring domain into a vacuum created by removal of lipids . furthermore , and most importantly , most of the computational studies investigating membrane binding of the anchoring domain at atomic resolutions are extremely costly , requiring at least 100s of nanoseconds of simulation time to observe relevant phenomena . thus , these ( md ) simulations are often too expensive to conduct multiple trials , collect sufficient statistics , and ensure the validity of the models proposed . in this study we have utilized the gla domain of human protein c ( hprc ) as a representative membrane anchoring domain to test the effectiveness and efficiency of a simple biphasic solvent , membrane - mimetic model in characterizing the nature of the membrane anchoring domain and in capturing and describing the process of hydrophobic insertion of membrane anchoring domains . during the coagulation cascade , activated protein c ( apc ) acts as an anticoagulant(40 ) helping to stop clot formation . hprc has a similar overall structure to other vitamin k - dependent coagulation proteins ( figure 1a ) containing a serine protease ( sp ) catalytic domain , two egf - like domains and the membrane - anchoring gla domain.(41 ) the structure of the hprc gla domain is very similar to the gla domains of other vitamin k - dependent hemostatic proteins such as factor viia ( fviia ) and prothrombin,(42 ) containing seven bound ca ions coordinated by nine gla residues ( figure 1b ) . binding of these ca ions is necessary for the formation of an active tertiary structure , as this causes the conformational change necessary to expose the 11-residue hydrophobic -loop and prepare the anchoring domain for insertion into the plasma membrane . recent studies have also demonstrated that membrane insertion involves mainly the -loop and the bound ca ions , with the hydrophobic triad known as the keel forming the penetrating surface . structure of human protein c. ( a ) a general representation of hprc showing the relative positions of the gla anchoring domain , the two egf - like domains , and the catalytic serine protease ( sp ) domain . ( b ) the gla domain of hprc . the backbone is shown in gray while the seven bound ca ions are shown as yellow spheres and numbered according to the crystal structure of the gla domain of fviia.(69 ) the nine gla residues of hprc - gla are colored in cyan , and the hydrophobic residues of the keel are shown in red . the arrow represents the vector connecting ca-4 and the c of phe40 which was used to represent the gla domain s axis in subsequent angle calculations . this structure is the starting point for all the simulations in the biphasic solvent system . this report proposes a conceptually simple , but very efficient md - based method for characterizing the depth of hydrophobic penetration and obtaining initial membrane - bound models of anchoring domains in peripheral membrane proteins . after construction of a water / dcle ( 1,1-dichloroethane ) biphasic system , multiple short simulation trials were performed to ensure the fidelity of the calculated properties of the organic phase to true experimental values.(49 ) the viability of this model as a membrane mimetic was then tested by placing the gla domain of hprc into the equilibrated biphasic system in multiple initial orientations with respect to the interface and performing unconstrained , equilibrium md simulations . these simulations consistently resulted in spontaneous penetration of the gla domain into the dcle phase with exactly the same anchor ( the keel ) , and achieving the same final height and angle of membrane insertion in a few nanoseconds . it is apparent from these simulations that the keel plays a pivotal role in the insertion dynamics of this anchoring domain much as it does in fviia . it appears that dcle provides an optimal organic phase for the devised simplified membrane model , as employing several other organic solvents , including dimethyl sulfide ( dms ) , ethyl propyl ether ( epe ) , or heptane did not successfully capture the process of membrane insertion of the anchoring domain . to our knowledge , this study represents the first use of a biphasic solvent system to investigate the atomistic details of membrane insertion of the anchoring domains . the gla domain of hprc was placed into the aqueous phase of organic / water biphasic solvent systems in different initial configurations and simulated with all - atom md , in order to describe the process of its hydrophobic insertion and to determine the depth of its penetration into this membrane - mimetic model . in this section the details of the modeling steps and the protocols used for the md simulations while several different organic solvents were tested , dcle turned out to be the most effective solvent in terms of capturing the process of membrane binding and insertion of the studied anchoring domain . therefore , we will describe the modeling steps in detail only for the dcle / water biphasic model in the following , noting that similar protocols and procedures were employed when using other organic solvents . a single dcle molecule was constructed by exchanging two hydrogen ( h ) atoms for two chlorine ( cl ) atoms in an ethane molecule . the resulting structure was then relaxed in vacuo for 100 ps . a 60 60 60 box of dcle was then generated using the packmol program(50 ) replicating the relaxed dcle structure 1,578 times , in order to produce the correct density of this organic solvent . a similar 60 60 60 box containing 6,845 waters was generated and placed on top of the dcle box using the solvate plugin in vmd(51 ) to create a biphasic solvent system with final dimensions of 60 60 120 containing 34,000 atoms and with the interface normal aligned along the z - axis . this structure was then energy minimized and equilibrated for 2 ns using an npt ensemble with a target pressure and temperature of 1.0 atm and 310 k , respectively . similar procedures were employed when constructing biphasic solvent boxes composed of water and either dms , heptane , or epe . the resulting biphasic solvent boxes were used for all subsequent simulations . a partial structure of the gla domain of hprc(52 ) was taken from the rcsb protein data bank ( pdb)(53 ) ( pdb entry 1lqv ) containing residues 133 of the protein segment and the seven bound ca ions . the missing eleven residues of hprc gla domain , comprising the c - terminal helix , were modeled using the bovine factor x gla domain(48 ) ( pdb entry 1iod ) and making the following mutations using the psfgen plugin of vmd:(51 ) a34v , 35d , q36d , d38l , 39a , and y44h ( where stands for gla ) . the complete 44-amino acid hprc gla domain was then solvated in 10,263 water molecules and neutralized with 3 na ions using the solvate and autoionize plugins of vmd(51 ) to give the system final dimensions of 56 48 45 containing 11,000 atoms . the system was energy minimized and simulated for 50 ps under npt conditions ( p = 1.0 atm , t = 310 k ) during which all c atoms were harmonically constrained ( k = 5.0 kcal mol ) . the resulting structure of hprc - gla was used as the initial structure for all insertion simulations . the equilibrated hprc gla domain was added to the aqueous phase of the equilibrated biphasic solvent system . the gla domain of hprc was then rotated and/or translated to produce a total of six different initial configurations for the dcle / water system . these initial configurations were chosen to represent a wide variety of height from the aqueous organic interface in addition to different orientations ranging from parallel with the aqueous organic interface to perpendicular to the aqueous organic interface . overlapping solvent molecules were then removed , and the resulting systems were simulated using the protocol below . for dms , epe , and heptane biphasic systems , only one initial configuration of the protein was used , namely , with the keel positioned 10 from the interface and the protein s axis ( figure 1b ) aligned with the interface normal . all simulations were performed using the namd2 molecular dynamics software,(54 ) utilizing both the charmm27 force field with / cross term map ( cmap ) corrections(55 ) and charmm36(56 ) ( cgenff ) set of force field parameters . all simulations were run under npt conditions with 1.0 atm as the target pressure , 310 k as the temperature , and the time step of 2 fs . constant temperature was maintained by langevin dynamics using a damping coefficient , , of 1 ps applied to all atoms . nonbonded interactions were cut off after 12 with a smoothing function applied after 10 . the particle mesh ewald ( pme ) method(60 ) was used for long - range electrostatic calculations with a grid density greater than 1 . an important aspect of all subsequent calculations was the determination of the water / dcle interface . this was done by first selecting a volume which is 5 on either side of the plane separating the aqueous and organic phases . the average z - coordinate for all dcle molecules within 2.5 of a water molecule was calculated and represented the interface . to calculate the depth of penetration of hprc - gla , the center of mass of the keel the depth of penetration of hprc - gla is defined as the z - coordinate difference between the center of mass of the keel and that of the interface . aqueous solubility was calculated by counting the number of dcle molecules that were both within 2.5 of water and farther than 2.5 from other dcle molecules in every frame . this was then averaged over the number of frames and divided by the equilibrated volume of water to give the number solubility , which was converted to g / ml of water to give the aqueous solubility . to determine the mobility of the various organic phases used , the self - diffusion constant , d , of each organic phase was calculated as the asymptotic value of the einstein relation : where r(t0 ) is the position of a molecule at time t0 and r(t0 + t ) is the position of the molecule after time t . although , in actuality , t is limited by the length of the simulation , the value of d converged quickly ( t < 10 ns ) . because the diffusion coefficient is calculated as a function of the time difference , t , the number of sample points decreases as t increases , thus making the final points in the diffusion calculation prone to error . therefore , the asymptotic value was calculated as the mean of the final 10 ns of the trajectory , excluding the last 5 ns . below the results obtained from modeling and simulation of various biphasic solvent models will be first presented . then , we turn our attention to the simulations in which insertion of an anchoring domain into the biphasic membrane was studied . as the dcle / water biphasic system was found to be the most effective model with regard to capturing the phenomenon of protein insertion , we will primarily discuss this model in the following section . the results obtained with other biphasic models and their performance will be discussed only briefly and using the dcle / water biphasic model as a reference . the main drive behind the design of a highly mobile membrane - mimetic model originates from a long - standing problem that we and others have been facing when studying protein insertion into lipid bilayers in simulation studies employing all - atom representations ; lipid molecules exhibit slow lateral diffusion , and respond very slowly to an inserting protein , resulting often in their insufficient relaxation within the time scales accessible to md simulations . one of the major factors contributing to the slow diffusion of lipid molecules within a bilayer environment is the long tails of the lipid molecules in a biological membrane , which tend to form highly disordered and entangled structures within the core of a bilayer . because organic solvent molecules are generally much smaller than lipid tails , they diffuse much faster , having a diffusion coefficient on the order of 10 cm s compared to 10 cm s for most lipids . faster diffusion will allow the organic phase to better accommodate the protein upon disruption of the interface , decreasing the time needed to simulate we therefore based our choice of a molecular representation of the hydrophobic core in our model on the criteria of size , aqueous solubility , phase at the desired simulation temperature ( room or body temperature ) , and availability of force field parameters . with the release of the charmm general force field , cgenff,(56 ) most of the organic solvents available in cgenff , however , are either volatile at 310 k ( e.g. , acetone , methylene chloride , and ether ) or are lengthy molecules which would not be optimal for constructing a highly mobile membrane - mimetic model . to determine the optimum solvent to be used in the biphasic solvent system , we tested four different organic species : dcle , dms , epe , and heptane . heptane was chosen due to its obvious similarity to the true membrane core and the fact that it is the smallest alkane which is liquid at the simulation temperature . additionally , the parameters were readily created by extending the existing cgenff parameters for pentane . dcle , dms , and epe were chosen due to their small size ( between 8 and 16 atoms ) , their nonpolar nature ( each with permanent dipole moments less than 1.83 d),(49 ) and the availability of parameters of easily adaptable analogues . since the fluidity of the solvent phase is a critical factor in determining the relaxation of the organic solvent in response to protein insertion , the self - diffusion constant ( d ) of the organic species was calculated ( figure 2 ) . out of the species tested , dcle clearly has the largest self - diffusion coefficient ( d = ( 1.35 0.02 ) 10 cm s ) with dms diffusing almost as rapidly ( d = ( 1.24 0.01 ) 10 cm s ) . the larger species , epe and heptane , had much smaller self - diffusion constants ( on the order of 10 cm s ) . the calculated diffusion constants suggest that the larger species will be less accommodating for the inserting protein . ( left ) plot of the self - diffusion coefficients d calculated for each species as a function of the time interval t ; asymptotic values were taken as the value of d. in the plot , the d at a specific time interval is given in green for dcle , orange for dms , purple for epe , and red for heptane . ( right ) molecular images of the organic species tested ; the outline of each molecule corresponds to the same color line on the plot . in fact , membrane insertion simulations clearly established the superiority of dcle over the other three tested organic solvents as the organic solvent in our biphasic membrane model . the anchoring domain was found to have difficulty inserting and/or staying inserted in these particular organic phases composed of heptane and epe , possibly due to the large size of the solvent molecules . for the case of the biphasic solvent system composed of dms , the protein only partially inserted into the dms phase ; full insertion and formation of a stable membrane - bound form was only observed in the case of dcle . we attribute this to the slight amphipathic nature of dcle , which includes both hydrophobic ( methyl ) and weak polar ( dichloromethyl ) groups in its structure , rendering it with the ability to establish either mode of interaction with various groups in a protein , thus representing a membrane more effectively in such a simplified model as the one used here . although , among the tested solvents , heptane is chemically the closest species to the core of a biological membrane , it completely lacks the ability of establishing partial polar interactions with the incoming protein , a factor that might have contributed to the inability of the biphasic heptane / water model in capturing membrane insertion of the anchoring domain . the distinct advantage that dcle has over a strictly nonpolar species is its permanent dipole moment that allows for charge dipole interactions between the solvent and the submerged portions of the protein backbone ( figure 3 ) . moreover , the methyl groups of dcle are able to surround the hydrophobic moieties of the keel ( i.e. , side chains of phe and leu in hprc gla domain ) , providing a locally hydrophobic environment ( figure 3 ) . the dipole moment together with the large diffusion coefficient allows dcle to adapt optimally and quickly to the insertion of protein . radial distribution functions g(r ) are shown for chloride ( purple ) and methyl ( black ) groups for ( a ) phe4 side chain , ( b ) leu5 side chain , ( c ) leu8 side chain , ( d ) backbone oxygen of phe4 , ( e ) backbone oxygen of leu5 , and ( f ) backbone oxygen of leu8 . arrows depict the position of the first peak for each distribution . after simulating the dcle / water biphasic solvent system for 2 ns , the density of the system was calculated to be 1.14 0.05 g / ml , which shows good agreement with the accepted value of 1.2 g / ml ( table 1).(49 ) for a consistent interface to be maintained , the aqueous solubility of dcle needed to be modeled very well by this system , and indeed , it was . the accepted value for the solubility of dcle in water is 8.6 10 g / ml.(49 ) over the 2 ns trajectory , we obtained an aqueous solubility of ( 9.89 2.9 ) 10 g / ml , which agrees with the accepted value reported in table 1.(49 ) in addition , figure 4 shows how well the interface is maintained ; the steep density gradients on either side clearly indicate the separation of dcle and water at the interface . thus , the physical properties of dcle vital to this study were reproduced in the simulations . during the equilibration , the dcle solvent box did experience a small amount of shrinkage , but equilibrated to 86% of its original size within 89 ps . the z - dimension shrank 5 and fluctuated 0.5 around its equilibrated value . the freedom of the organic phase in modulating its dimension will be an added advantage of the biphasic system when used for integral proteins / peptides , as it will ensure an optimal match of the thickness of the hydrophobic layer to the inserted protein . calculated every 2 ps for the duration of the trajectory with the arithmetic mean and standard deviation displayed . experimental value found in lide.(49 ) the density of dcle ( solid curve ) and water ( dashed curve ) phases averaged over the simulation period of 2 ns . the dotted line at x = 0 represents the approximate location of the dcle / water interface . a common problem encountered when studying the membrane - bound forms of anchoring domains is the multitude of atomic models proposed , which seem to depend heavily on the initial orientation and position of the anchoring domain with respect to the membrane . thus , we wished to test the ability of the reduced membrane model ( dcle / water biphasic model ) to consistently reproduce a single equilibrium structure by simulating a variety of initial orientations . the six initial configurations chosen to test the viability of the biphasic solvent system as a model for membrane insertion are shown in figure 5 . these particular initial configurations display a variety of heights from the aqueous organic interface , as well as a spectrum of angles ranging from parallel with the interface normal to perpendicular to the normal . the green surface represents the dcle volume while the water is implied in the empty space . the labels found in each box will be adopted throughout the text to simplify discussion of these systems . the time evolution of the height of the gla domain from the interface over the entire 30 ns course of the simulations is displayed in figure 6 . it is clear from the plot that each initial configuration converges to a height of 2.20 1.04 ( table 2 ) within a few nanoseconds and fluctuates slightly around this equilibrium point for the remainder of the simulation ( figure 6 ) . the angle with respect to the interface normal also converges at approximately the same rate to 23.37 12.48 ( figure 6 ) ; figure 7 displays the converged structure of hprc gla domain ( snapshot taken at t = 29 ns ) . as expected , the 60 and 90 trials were the slowest to insert , since they needed more time in solution to explore and find an optimal orientation toward the interface . this rate of insertion corresponds to at least 1 order of magnitude increase in the speed of insertion over previous studies , making this method more accessible to md simulations . in the simulation study of fviia anchoring,(21 ) the gla domain fluctuates 5 around its equilibrium height ; in this study , we see a similar degree of fluctuation around the equilibrium height . the plot of the domain s angle to the interface normal , however , shows a greater variability which is likely due to the absence of headgroup specific interactions with the anchoring domain . ohkubo and tajkhorshid(21 ) have demonstrated that the seven bound ca ions of the hfvii gla domain interact significantly with the phosphate groups of the lipid headgroups resulting in the stabilization of the membrane - bound complex . this effect is clearly missing from our model , resulting in a larger degree of fluctuation of the domain at the interface . we note that specific contacts between the lipid headgroups and peripheral proteins are of utmost importance in interaction of proteins such as the ras family and ferm domain(66 ) with the membrane . these interactions are not represented by the simple model used in our simulations , and while the model appears to be very efficient in determining mostly hydrophobic interactions , it is not able to describe specific lipid protein interactions . ( top ) the height of the keel c atoms above the dcle / water interface , which was assumed to be the average position of the first layer of dcle molecules in contact with water . ( bottom ) the angle between the gla domain s axis , determined by the vector traced from the ca-4 and the c of phe40 ( see figure 1 ) , and the interface normal ( z - axis ) . the colors used in these plots correspond to the outline around each orientation found in figure 5 . height was determined by taking difference of the center of mass of keel residue backbone atoms and instantaneous interface z - coordinate . interface normal was taken to be z - axis ; gla domain axis was chosen to be the vector from ca-4 to the c of phe40 ( figures 1b and 7b ) . depiction of the hprc gla domain at t = 29 ns . in this figure , the green represents dcle , the white represents water , the protein backbone is depicted in gray , and the keel residues are shown in red . ( a ) figure shows a side view of the gla domain with its average height above the interface of 2.20 1.04 . ( b ) figure shows a front view of the gla domain together with the interface normal and the defined protein axis ; the equilibrium angle of the protein was found to be 23.37 12.48. ( c ) a front view of the fully inserted hfviia gla domain.(21 ) ( b ) and ( c ) demonstrate that the equilibrium structure of the inserted gla domain using the biphasic solvent system is comparable to the structure obtained using an all - atom representation of the membrane . in close agreement with previous simulations of the fvii gla domain,(21 ) we observed a similar mechanism by which both fviia and hprc gla domains insert into the hydrophobic layer . no matter what angle the anchoring domain initially makes with the interface normal , it has an apparent grab - and - pull motion . the keel approaches the interface at an angle until phe4 inserts itself into the hydrophobic volume ; this is followed by a domino - like falling of the other two keel residues into the organic phase which coincides with the domain standing up and the angle between the domain and the interface normal decreasing . capturing the insertion dynamics is important to the study of anchoring domains , and the observed dynamics of hprc - gla provides further qualitative support for the ability of the model to capture similar phenomena to those obtained from full membrane simulations . energetic analysis of the interaction between the gla domain and the organic phase of the membrane mimetic model clearly indicates that nonpolar interactions are the main driving force for association and insertion of the gla domain into the organic phase . using the last 5 ns of each of the six membrane insertion simulation of the gla domain into the dcle / water biphasic system , an average electrostatic interaction of only 11 kcal / mol between the gla domain and the organic phase is calculated , whereas the van der waals component of the interaction energy amounts to more than 47 kcal / mol . membrane binding of peripheral proteins is a key step in regulating their activity in physiological processes as diverse as endosomal trafficking and hemostasis . these proteins have evolved specialized anchoring domains , which play a pivotal role in recognition of specific membrane regions and the proteins subsequent binding and insertion . however , given the fluid nature of the membrane and the reversible nature of the binding process , current experimental techniques have not been able to conclusively determine the relative depth and orientation of the membrane - bound forms of these anchoring domains . there still remains an ongoing debate on depth of penetration , equilibrium angle , and even the identity of the domains involved in anchoring these proteins . limitations to the time scales accessible by md simulations have also made computational studies of membrane anchoring domains very challenging . in this paper , we present , to our knowledge , the first use of a biphasic solvent model as a simple representation of a membrane to identify the membrane - interacting portion and to investigate the membrane - bound orientations of anchoring domains . the developed biphasic solvent model is demonstrated to be an efficient method to probe a protein and to determine the specific moieties involved in membrane penetration . the model is computationally very efficient , owing to purposefully keeping the size of the molecular species composing the hydrophobic core ( organic phase ) small , an aspect which is in contrast to previously reported models used to represent simplified membranes , e.g. , octanol / water phases . at the same time , since the model remains atomistic , one does not have to be concerned with the potential complications of coarse - grained and/or multiscale representations , or shortcomings arising from implicit representations of membrane and water . throughout our multiple trials sampling a variety of initial orientations and positions of a gla domain ( the anchoring domain of vitamin k - dependent coagulation proteins ) , the keel residues of the domain , known to be the membrane penetrating residues , were consistently found to be the only residues which spontaneously inserted into the organic solvent within a few nanoseconds , demonstrating their unique affinity for the hydrophobic core . in addition to identifying the anchor , this model also allows for rapid analysis of the depth of penetration of the anchoring domain and equilibrium angle relative to the interface normal . a clear advantage of the method over static analysis of hydrophobic surfaces of proteins is related to the dynamical description of the process during which conformational changes of the protein can be induced due to interaction with the hydrophobic phase . in addition to hydrophobic insertion , which constitutes an important and necessary mode of interaction for membrane - anchoring domains , it is well - known that specific interactions with lipid head groups form another critical set of interactions for these proteins . lipid head groups are very important in the process of reversible membrane binding by membrane anchors , and in many cases they control whether or not a peripheral protein should bind the membrane . such specific protein we note , however , that the final structures obtained from the biphasic solvent model simulations can be used to construct models of membrane - bound proteins with accurate full membrane representations . the biphasic system can be used to efficiently identify how deep the protein penetrates into the membrane s hydrophobic core , thereby providing a guide for placement and modeling of the protein in a full membrane system . by aligning the interfacial region of the full lipid bilayer to the aqueous organic interface in the biphasic model , one can maximize the overlap between the hydrophobic regions of the two representations , thus ensuring an optimal initial insertion of the protein into the hydrophobic core of the full membrane . work is in progress to add specific representations of the lipid headgroup to our biphasic solvent model while keeping the simulation times necessary to capture spontaneous membrane binding still on the nanosecond time scale.(67 ) once completed , the model will provide a platform for more detailed computational studies on the variety of molecular mechanisms used by anchoring domains to bind to the membrane .

membrane binding of peripheral proteins , mediated by specialized anchoring domains , is a crucial step for their biological function . computational studies of membrane insertion , however , have proven challenging and largely inaccessible , due to the time scales required for the complete description of the process , mainly caused by the slow diffusion of the lipid molecules composing the membrane . furthermore , in many cases , the nature of the membrane anchor , i.e. , the part of the protein that inserts into the membrane , is also unknown . here , we address some of these issues by developing and employing a simplified representation of the membrane by a biphasic solvent model which we demonstrate can be used efficiently to capture and describe the process of hydrophobic insertion of membrane anchoring domains in all - atom molecular dynamics simulations . applying the model , we have studied the insertion of the anchoring domain of a coagulation protein ( the gla domain of human protein c ) , starting from multiple initial configurations varying with regard to the initial orientation and height of the protein with respect to the membrane . in addition to efficiently and consistently identifying the keel region as the hydrophobic membrane anchor , within a few nanoseconds each configuration simulated showed a convergent height ( 2.20 1.04 ) and angle with respect to the interface normal ( 23.37 12.48 ) . we demonstrate that the model can produce the same results as those obtained from a full representation of a membrane , in terms of both the depth of penetration and the orientation of the protein in the final membrane - bound form with an order of magnitude decrease in the required computational time compared to previous models , allowing for a more exhaustive search for the correct membrane - bound configuration .

Introduction Methods Results and Discussion Discussion

as the association of peripheral membrane proteins is often intended to be transient , these proteins have evolved specialized domains , termed membrane anchoring domains , which are responsible for reversibly binding to the membrane . these membrane - anchoring domains take advantage of different molecular strategies ( various types of specific lipid protein interactions ) in achieving membrane affinities tuned to the function of the proteins , although the binding can be broadly viewed as a combination of electrostatic interactions with ( often anionic ) lipids and hydrophobic interactions with the apolar core of the membrane . in many cases , membrane binding constitutes a step in the activation of the protein , a process which is chiefly controlled through modulation of the lipid composition of the membrane in specific areas . developing a molecular view of how membrane - anchoring domains interact with the membrane is therefore crucial to our understanding of peripheral proteins function . despite the high relevance of the phenomenon , due to technical challenges involved in obtaining high - resolution information on the process of membrane insertion / binding , there have been relatively few studies achieving atomistic details on the membrane - bound forms of peripheral proteins. (9 ) furthermore , different experimental approaches have often resulted in different membrane - bound models for the same anchoring domain , adding to the uncertainty of the problem . for instance , a variety of experimental techniques , including epr , fluorescent labeling,(12 ) and nmr,(14 ) have been utilized to characterize the membrane - bound forms of the c2 domain , a common anchoring domain in signal transduction and membrane trafficking of proteins . these experiments , however , have resulted in membrane - bound models ranging from 5.2 insertion and making an angle of 77 with the membrane normal to the domain being inserted 10 and making an angle of 52 with the membrane normal . (16 ) similar discrepancies are found in the studies of the gla domains , the anchoring domain of vitamin k - dependent coagulation factors , where the relative orientation and depth of penetration into the membrane are largely disputed , and the fyve domain , where the membrane binding motif has not even been characterized . computational studies on membrane anchoring domains have proven equally difficult . in contrast to integral membrane proteins , whose membrane binding mode is fairly well understood , and for which even semiautomated insertion protocols exist , there are currently no robust methods to determine how deep and in what orientation a peripheral membrane protein will insert into a biological membrane , or even in some cases which residues comprise the membrane anchor . compounded further by the need of describing the protein s structural changes that are induced / accompanied by insertion into the membrane , especially with regard to the side chains in and around the anchor region , and the slow diffusion of lipid molecules around the insertion point , describing the process of membrane insertion has proven extremely challenging in simulation studies . of the small number of computational studies conducted on peripheral proteins , a majority have focused on only a handful of anchoring domains , namely , the bar , c2,(31 ) gla,(21 ) fyve,(32 ) px , and membrane binding domain of prostaglandin h2 synthase . naturally , most of these studies had to rely on initial embedding ( partially or fully ) of the anchoring domain into the lipid bilayer . while these studies have provided important information on how the anchoring domains might be stabilized within the membrane , they give little detail on the extremely dynamic process of insertion . modeling the membrane - bound form in these studies also requires a priori knowledge of the anchoring region ( the anchor ) . in addition , manually embedding the protein in the membrane introduces biased sampling and might restrict the states available to the protein . however , in order to observe membrane insertion within the time scales accessible to molecular dynamics ( md ) simulations , these studies had to make use of methods such as implicit membranes , steered molecular dynamics ( smd)(21 ) or relaxing the anchoring domain into a vacuum created by removal of lipids . furthermore , and most importantly , most of the computational studies investigating membrane binding of the anchoring domain at atomic resolutions are extremely costly , requiring at least 100s of nanoseconds of simulation time to observe relevant phenomena . in this study we have utilized the gla domain of human protein c ( hprc ) as a representative membrane anchoring domain to test the effectiveness and efficiency of a simple biphasic solvent , membrane - mimetic model in characterizing the nature of the membrane anchoring domain and in capturing and describing the process of hydrophobic insertion of membrane anchoring domains . hprc has a similar overall structure to other vitamin k - dependent coagulation proteins ( figure 1a ) containing a serine protease ( sp ) catalytic domain , two egf - like domains and the membrane - anchoring gla domain. (41 ) the structure of the hprc gla domain is very similar to the gla domains of other vitamin k - dependent hemostatic proteins such as factor viia ( fviia ) and prothrombin,(42 ) containing seven bound ca ions coordinated by nine gla residues ( figure 1b ) . binding of these ca ions is necessary for the formation of an active tertiary structure , as this causes the conformational change necessary to expose the 11-residue hydrophobic -loop and prepare the anchoring domain for insertion into the plasma membrane . recent studies have also demonstrated that membrane insertion involves mainly the -loop and the bound ca ions , with the hydrophobic triad known as the keel forming the penetrating surface . structure of human protein c. ( a ) a general representation of hprc showing the relative positions of the gla anchoring domain , the two egf - like domains , and the catalytic serine protease ( sp ) domain . the backbone is shown in gray while the seven bound ca ions are shown as yellow spheres and numbered according to the crystal structure of the gla domain of fviia. (69 ) the nine gla residues of hprc - gla are colored in cyan , and the hydrophobic residues of the keel are shown in red . this report proposes a conceptually simple , but very efficient md - based method for characterizing the depth of hydrophobic penetration and obtaining initial membrane - bound models of anchoring domains in peripheral membrane proteins . (49 ) the viability of this model as a membrane mimetic was then tested by placing the gla domain of hprc into the equilibrated biphasic system in multiple initial orientations with respect to the interface and performing unconstrained , equilibrium md simulations . these simulations consistently resulted in spontaneous penetration of the gla domain into the dcle phase with exactly the same anchor ( the keel ) , and achieving the same final height and angle of membrane insertion in a few nanoseconds . it is apparent from these simulations that the keel plays a pivotal role in the insertion dynamics of this anchoring domain much as it does in fviia . it appears that dcle provides an optimal organic phase for the devised simplified membrane model , as employing several other organic solvents , including dimethyl sulfide ( dms ) , ethyl propyl ether ( epe ) , or heptane did not successfully capture the process of membrane insertion of the anchoring domain . to our knowledge , this study represents the first use of a biphasic solvent system to investigate the atomistic details of membrane insertion of the anchoring domains . the gla domain of hprc was placed into the aqueous phase of organic / water biphasic solvent systems in different initial configurations and simulated with all - atom md , in order to describe the process of its hydrophobic insertion and to determine the depth of its penetration into this membrane - mimetic model . in this section the details of the modeling steps and the protocols used for the md simulations while several different organic solvents were tested , dcle turned out to be the most effective solvent in terms of capturing the process of membrane binding and insertion of the studied anchoring domain . therefore , we will describe the modeling steps in detail only for the dcle / water biphasic model in the following , noting that similar protocols and procedures were employed when using other organic solvents . a similar 60 60 60 box containing 6,845 waters was generated and placed on top of the dcle box using the solvate plugin in vmd(51 ) to create a biphasic solvent system with final dimensions of 60 60 120 containing 34,000 atoms and with the interface normal aligned along the z - axis . a partial structure of the gla domain of hprc(52 ) was taken from the rcsb protein data bank ( pdb)(53 ) ( pdb entry 1lqv ) containing residues 133 of the protein segment and the seven bound ca ions . the equilibrated hprc gla domain was added to the aqueous phase of the equilibrated biphasic solvent system . the gla domain of hprc was then rotated and/or translated to produce a total of six different initial configurations for the dcle / water system . these initial configurations were chosen to represent a wide variety of height from the aqueous organic interface in addition to different orientations ranging from parallel with the aqueous organic interface to perpendicular to the aqueous organic interface . for dms , epe , and heptane biphasic systems , only one initial configuration of the protein was used , namely , with the keel positioned 10 from the interface and the protein s axis ( figure 1b ) aligned with the interface normal . to calculate the depth of penetration of hprc - gla , the center of mass of the keel the depth of penetration of hprc - gla is defined as the z - coordinate difference between the center of mass of the keel and that of the interface . to determine the mobility of the various organic phases used , the self - diffusion constant , d , of each organic phase was calculated as the asymptotic value of the einstein relation : where r(t0 ) is the position of a molecule at time t0 and r(t0 + t ) is the position of the molecule after time t . because the diffusion coefficient is calculated as a function of the time difference , t , the number of sample points decreases as t increases , thus making the final points in the diffusion calculation prone to error . therefore , the asymptotic value was calculated as the mean of the final 10 ns of the trajectory , excluding the last 5 ns . then , we turn our attention to the simulations in which insertion of an anchoring domain into the biphasic membrane was studied . as the dcle / water biphasic system was found to be the most effective model with regard to capturing the phenomenon of protein insertion , we will primarily discuss this model in the following section . the main drive behind the design of a highly mobile membrane - mimetic model originates from a long - standing problem that we and others have been facing when studying protein insertion into lipid bilayers in simulation studies employing all - atom representations ; lipid molecules exhibit slow lateral diffusion , and respond very slowly to an inserting protein , resulting often in their insufficient relaxation within the time scales accessible to md simulations . one of the major factors contributing to the slow diffusion of lipid molecules within a bilayer environment is the long tails of the lipid molecules in a biological membrane , which tend to form highly disordered and entangled structures within the core of a bilayer . faster diffusion will allow the organic phase to better accommodate the protein upon disruption of the interface , decreasing the time needed to simulate we therefore based our choice of a molecular representation of the hydrophobic core in our model on the criteria of size , aqueous solubility , phase at the desired simulation temperature ( room or body temperature ) , and availability of force field parameters . ( left ) plot of the self - diffusion coefficients d calculated for each species as a function of the time interval t ; asymptotic values were taken as the value of d. in the plot , the d at a specific time interval is given in green for dcle , orange for dms , purple for epe , and red for heptane . the anchoring domain was found to have difficulty inserting and/or staying inserted in these particular organic phases composed of heptane and epe , possibly due to the large size of the solvent molecules . for the case of the biphasic solvent system composed of dms , the protein only partially inserted into the dms phase ; full insertion and formation of a stable membrane - bound form was only observed in the case of dcle . we attribute this to the slight amphipathic nature of dcle , which includes both hydrophobic ( methyl ) and weak polar ( dichloromethyl ) groups in its structure , rendering it with the ability to establish either mode of interaction with various groups in a protein , thus representing a membrane more effectively in such a simplified model as the one used here . although , among the tested solvents , heptane is chemically the closest species to the core of a biological membrane , it completely lacks the ability of establishing partial polar interactions with the incoming protein , a factor that might have contributed to the inability of the biphasic heptane / water model in capturing membrane insertion of the anchoring domain . moreover , the methyl groups of dcle are able to surround the hydrophobic moieties of the keel ( i.e. a common problem encountered when studying the membrane - bound forms of anchoring domains is the multitude of atomic models proposed , which seem to depend heavily on the initial orientation and position of the anchoring domain with respect to the membrane . the time evolution of the height of the gla domain from the interface over the entire 30 ns course of the simulations is displayed in figure 6 . it is clear from the plot that each initial configuration converges to a height of 2.20 1.04 ( table 2 ) within a few nanoseconds and fluctuates slightly around this equilibrium point for the remainder of the simulation ( figure 6 ) . the angle with respect to the interface normal also converges at approximately the same rate to 23.37 12.48 ( figure 6 ) ; figure 7 displays the converged structure of hprc gla domain ( snapshot taken at t = 29 ns ) . the plot of the domain s angle to the interface normal , however , shows a greater variability which is likely due to the absence of headgroup specific interactions with the anchoring domain . ohkubo and tajkhorshid(21 ) have demonstrated that the seven bound ca ions of the hfvii gla domain interact significantly with the phosphate groups of the lipid headgroups resulting in the stabilization of the membrane - bound complex . ( bottom ) the angle between the gla domain s axis , determined by the vector traced from the ca-4 and the c of phe40 ( see figure 1 ) , and the interface normal ( z - axis ) . ( a ) figure shows a side view of the gla domain with its average height above the interface of 2.20 1.04 . ( b ) figure shows a front view of the gla domain together with the interface normal and the defined protein axis ; the equilibrium angle of the protein was found to be 23.37 12.48. (21 ) ( b ) and ( c ) demonstrate that the equilibrium structure of the inserted gla domain using the biphasic solvent system is comparable to the structure obtained using an all - atom representation of the membrane . the keel approaches the interface at an angle until phe4 inserts itself into the hydrophobic volume ; this is followed by a domino - like falling of the other two keel residues into the organic phase which coincides with the domain standing up and the angle between the domain and the interface normal decreasing . capturing the insertion dynamics is important to the study of anchoring domains , and the observed dynamics of hprc - gla provides further qualitative support for the ability of the model to capture similar phenomena to those obtained from full membrane simulations . energetic analysis of the interaction between the gla domain and the organic phase of the membrane mimetic model clearly indicates that nonpolar interactions are the main driving force for association and insertion of the gla domain into the organic phase . using the last 5 ns of each of the six membrane insertion simulation of the gla domain into the dcle / water biphasic system , an average electrostatic interaction of only 11 kcal / mol between the gla domain and the organic phase is calculated , whereas the van der waals component of the interaction energy amounts to more than 47 kcal / mol . membrane binding of peripheral proteins is a key step in regulating their activity in physiological processes as diverse as endosomal trafficking and hemostasis . however , given the fluid nature of the membrane and the reversible nature of the binding process , current experimental techniques have not been able to conclusively determine the relative depth and orientation of the membrane - bound forms of these anchoring domains . limitations to the time scales accessible by md simulations have also made computational studies of membrane anchoring domains very challenging . in this paper , we present , to our knowledge , the first use of a biphasic solvent model as a simple representation of a membrane to identify the membrane - interacting portion and to investigate the membrane - bound orientations of anchoring domains . the model is computationally very efficient , owing to purposefully keeping the size of the molecular species composing the hydrophobic core ( organic phase ) small , an aspect which is in contrast to previously reported models used to represent simplified membranes , e.g. throughout our multiple trials sampling a variety of initial orientations and positions of a gla domain ( the anchoring domain of vitamin k - dependent coagulation proteins ) , the keel residues of the domain , known to be the membrane penetrating residues , were consistently found to be the only residues which spontaneously inserted into the organic solvent within a few nanoseconds , demonstrating their unique affinity for the hydrophobic core . in addition to identifying the anchor , this model also allows for rapid analysis of the depth of penetration of the anchoring domain and equilibrium angle relative to the interface normal . a clear advantage of the method over static analysis of hydrophobic surfaces of proteins is related to the dynamical description of the process during which conformational changes of the protein can be induced due to interaction with the hydrophobic phase . in addition to hydrophobic insertion , which constitutes an important and necessary mode of interaction for membrane - anchoring domains , it is well - known that specific interactions with lipid head groups form another critical set of interactions for these proteins . lipid head groups are very important in the process of reversible membrane binding by membrane anchors , and in many cases they control whether or not a peripheral protein should bind the membrane . such specific protein we note , however , that the final structures obtained from the biphasic solvent model simulations can be used to construct models of membrane - bound proteins with accurate full membrane representations . the biphasic system can be used to efficiently identify how deep the protein penetrates into the membrane s hydrophobic core , thereby providing a guide for placement and modeling of the protein in a full membrane system . by aligning the interfacial region of the full lipid bilayer to the aqueous organic interface in the biphasic model , one can maximize the overlap between the hydrophobic regions of the two representations , thus ensuring an optimal initial insertion of the protein into the hydrophobic core of the full membrane . work is in progress to add specific representations of the lipid headgroup to our biphasic solvent model while keeping the simulation times necessary to capture spontaneous membrane binding still on the nanosecond time scale. (67 ) once completed , the model will provide a platform for more detailed computational studies on the variety of molecular mechanisms used by anchoring domains to bind to the membrane .

acute myocardial infarction ( ami ) is a common manifestation of coronary heart disease that affected > 800 000 adults in the united states in 2010.1 concomitant with advances in prehospital and hospital treatment , inhospital survival after ami has dramatically improved.2 many patients are being discharged from the hospital into the community who are at risk for being readmitted to the hospital due to a variety of contributory factors and reasons.3 , 4 although not all hospital readmissions can be prevented , excess readmissions within a short time frame can be a marker of poor quality of care and efficiency . since the centers for medicare and medicaid services ( cms ) began publicly reporting 30day riskstandardized readmission rates for heart failure , ami , and pneumonia as performance measures,5 30day hospital readmission rates have become a quality performance measure for patients hospitalized with ami.5 , 6 there has been considerable interest from hospitals and clinicians to better understand and improve modifiable factors associated with 30day hospital readmissions , which are increasingly being linked to hospital reimbursement.7 although several studies have reported 30day rehospitalization rates among patients surviving hospitalization for ami,4 , 8 , 9 few have examined risk factors for being readmitted to the hospital during the following month by using multivariable regression analyses . moreover , there are little contemporary data that describe longterm trends in 30day rehospitalization rates , the reasons for rehospitalization , as well as sociodemographic , clinical , and treatmentrelated factors that may affect 30day rehospitalization rates among patients surviving an ami . our primary study objective was to describe relatively contemporary decade long ( 20012011 ) trends in the frequency of 30day rehospitalizations among patients surviving hospitalization for an ami . our secondary study objective was to describe patient characteristics , clinical factors , and treatment practices associated with an increased risk of 30day rehospitalizations among residents of central massachusetts discharged from the 3 principal medical centers in central massachusetts after an ami . data from the worcester heart attack study were used in this study.10 , 11 , 12 , 13 described elsewhere in detail,10 , 11 , 12 , 13 the worcester heart attack study is an ongoing populationbased investigation examining longterm trends in the descriptive epidemiology of ami in residents of the worcester , ma , metropolitan area ( 2010 census 518 000 ) hospitalized at all 16 medical centers in central massachusetts on an approximate biennial basis between 1975 and 2011.10 , 11 , 12 , 13 due to hospital closures , mergers , or conversion to longterm care or rehabilitation facilities , fewer hospitals ( n=11 ) have been providing care to greater worcester residents during recent years . computerized printouts of patients discharged from all greater worcester hospitals with possible ami ( international classification of disease , ninth revision [ icd ] codes : 410 to 414 , 786.5 ) were identified , and cases of possible ami were independently validated by using predefined criteria for ami.10 , 11 , 12 , 13 these criteria included a suggestive clinical history , increases in several serum biomarkers ( eg , creatine kinase [ ck ] , ckmb , and troponin values ) , and serial electrocardiographic findings during hospitalization consistent with the presence of ami . patients who satisfied at least 2 of these 3 criteria and were residents of the worcester metropolitan area , because this study is population based , were included . because the focus of the current study was rehospitalization after hospital discharge for ami , we included adult residents of the worcester metropolitan area who survived their index hospitalization for ami in 2001 , 2003 , 2005 , 2007 , 2009 , and 2011 . we further restricted our study population to patients hospitalized at the 3 largest tertiary care and community medical centers in central massachusetts ( umassmemorial health care and st . this was done since the majority ( 90% ) of patients hospitalized for ami in central massachusetts were discharged from these facilities , which also have excellent electronic medical records . the patient 's index hospitalization occurred in any of the 3 study hospitals , as did any subsequent rehospitalization . patients who had their index hospitalization or their rehospitalization outside of these major medical centers were not included in this study . this study was approved by the institutional review board at the university of massachusetts medical school and that informed consent was waived . trained nurses and physicians abstracted information on patient demographic characteristics , medical history , clinical data , and treatment practices through the review of hospital medical records . these factors included patient 's sociodemographic characteristics ( eg , age , sex , race , marital status ) , year of hospitalization , hospital length of stay , history of previously diagnosed comorbidities ( eg , stroke , diabetes , heart failure ) , ami order ( initial versus prior ) and ami type ( stsegment elevation myocardial infarction [ stemi ] versus nonstemi [ nstemi]).14 , 15 information on the development of important inhospital complications including atrial fibrillation,16 cardiogenic shock,17 heart failure,18 and stroke19 was also collected . data on the receipt of thrombolytic therapy and 3 coronary diagnostic and interventional procedures ( cardiac catheterization , percutaneous coronary intervention [ pci ] , and coronary artery bypass grafting [ cabg ] ) during hospitalization , and pharmacotherapies at the time of hospital discharge , including the prescribing of 4 effective cardiac medications ( angiotensin converting inhibitors [ aceis]/angiotensin type ii receptor blockers [ arbs ] , aspirin , blockers , and lipidlowering agents ) , were obtained . a rehospitalization was defined as the patient 's first admission to a study hospital within 30 days of discharge after their index hospitalization for ami during the years under study . two of the study investigators adjudicated whether the principal reason for readmission was due to either an ami , cardiovascular disease ( cvd ) ( excluding ami)related , or non cvdrelated readmission based on the review of information contained in hospital medical records . indications for cvdrelated hospitalizations included unstable angina , heart failure , type ii diabetes mellitus , and chronic ischemic heart disease . for ease of analysis and interpretation , we aggregated the 6 individual study years into 2year strata of 20012003 ( earliest ) , 20052007 ( middle ) , and 20092011 ( most recent ) for purposes of examining trends in our principal study outcomes . differences in the distribution of various patient demographic and clinical characteristics between patients hospitalized during the 3 aggregated periods were examined using the anova test for continuous variables and the test for categorical variables . the cochranarmitage tests and linear regression models were used to assess for linear trends over time among categorical and continuous variables , respectively . shortterm rehospitalization rates were examined by calculating the frequency of having a first rehospitalization within 30 days among patients discharged from the hospital after their index ami during the years under study . we examined the reasons for being rehospitalized during this period and calculated the causespecific 30day rehospitalization rates . multivariableadjusted logistic regression analyses were performed to examine the association between the main explanatory variable of period of hospitalization ( 20012003 , earliest ; 20052007 , middle ; and 20092011 , most recent ) and the outcome of whether the patient was rehospitalized during the following 30 days while adjusting for several potentially confounding variables of prognostic importance . we dummy coded the variable of time period of hospitalization with the earliest period ( 20012003 ) serving as the reference group . several covariates associated with rehospitalization after ami in prior studies3 , 4 were examined including age , sex , race ( white versus nonwhite ) , marital status ( married versus not married ) , ami order ( initial versus prior ) , ami type ( stemi versus nstemi ) , previously diagnosed comorbid conditions ( angina , atrial fibrillation , heart failure , hypertension , peripheral vascular disease , stroke , diabetes , chronic obstructive pulmonary disease , depression , and chronic kidney disease ) , hospital clinical complications ( atrial fibrillation , heart failure , cardiogenic shock , and stroke ) , and hospital length of stay . we further adjusted for hospital treatment practices , including the receipt of thrombolytic therapy and 3 coronary diagnostic and interventional procedures ( cardiac catheterization , pci , and cabg ) , and the prescribing of 4 guideline recommended cardiac medications ( aceis / arbs , aspirin , blockers , and lipidlowering agents ) at the time of hospital discharge during the patient 's index hospitalization in our regression analyses to examine the potential effects of hospital treatment practices on 30day rehospitalization trends . we repeated the same analyses after excluding patients ( n=165 ) who were not rehospitalized but died within the 30day postdischarge period . in addition , we repeated the same analyses by treating patients who were not rehospitalized but died within the 30day postdischarge period as those who had a 30day readmission . the results of our logistic regression analyses were presented as multivariableadjusted odds ratios ( or ) and accompanying 95% cis , which were calculated based on standard errors clustered at the hospital level to account for potential withinhospital correlation with variance adjustment through the use of morel 's small sample bias correction.20 all statistical analyses were conducted by using sas version 9.3 ( sas institute , inc ) . trained nurses and physicians abstracted information on patient demographic characteristics , medical history , clinical data , and treatment practices through the review of hospital medical records . these factors included patient 's sociodemographic characteristics ( eg , age , sex , race , marital status ) , year of hospitalization , hospital length of stay , history of previously diagnosed comorbidities ( eg , stroke , diabetes , heart failure ) , ami order ( initial versus prior ) and ami type ( stsegment elevation myocardial infarction [ stemi ] versus nonstemi [ nstemi]).14 , 15 information on the development of important inhospital complications including atrial fibrillation,16 cardiogenic shock,17 heart failure,18 and stroke19 was also collected . data on the receipt of thrombolytic therapy and 3 coronary diagnostic and interventional procedures ( cardiac catheterization , percutaneous coronary intervention [ pci ] , and coronary artery bypass grafting [ cabg ] ) during hospitalization , and pharmacotherapies at the time of hospital discharge , including the prescribing of 4 effective cardiac medications ( angiotensin converting inhibitors [ aceis]/angiotensin type ii receptor blockers [ arbs ] , aspirin , blockers , and lipidlowering agents ) , were obtained . a rehospitalization was defined as the patient 's first admission to a study hospital within 30 days of discharge after their index hospitalization for ami during the years under study . two of the study investigators adjudicated whether the principal reason for readmission was due to either an ami , cardiovascular disease ( cvd ) ( excluding ami)related , or non cvdrelated readmission based on the review of information contained in hospital medical records . indications for cvdrelated hospitalizations included unstable angina , heart failure , type ii diabetes mellitus , and chronic ischemic heart disease . for ease of analysis and interpretation , we aggregated the 6 individual study years into 2year strata of 20012003 ( earliest ) , 20052007 ( middle ) , and 20092011 ( most recent ) for purposes of examining trends in our principal study outcomes . differences in the distribution of various patient demographic and clinical characteristics between patients hospitalized during the 3 aggregated periods were examined using the anova test for continuous variables and the test for categorical variables . the cochranarmitage tests and linear regression models were used to assess for linear trends over time among categorical and continuous variables , respectively . shortterm rehospitalization rates were examined by calculating the frequency of having a first rehospitalization within 30 days among patients discharged from the hospital after their index ami during the years under study . we examined the reasons for being rehospitalized during this period and calculated the causespecific 30day rehospitalization rates . multivariableadjusted logistic regression analyses were performed to examine the association between the main explanatory variable of period of hospitalization ( 20012003 , earliest ; 20052007 , middle ; and 20092011 , most recent ) and the outcome of whether the patient was rehospitalized during the following 30 days while adjusting for several potentially confounding variables of prognostic importance . we dummy coded the variable of time period of hospitalization with the earliest period ( 20012003 ) serving as the reference group . several covariates associated with rehospitalization after ami in prior studies3 , 4 were examined including age , sex , race ( white versus nonwhite ) , marital status ( married versus not married ) , ami order ( initial versus prior ) , ami type ( stemi versus nstemi ) , previously diagnosed comorbid conditions ( angina , atrial fibrillation , heart failure , hypertension , peripheral vascular disease , stroke , diabetes , chronic obstructive pulmonary disease , depression , and chronic kidney disease ) , hospital clinical complications ( atrial fibrillation , heart failure , cardiogenic shock , and stroke ) , and hospital length of stay . we further adjusted for hospital treatment practices , including the receipt of thrombolytic therapy and 3 coronary diagnostic and interventional procedures ( cardiac catheterization , pci , and cabg ) , and the prescribing of 4 guideline recommended cardiac medications ( aceis / arbs , aspirin , blockers , and lipidlowering agents ) at the time of hospital discharge during the patient 's index hospitalization in our regression analyses to examine the potential effects of hospital treatment practices on 30day rehospitalization trends . we repeated the same analyses after excluding patients ( n=165 ) who were not rehospitalized but died within the 30day postdischarge period . in addition , we repeated the same analyses by treating patients who were not rehospitalized but died within the 30day postdischarge period as those who had a 30day readmission . the results of our logistic regression analyses were presented as multivariableadjusted odds ratios ( or ) and accompanying 95% cis , which were calculated based on standard errors clustered at the hospital level to account for potential withinhospital correlation with variance adjustment through the use of morel 's small sample bias correction.20 all statistical analyses were conducted by using sas version 9.3 ( sas institute , inc ) . the study population consisted of 4810 adult residents of the worcester metropolitan area who survived their hospitalization for ami at the 3 major central massachusetts medical centers between 2001 and 2011 ( table 1 ) . overall , the average age of this population was 68.9 years , 41.8% were women , 92.4% were white , and 54.6% were married . characteristics of patients who survived an ami : worcester heart attack study , 20012011 ami indicates acute myocardial infarction . pvalues derived from anova tests for continuous variables and tests for categorical variables . p values derived from cochranarmitage tests for categorical variables and linear regression models for continuous variables . during the most recent years under study , patients who survived their ami were more likely to be younger and have a history of hypertension , peripheral vascular disease , diabetes , or chronic kidney disease than were patients who were hospitalized during earlier study periods ( table 1 ) . between 2001 and 2011 , 32.6% of our study sample was diagnosed with a stemi , which declined from 34.0% in 20012003 to 31.1% in 20092011 . the average hospital length of stay in this study population was 5.5 days , which declined from 6.4 days in 20012003 to 4.7 days in 20092011 ( table 1 ) . in addition , the likelihood of developing cardiogenic shock and stroke during hospitalization remained relatively low ( 3.5% and 1.4% overall , respectively ) , whereas the incidence rates of inhospital heart failure and atrial fibrillation were considerably higher ( 34.4% and 18.8% overall , respectively ) during the years under study ( table 1 ) . the average postdischarge 30day rehospitalization rates decreased from 20012003 ( 20.5% ) to 20092011 ( 15.8% ) ( p for trend=0.001 ) ( table 2 ) . the proportion of patients who were rehospitalized was the highest ( 6.6% ) during the first week ( 0 to 7 days ) after hospital discharge and continued to decrease as the length of postdischarge time increased ( figure 1 ) . rehospitalizations that occurred during the first week after hospital discharge accounted for 35.6% of all 30day rehospitalizations ; this proportion decreased from 39.2% in 20012003 to 27.2% in 20092011 . association between time period of hospitalization and 30day allcause rehospitalizations among patients who survived an ami : worcester heart attack study , 20012011 ( n=4810 ) acei indicates angiotensinconverting inhibitors ; ami , acute myocardial infarction ; arbs , angiotensin type ii receptor blockers ; cabg , coronary artery bypass grafting ; or , odds ratio ; pci , percutaneous coronary intervention . adjusted for age , sex , race , marital status , and previously diagnosed comorbid conditions . adjusted for sociodemographic characteristics , comorbid conditions , ami order , ami type , inhospital complications , and hospital length of stay . adjusted for sociodemographic characteristics , comorbid conditions , inhospital factors , and inhospital management as represented by thrombolytic therapy and receipt of 3 coronary interventional procedures ( cardiac catheterization , pci , and cabg ) and prescribing of 4 guidelinerecommended cardiac medications ( aceis / arbs , lipidlowering agents , blockers , and aspirin ) at the time of hospital discharge . rates of rehospitalizations within 30 days after hospital discharge among patients who survived an acute myocardial infarction : worcester heart attack study , 20012011 ( n=4810 ) . in examining the specific causes of 30day rehospitalizations , 53.9% were cvd related ( excluding ami ) , 38.1% were non cvd related , and 8.0% were due to a recurrent ami during the years under study . the overall causespecific 30day rehospitalization rates due to cvd , noncvd , and ami were 10.0% , 7.0% , and 1.5% , respectively , during the years under study ( figure 2 ) . cvdrelated reasons decreased ( p for trend < 0.001 ) during the years under study , while no significant changing trends in 30day rehospitalization rates due to cvd ( excluding ami ) or ami were observed ( figure 2 ) . causespecific 30day rehospitalization rates after hospital discharge among patients who survived an ami : worcester heart attack study , 20012011 ( n=4810 ) . ami indicates acute myocardial infarction ; cvd , cardiovascular diseases exclude ami . in examining changing trends in 30day rehospitalizations after adjusting for several demographic characteristics , comorbidities , and inhospital clinical factors , there was no significant difference in the odds of having a 30day rehospitalization in 20052007 ( or 0.88 , 95% ci 0.74 to 1.04 ) , but there was a reduced odds of being rehospitalized during the subsequent 30 days ( or 0.74 , 95% ci 0.61 to 0.91 ) among patients who survived an ami in 20092011 , in comparison with those discharged in 20012003 ( table 2 ) . after further adjustment for medical procedures and treatments received during hospitalization , there was a significantly reduced odds of being rehospitalized during the subsequent 30 days ( or 0.78 , 95% ci 0.65 to 0.93 ) in 20092011 compared with those discharged from the hospital in 20012003 ( table 2 ) . we observed similar results after excluding patients who were not rehospitalized but died during the 30day postdischarge period . we also observed similar results when we analyzed these patients as those who had a 30day hospital readmission . using multivariableadjusted regression analyses , we examined the role of various prognostic factors associated with 30day rehospitalizations in all study patients ( table 3 ) . female sex , having previously diagnosed heart failure and chronic kidney disease , and the development of inhospital cardiogenic shock and heart failure were significantly associated with an increased odds of being rehospitalized for any reason within 30 days after hospital discharge . on the other hand , patients who received various coronary diagnostic and interventional procedures ( cardiac catheterization and pci and/or cabg ) had a reduced odds for being rehospitalized within 30 days after hospital discharge compared with those who did not undergo these procedures ( table 3 ) . we observed similar factors that were significantly associated with 30day rehospitalizations after excluding patients who were not rehospitalized but died during the 30day postdischarge period . we also observed similar factors associated with hospital readmission when we analyzed these patients as having had a 30day hospital readmission . association between various prognostic factors and 30day allcause rehospitalizations among patients who survived an ami : worcester heart attack study , 20012011 ( n=4810 ) acei indicates angiotensinconverting inhibitor ; ami , acute myocardial infarction ; arb , angiotensin type ii receptor blocker ; cabg , coronary artery bypass grafting ; or , odds ratios ; pci , percutaneous coronary intervention . adjusted for study period , sociodemographic characteristics , and comorbidities . adjusted for study period , sociodemographic characteristics , comorbid conditions , ami order , ami type , inhospital clinical complications , and hospital length of stay . adjusted for study period , sociodemographic characteristics , comorbid conditions , inhospital factors , and inhospital management as represented by thrombolytic therapy and receipt of 3 coronary interventional procedures ( cardiac catheterization , pci , and cabg ) and prescribing of 4 guidelinerecommended cardiac medications ( aceis / arbs , lipidlowering agents , blockers , and aspirin ) at the time of hospital discharge . the study population consisted of 4810 adult residents of the worcester metropolitan area who survived their hospitalization for ami at the 3 major central massachusetts medical centers between 2001 and 2011 ( table 1 ) . overall , the average age of this population was 68.9 years , 41.8% were women , 92.4% were white , and 54.6% were married . characteristics of patients who survived an ami : worcester heart attack study , 20012011 ami indicates acute myocardial infarction . pvalues derived from anova tests for continuous variables and tests for categorical variables . p values derived from cochranarmitage tests for categorical variables and linear regression models for continuous variables . during the most recent years under study , patients who survived their ami were more likely to be younger and have a history of hypertension , peripheral vascular disease , diabetes , or chronic kidney disease than were patients who were hospitalized during earlier study periods ( table 1 ) . between 2001 and 2011 , 32.6% of our study sample was diagnosed with a stemi , which declined from 34.0% in 20012003 to 31.1% in 20092011 . the average hospital length of stay in this study population was 5.5 days , which declined from 6.4 days in 20012003 to 4.7 days in 20092011 ( table 1 ) . in addition , the likelihood of developing cardiogenic shock and stroke during hospitalization remained relatively low ( 3.5% and 1.4% overall , respectively ) , whereas the incidence rates of inhospital heart failure and atrial fibrillation were considerably higher ( 34.4% and 18.8% overall , respectively ) during the years under study ( table 1 ) . the average postdischarge 30day rehospitalization rates decreased from 20012003 ( 20.5% ) to 20092011 ( 15.8% ) ( p for trend=0.001 ) ( table 2 ) . the proportion of patients who were rehospitalized was the highest ( 6.6% ) during the first week ( 0 to 7 days ) after hospital discharge and continued to decrease as the length of postdischarge time increased ( figure 1 ) . rehospitalizations that occurred during the first week after hospital discharge accounted for 35.6% of all 30day rehospitalizations ; this proportion decreased from 39.2% in 20012003 to 27.2% in 20092011 . association between time period of hospitalization and 30day allcause rehospitalizations among patients who survived an ami : worcester heart attack study , 20012011 ( n=4810 ) acei indicates angiotensinconverting inhibitors ; ami , acute myocardial infarction ; arbs , angiotensin type ii receptor blockers ; cabg , coronary artery bypass grafting ; or , odds ratio ; pci , percutaneous coronary intervention . adjusted for age , sex , race , marital status , and previously diagnosed comorbid conditions . adjusted for sociodemographic characteristics , comorbid conditions , ami order , ami type , inhospital complications , and hospital length of stay . adjusted for sociodemographic characteristics , comorbid conditions , inhospital factors , and inhospital management as represented by thrombolytic therapy and receipt of 3 coronary interventional procedures ( cardiac catheterization , pci , and cabg ) and prescribing of 4 guidelinerecommended cardiac medications ( aceis / arbs , lipidlowering agents , blockers , and aspirin ) at the time of hospital discharge . rates of rehospitalizations within 30 days after hospital discharge among patients who survived an acute myocardial infarction : worcester heart attack study , 20012011 ( n=4810 ) . in examining the specific causes of 30day rehospitalizations , 53.9% were cvd related ( excluding ami ) cvd related , and 8.0% were due to a recurrent ami during the years under study . the overall causespecific 30day rehospitalization rates due to cvd , noncvd , and ami were 10.0% , 7.0% , and 1.5% , respectively , during the years under study ( figure 2 ) . the average postdischarge 30day rehospitalization rates due to non cvdrelated reasons decreased ( p for trend < 0.001 ) during the years under study , while no significant changing trends in 30day rehospitalization rates due to cvd ( excluding ami ) or ami were observed ( figure 2 ) . causespecific 30day rehospitalization rates after hospital discharge among patients who survived an ami : worcester heart attack study , 20012011 ( n=4810 ) . ami indicates acute myocardial infarction ; cvd , cardiovascular diseases exclude ami . in examining changing trends in 30day rehospitalizations after adjusting for several demographic characteristics , comorbidities , and inhospital clinical factors , there was no significant difference in the odds of having a 30day rehospitalization in 20052007 ( or 0.88 , 95% ci 0.74 to 1.04 ) , but there was a reduced odds of being rehospitalized during the subsequent 30 days ( or 0.74 , 95% ci 0.61 to 0.91 ) among patients who survived an ami in 20092011 , in comparison with those discharged in 20012003 ( table 2 ) . after further adjustment for medical procedures and treatments received during hospitalization , there was a significantly reduced odds of being rehospitalized during the subsequent 30 days ( or 0.78 , 95% ci 0.65 to 0.93 ) in 20092011 compared with those discharged from the hospital in 20012003 ( table 2 ) . we observed similar results after excluding patients who were not rehospitalized but died during the 30day postdischarge period . we also observed similar results when we analyzed these patients as those who had a 30day hospital readmission . using multivariableadjusted regression analyses , we examined the role of various prognostic factors associated with 30day rehospitalizations in all study patients ( table 3 ) . female sex , having previously diagnosed heart failure and chronic kidney disease , and the development of inhospital cardiogenic shock and heart failure were significantly associated with an increased odds of being rehospitalized for any reason within 30 days after hospital discharge . on the other hand , patients who received various coronary diagnostic and interventional procedures ( cardiac catheterization and pci and/or cabg ) had a reduced odds for being rehospitalized within 30 days after hospital discharge compared with those who did not undergo these procedures ( table 3 ) . we observed similar factors that were significantly associated with 30day rehospitalizations after excluding patients who were not rehospitalized but died during the 30day postdischarge period . we also observed similar factors associated with hospital readmission when we analyzed these patients as having had a 30day hospital readmission . association between various prognostic factors and 30day allcause rehospitalizations among patients who survived an ami : worcester heart attack study , 20012011 ( n=4810 ) acei indicates angiotensinconverting inhibitor ; ami , acute myocardial infarction ; arb , angiotensin type ii receptor blocker ; cabg , coronary artery bypass grafting ; or , odds ratios ; pci , percutaneous coronary intervention . adjusted for study period , sociodemographic characteristics , and comorbidities . adjusted for study period , sociodemographic characteristics , comorbid conditions , ami order , ami type , inhospital clinical complications , and hospital length of stay . adjusted for study period , sociodemographic characteristics , comorbid conditions , inhospital factors , and inhospital management as represented by thrombolytic therapy and receipt of 3 coronary interventional procedures ( cardiac catheterization , pci , and cabg ) and prescribing of 4 guidelinerecommended cardiac medications ( aceis / arbs , lipidlowering agents , blockers , and aspirin ) at the time of hospital discharge . the results of this large observational study suggest that , among greater worcester residents who survived their hospitalization for an ami at the major medical centers in central massachusetts between 2001 and 2011 , nearly 1 in 5 patients remained at risk for being rehospitalized within 30 days and 36% of all 30day rehospitalizations occurred during the first week after hospital discharge during the years under study . our findings suggest a decline in the odds of being rehospitalized during the first 30 days after hospital discharge in the most recent years under study , though this odds was slightly attenuated after further adjustment for hospital treatment practices . in addition , we identified several demographic and clinical factors associated with an increased odds for being rehospitalized during the first month after hospital discharge for ami . reducing hospital readmissions is a national priority to improve the quality of patient care and lower health care spending.5 , 7 , 21 this is because excess hospital readmissions indicate potentially poor health care quality or inadequate coordination of postdischarge care , represent a significant burden to both patients and the health care system , and are costly.5 , 7 , 21 several prior studies have examined the frequency of 30day rehospitalizations after ami.4 , 8 , 9 between 2007 and 2009 , nearly 1 in every 5 medicare feeforservice patients discharged from all acute care hospitals in the united states after an ami was readmitted within 30 days after hospital discharge.8 in a recent study that used an allpayer administrative data set from california , which consisted of 107 256 hospitalizations for ami among adults younger than 65 years between 2007 and 2009 , the 30day rehospitalization rate was 15%.9 a retrospective cohort study conducted in 3 hospitals in olmsted county , mn , during 19872010 found that the 30day readmission rates among adult patients who survived their hospitalization for a first ami were 23% during 19871992 , 22% during 19932004 , and 19% during the most recent period under study ( 20052010).4 in our study , we found similar results in that nearly onefifth of adult greater worcester residents who survived their hospitalization for an ami on a biennial basis between 2001 and 2011 were readmitted to the hospital within 30 days after hospital discharge . in addition , our study observed an encouraging decline in the odds of having a 30day rehospitalization during the most recent years ( 20092011 ) under study after adjustment for several potentially confounding variables of prognostic importance . since june 2009 , the cms began publicly reporting 30day riskstandardized readmission rates for ami as one of the major hospital performance measures.5 further , the patient protection affordable care act of 2010 , through the establishment of the medicare hospital readmissions reduction program , has created new payment penalties ( effective october 1 , 2012 ) to reduce readmissions because hospitals with excess readmissions can lose up to 3% of their medicare reimbursement by 2015.7 although some early evidence suggests that the medicare hospital readmissions reduction program has had a positive impact on reducing the rates of 30day rehospitalization among medicare beneficiaries,22 there remains no clear consensus on how many hospital readmissions may in all actuality be preventable . moreover , there are also concerns about potential flaws in the methodology for determining excess readmissions and computation of the penalty to hospitals.21 thus , it remains of considerable public health and clinical importance to continue monitoring contemporary trends in 30day rehospitalization rates after ami given ongoing refinement of the methodological approach by the cms to predict the risk of hospital readmissions according to various patient , provider , and health systems characteristics . with regard to the timing of hospital readmissions , a recent study that analyzed medicare feeforservice claims data ( 20072009 ) on 30day readmissions after hospitalization for ami showed that 40% of all 30day readmissions occurred during the first week after hospital discharge.8 another recent study using 20072009 administrative data from the state of california found that 19% of readmissions occurred within 0 to 3 days , and 21% occurred during 4 to 7 days , after hospital discharge for ami.9 similar to these results , our study found that 36% of all 30day rehospitalizations occurred during the first week after hospital discharge for ami . these findings suggest that proper arrangement of transitional care and continuing followup with patients during the first several days to first week post hospital discharge can be beneficial in reducing hospital readmission rates within 30 days among these patients . encouragingly , we also observed a decline in the proportion of patients who were readmitted during this particularly highrisk period during the years under study , which may suggest that efforts at reducing hospital readmissions may be paying dividends in reducing shortterm hospital readmissions . while we did not have specific information available to more fully describe and characterize any efforts that were being carried out at participating study hospitals to reduce their hospital readmission rates for patients discharged after an ami , moreaggressive systemwide efforts were being put in place in 2011 at umassmemorial health care to systematically measure and reduce the rates of hospital readmissions at these 2 medical centers . a retrospective cohort study of adult patients in olmsted county , mn,4 who were discharged from the hospital after a first ami found that 43% of 30day rehospitalizations after ami were related to the incident ami , 30% were unrelated , and 27% had an unclear relationship . the investigators also reported that about 8% of all 30day rehospitalizations were due to a recurrent ami.4 a recent study of medicare feeforservice beneficiaries hospitalized for ami at all acute care hospitals in the united states between 2007 and 2009 showed that 10% of patients were readmitted for the same condition after their index ami hospitalization.8 we observed similar results in that 8% of all 30day rehospitalizations were due to ami , and a significant proportion ( 38% ) of all 30day rehospitalizations were non cvd related . as the prevalence of comorbid conditions and aging of the american population increase over time , and efforts continue to be focused on the enhanced use of effective secondary prevention strategies to improve the postdischarge outcomes of patients with ami , 30day rehospitalizations after ami due to noncvd causes require further attention . indeed , declining lengths of hospital stay , number and complexities of evidencebased medications , older age of this patient population , increased use of observation units , and primary focus on the patient 's underlying heart condition , but not on other important comorbidities these patients have been previously diagnosed with , may have in part contributed to the high rate of non recent research has also suggested that patients discharged from the hospital may be vulnerable to posthospitalization syndrome , which puts them at risk for rehospitalization for conditions unrelated to their initial hospitalization.23 further research is needed to confirm the association between this syndrome and other hospital and postdischarge factors that may place patients at risk for non cvdrelated hospital readmissions and identify effective strategies for reducing readmissions . although efforts remain ongoing to find strategies that hospitals can use to prevent many readmissions , there is an ongoing debate on whether the hospital is the appropriate entity to be held accountable for all shortterm readmissions , particularly when many of the events and circumstances that are associated with readmission may take place outside of the hospital setting and after the administration of effective acute care . these factors include patients lifestyle behaviors and practices ; employment , marital , and financial status ; adherence to discharge instructions and medications ; and the availability and quality of postdischarge care . thus , reducing the frequency of hospital readmissions requires considerable collaborations , not only from hospitals but also from patients and their caregivers and other community professionals and providers across the continuum of health . due to ongoing and planned changes in national reimbursement policies,7 there has been great interest from health care providers to better understand and improve modifiable factors associated with 30day rehospitalizations . a recent study in olmsted county , mn , examined factors associated with 30day rehospitalizations after an incident ami.4 the investigators found that certain comorbid conditions , a longer hospital stay , and complications of coronary angiography and revascularization or reperfusion were associated with an increased risk of being rehospitalized.4 our study observed that patients with a history of previously diagnosed heart failure and chronic kidney disease and the development of inhospital cardiogenic shock and heart failure were significantly associated with an increased odds of being rehospitalized during the first month after hospital discharge for ami . although most of these factors are not modifiable , these findings suggest that health care providers should pay extra attention to these highrisk groups of vulnerable patients to prevent potential early readmissions when planning hospital discharge and postdischarge management . future studies examining the postdischarge transitions of care in higherrisk patients , including those with multiple comorbid conditions and hospital clinical complications , remain needed to achieve greater declines in 30day rehospitalizations in this patient population . despite the potential for confounding by indication given the nonrandomized nature of the present investigation , our multivariable regression analyses adjusting for the use of various hospital treatment practices showed that the use of invasive coronary interventions was associated with a reduced odds of being rehospitalized among patients hospitalized with ami during the decadelong period under study . furthermore , encouraging declines in 30 day rehospitalizations during the years under study were slightly attenuated after adjustment for hospital treatment practices , suggesting the beneficial effects of various cardiac treatment practices on 30day readmission rates . a recent study examining 30day rehospitalizations after an acute coronary syndrome among 5219 patients enrolled in the australian and new zealand populations of the global registry of acute coronary events ( grace ) between 1999 and 2007 also observed similar results in that coronary revascularization during the acute hospital stay was associated with a reduced odds of being rehospitalized during the next month.24 the strengths of the present study include its large population of residents of all ages and both sexes from a major central massachusetts metropolitan area who were hospitalized with a confirmed ami and examination of relatively contemporary decadelong trends in 30day rehospitalization rates among hospital survivors of an ami . several limitations need to be acknowledged , however , in the interpretation of the present findings . because our study population included only patients who had been hospitalized and discharged at 3 central massachusetts medical centers , one needs to be careful in extrapolating our findings to those who reside in other geographic areas . if a rehospitalization occurred outside of the worcester metropolitan area or at other medical centers in central massachusetts , it was not captured , although it is expected that this number would be quite small and unlikely to have changed during the years under study . this is because most patients typically return to their hospital of admission and the large tertiary care and community hospitals included in the present investigation capture the vast majority of hospitalizations that occur among residents of the worcester metropolitan area . because study patients were predominantly white , the generalizability of our findings to other race / ethnicity groups may be limited . there is also the potential for unmeasured confounding in our observed associations because we did not have information available on several patientassociated characteristics , such as income , education , psychosocial factors , and treatment preference , that may have affected the end points examined . we were unable to collect information on other factors that have been shown to affect 30day rehospitalization after ami , including transitions of care and patients adherence to various postdischarge treatment regimens . finally , although our study observed an encouraging decline in 30day rehospitalizations during the most recent years ( 20092011 ) under study , future studies remain warranted to continue monitoring changes in 30day rehospitalization rates after the implementation of financial penalties to hospitals due to excess readmissions in 2012.7 reducing hospital readmissions is a national priority to improve the quality of patient care and lower health care spending.5 , 7 , 21 this is because excess hospital readmissions indicate potentially poor health care quality or inadequate coordination of postdischarge care , represent a significant burden to both patients and the health care system , and are costly.5 , 7 , 21 several prior studies have examined the frequency of 30day rehospitalizations after ami.4 , 8 , 9 between 2007 and 2009 , nearly 1 in every 5 medicare feeforservice patients discharged from all acute care hospitals in the united states after an ami was readmitted within 30 days after hospital discharge.8 in a recent study that used an allpayer administrative data set from california , which consisted of 107 256 hospitalizations for ami among adults younger than 65 years between 2007 and 2009 , the 30day rehospitalization rate was 15%.9 a retrospective cohort study conducted in 3 hospitals in olmsted county , mn , during 19872010 found that the 30day readmission rates among adult patients who survived their hospitalization for a first ami were 23% during 19871992 , 22% during 19932004 , and 19% during the most recent period under study ( 20052010).4 in our study , we found similar results in that nearly onefifth of adult greater worcester residents who survived their hospitalization for an ami on a biennial basis between 2001 and 2011 were readmitted to the hospital within 30 days after hospital discharge . in addition , our study observed an encouraging decline in the odds of having a 30day rehospitalization during the most recent years ( 20092011 ) under study after adjustment for several potentially confounding variables of prognostic importance . since june 2009 , the cms began publicly reporting 30day riskstandardized readmission rates for ami as one of the major hospital performance measures.5 further , the patient protection affordable care act of 2010 , through the establishment of the medicare hospital readmissions reduction program , has created new payment penalties ( effective october 1 , 2012 ) to reduce readmissions because hospitals with excess readmissions can lose up to 3% of their medicare reimbursement by 2015.7 although some early evidence suggests that the medicare hospital readmissions reduction program has had a positive impact on reducing the rates of 30day rehospitalization among medicare beneficiaries,22 there remains no clear consensus on how many hospital readmissions may in all actuality be preventable . moreover , there are also concerns about potential flaws in the methodology for determining excess readmissions and computation of the penalty to hospitals.21 thus , it remains of considerable public health and clinical importance to continue monitoring contemporary trends in 30day rehospitalization rates after ami given ongoing refinement of the methodological approach by the cms to predict the risk of hospital readmissions according to various patient , provider , and health systems characteristics . with regard to the timing of hospital readmissions , a recent study that analyzed medicare feeforservice claims data ( 20072009 ) on 30day readmissions after hospitalization for ami showed that 40% of all 30day readmissions occurred during the first week after hospital discharge.8 another recent study using 20072009 administrative data from the state of california found that 19% of readmissions occurred within 0 to 3 days , and 21% occurred during 4 to 7 days , after hospital discharge for ami.9 similar to these results , our study found that 36% of all 30day rehospitalizations occurred during the first week after hospital discharge for ami . these findings suggest that proper arrangement of transitional care and continuing followup with patients during the first several days to first week post hospital discharge can be beneficial in reducing hospital readmission rates within 30 days among these patients . encouragingly , we also observed a decline in the proportion of patients who were readmitted during this particularly highrisk period during the years under study , which may suggest that efforts at reducing hospital readmissions may be paying dividends in reducing shortterm hospital readmissions . while we did not have specific information available to more fully describe and characterize any efforts that were being carried out at participating study hospitals to reduce their hospital readmission rates for patients discharged after an ami , moreaggressive systemwide efforts were being put in place in 2011 at umassmemorial health care to systematically measure and reduce the rates of hospital readmissions at these 2 medical centers . a retrospective cohort study of adult patients in olmsted county , mn,4 who were discharged from the hospital after a first ami found that 43% of 30day rehospitalizations after ami were related to the incident ami , 30% were unrelated , and 27% had an unclear relationship . the investigators also reported that about 8% of all 30day rehospitalizations were due to a recurrent ami.4 a recent study of medicare feeforservice beneficiaries hospitalized for ami at all acute care hospitals in the united states between 2007 and 2009 showed that 10% of patients were readmitted for the same condition after their index ami hospitalization.8 we observed similar results in that 8% of all 30day rehospitalizations were due to ami , and a significant proportion ( 38% ) of all 30day rehospitalizations were non cvd related . as the prevalence of comorbid conditions and aging of the american population increase over time , and efforts continue to be focused on the enhanced use of effective secondary prevention strategies to improve the postdischarge outcomes of patients with ami , 30day rehospitalizations after ami due to noncvd causes require further attention . indeed , declining lengths of hospital stay , number and complexities of evidencebased medications , older age of this patient population , increased use of observation units , and primary focus on the patient 's underlying heart condition , but not on other important comorbidities these patients have been previously diagnosed with , may have in part contributed to the high rate of non recent research has also suggested that patients discharged from the hospital may be vulnerable to posthospitalization syndrome , which puts them at risk for rehospitalization for conditions unrelated to their initial hospitalization.23 further research is needed to confirm the association between this syndrome and other hospital and postdischarge factors that may place patients at risk for non cvdrelated hospital readmissions and identify effective strategies for reducing readmissions . although efforts remain ongoing to find strategies that hospitals can use to prevent many readmissions , there is an ongoing debate on whether the hospital is the appropriate entity to be held accountable for all shortterm readmissions , particularly when many of the events and circumstances that are associated with readmission may take place outside of the hospital setting and after the administration of effective acute care . these factors include patients lifestyle behaviors and practices ; employment , marital , and financial status ; adherence to discharge instructions and medications ; and the availability and quality of postdischarge care . thus , reducing the frequency of hospital readmissions requires considerable collaborations , not only from hospitals but also from patients and their caregivers and other community professionals and providers across the continuum of health . due to ongoing and planned changes in national reimbursement policies,7 there has been great interest from health care providers to better understand and improve modifiable factors associated with 30day rehospitalizations . a recent study in olmsted county , mn , examined factors associated with 30day rehospitalizations after an incident ami.4 the investigators found that certain comorbid conditions , a longer hospital stay , and complications of coronary angiography and revascularization or reperfusion were associated with an increased risk of being rehospitalized.4 our study observed that patients with a history of previously diagnosed heart failure and chronic kidney disease and the development of inhospital cardiogenic shock and heart failure were significantly associated with an increased odds of being rehospitalized during the first month after hospital discharge for ami . although most of these factors are not modifiable , these findings suggest that health care providers should pay extra attention to these highrisk groups of vulnerable patients to prevent potential early readmissions when planning hospital discharge and postdischarge management . future studies examining the postdischarge transitions of care in higherrisk patients , including those with multiple comorbid conditions and hospital clinical complications , remain needed to achieve greater declines in 30day rehospitalizations in this patient population . despite the potential for confounding by indication given the nonrandomized nature of the present investigation , our multivariable regression analyses adjusting for the use of various hospital treatment practices showed that the use of invasive coronary interventions was associated with a reduced odds of being rehospitalized among patients hospitalized with ami during the decadelong period under study . furthermore , encouraging declines in 30 day rehospitalizations during the years under study were slightly attenuated after adjustment for hospital treatment practices , suggesting the beneficial effects of various cardiac treatment practices on 30day readmission rates . a recent study examining 30day rehospitalizations after an acute coronary syndrome among 5219 patients enrolled in the australian and new zealand populations of the global registry of acute coronary events ( grace ) between 1999 and 2007 also observed similar results in that coronary revascularization during the acute hospital stay the strengths of the present study include its large population of residents of all ages and both sexes from a major central massachusetts metropolitan area who were hospitalized with a confirmed ami and examination of relatively contemporary decadelong trends in 30day rehospitalization rates among hospital survivors of an ami . several limitations need to be acknowledged , however , in the interpretation of the present findings . because our study population included only patients who had been hospitalized and discharged at 3 central massachusetts medical centers , one needs to be careful in extrapolating our findings to those who reside in other geographic areas . if a rehospitalization occurred outside of the worcester metropolitan area or at other medical centers in central massachusetts , it was not captured , although it is expected that this number would be quite small and unlikely to have changed during the years under study . this is because most patients typically return to their hospital of admission and the large tertiary care and community hospitals included in the present investigation capture the vast majority of hospitalizations that occur among residents of the worcester metropolitan area . because study patients were predominantly white , the generalizability of our findings to other race / ethnicity groups may be limited . there is also the potential for unmeasured confounding in our observed associations because we did not have information available on several patientassociated characteristics , such as income , education , psychosocial factors , and treatment preference , that may have affected the end points examined . we were unable to collect information on other factors that have been shown to affect 30day rehospitalization after ami , including transitions of care and patients adherence to various postdischarge treatment regimens . finally , although our study observed an encouraging decline in 30day rehospitalizations during the most recent years ( 20092011 ) under study , future studies remain warranted to continue monitoring changes in 30day rehospitalization rates after the implementation of financial penalties to hospitals due to excess readmissions in 2012.7 the results of this large observational investigation provide insights into trends and causes of 30day rehospitalizations , and factors associated with an increased risk of 30day rehospitalizations , among patients who survived hospitalization for an ami between 2001 and 2011 . the likelihood of subsequent rehospitalizations during the following month remained frequent ; however , we observed an encouraging decline in the 30day rehospitalization rate during the most recent years under study . although most of the identified risk factors were not readily modifiable , our findings can , we hope , lead to better development of innovative , patientcentered , intervention strategies that can improve inhospital management and followup care that will further reduce the 30day rehospitalization rates of patients discharged from the hospital after an ami . this research was made possible by the cooperation of participating hospitals in the worcester metropolitan area . partial salary support for drs gore and goldberg was provided for by national institutes of health grant 1u01hl10526801 .

backgroundthere are limited data available describing relatively contemporary trends in 30day rehospitalizations among patients who survive hospitalization after an acute myocardial infarction ( ami ) in the community setting . we examined decadelong ( 20012011 ) trends in , and factors associated with , 30day rehospitalizations in patients discharged from 3 central massachusetts hospitals after ami.methods and resultsresidents of the worcester , ma , metropolitan area discharged after ami from 3 central massachusetts hospitals on a biennial basis between 2001 and 2011 comprised the study population ( n=4810 ) . logistic regression analyses were used to examine the association between selected factors and 30day rehospitalizations . the average age of this population was 69 years , 42% were women , and 92% were white . during the years under study , 18.5% of patients were rehospitalized within 30 days after hospital discharge . crude 30day rehospitalization rates decreased from 20.5% in 20012003 to 15.8% in 20092011 . after adjusting for several patient characteristics , there was a reduced odds of being rehospitalized in 20092011 ( odds ratio 0.74 , 95% ci 0.610.91 ) compared with 20012003 ; this trend was slightly attenuated after further adjustment for hospital treatment practices . female sex , having previously diagnosed heart failure and chronic kidney disease , and the development of inhospital cardiogenic shock and heart failure were associated with an increased odds of being rehospitalized.conclusionswhile the likelihood of subsequent shortterm rehospitalizations remained frequent , we observed an encouraging decline during the most recent years under study . several highrisk groups were identified for purposes of heightened surveillance and intervention efforts to reduce the likelihood of being readmitted .

Introduction Methods Data Collection Data Analysis Results Study Population Characteristics ThirtyDay Rehospitalization Rates Factors Associated With AllCause 30Day Rehospitalizations Discussion Trends in and Magnitude of 30Day Rehospitalization Rates Causes and Predictors for 30Day Rehospitalizations Study Strengths and Limitations Conclusions Sources of Funding Disclosures

acute myocardial infarction ( ami ) is a common manifestation of coronary heart disease that affected > 800 000 adults in the united states in 2010.1 concomitant with advances in prehospital and hospital treatment , inhospital survival after ami has dramatically improved.2 many patients are being discharged from the hospital into the community who are at risk for being readmitted to the hospital due to a variety of contributory factors and reasons.3 , 4 although not all hospital readmissions can be prevented , excess readmissions within a short time frame can be a marker of poor quality of care and efficiency . our secondary study objective was to describe patient characteristics , clinical factors , and treatment practices associated with an increased risk of 30day rehospitalizations among residents of central massachusetts discharged from the 3 principal medical centers in central massachusetts after an ami . data from the worcester heart attack study were used in this study.10 , 11 , 12 , 13 described elsewhere in detail,10 , 11 , 12 , 13 the worcester heart attack study is an ongoing populationbased investigation examining longterm trends in the descriptive epidemiology of ami in residents of the worcester , ma , metropolitan area ( 2010 census 518 000 ) hospitalized at all 16 medical centers in central massachusetts on an approximate biennial basis between 1975 and 2011.10 , 11 , 12 , 13 due to hospital closures , mergers , or conversion to longterm care or rehabilitation facilities , fewer hospitals ( n=11 ) have been providing care to greater worcester residents during recent years . multivariableadjusted logistic regression analyses were performed to examine the association between the main explanatory variable of period of hospitalization ( 20012003 , earliest ; 20052007 , middle ; and 20092011 , most recent ) and the outcome of whether the patient was rehospitalized during the following 30 days while adjusting for several potentially confounding variables of prognostic importance . several covariates associated with rehospitalization after ami in prior studies3 , 4 were examined including age , sex , race ( white versus nonwhite ) , marital status ( married versus not married ) , ami order ( initial versus prior ) , ami type ( stemi versus nstemi ) , previously diagnosed comorbid conditions ( angina , atrial fibrillation , heart failure , hypertension , peripheral vascular disease , stroke , diabetes , chronic obstructive pulmonary disease , depression , and chronic kidney disease ) , hospital clinical complications ( atrial fibrillation , heart failure , cardiogenic shock , and stroke ) , and hospital length of stay . we further adjusted for hospital treatment practices , including the receipt of thrombolytic therapy and 3 coronary diagnostic and interventional procedures ( cardiac catheterization , pci , and cabg ) , and the prescribing of 4 guideline recommended cardiac medications ( aceis / arbs , aspirin , blockers , and lipidlowering agents ) at the time of hospital discharge during the patient 's index hospitalization in our regression analyses to examine the potential effects of hospital treatment practices on 30day rehospitalization trends . shortterm rehospitalization rates were examined by calculating the frequency of having a first rehospitalization within 30 days among patients discharged from the hospital after their index ami during the years under study . multivariableadjusted logistic regression analyses were performed to examine the association between the main explanatory variable of period of hospitalization ( 20012003 , earliest ; 20052007 , middle ; and 20092011 , most recent ) and the outcome of whether the patient was rehospitalized during the following 30 days while adjusting for several potentially confounding variables of prognostic importance . several covariates associated with rehospitalization after ami in prior studies3 , 4 were examined including age , sex , race ( white versus nonwhite ) , marital status ( married versus not married ) , ami order ( initial versus prior ) , ami type ( stemi versus nstemi ) , previously diagnosed comorbid conditions ( angina , atrial fibrillation , heart failure , hypertension , peripheral vascular disease , stroke , diabetes , chronic obstructive pulmonary disease , depression , and chronic kidney disease ) , hospital clinical complications ( atrial fibrillation , heart failure , cardiogenic shock , and stroke ) , and hospital length of stay . we further adjusted for hospital treatment practices , including the receipt of thrombolytic therapy and 3 coronary diagnostic and interventional procedures ( cardiac catheterization , pci , and cabg ) , and the prescribing of 4 guideline recommended cardiac medications ( aceis / arbs , aspirin , blockers , and lipidlowering agents ) at the time of hospital discharge during the patient 's index hospitalization in our regression analyses to examine the potential effects of hospital treatment practices on 30day rehospitalization trends . overall , the average age of this population was 68.9 years , 41.8% were women , 92.4% were white , and 54.6% were married . during the most recent years under study , patients who survived their ami were more likely to be younger and have a history of hypertension , peripheral vascular disease , diabetes , or chronic kidney disease than were patients who were hospitalized during earlier study periods ( table 1 ) . in addition , the likelihood of developing cardiogenic shock and stroke during hospitalization remained relatively low ( 3.5% and 1.4% overall , respectively ) , whereas the incidence rates of inhospital heart failure and atrial fibrillation were considerably higher ( 34.4% and 18.8% overall , respectively ) during the years under study ( table 1 ) . association between time period of hospitalization and 30day allcause rehospitalizations among patients who survived an ami : worcester heart attack study , 20012011 ( n=4810 ) acei indicates angiotensinconverting inhibitors ; ami , acute myocardial infarction ; arbs , angiotensin type ii receptor blockers ; cabg , coronary artery bypass grafting ; or , odds ratio ; pci , percutaneous coronary intervention . rates of rehospitalizations within 30 days after hospital discharge among patients who survived an acute myocardial infarction : worcester heart attack study , 20012011 ( n=4810 ) . in examining changing trends in 30day rehospitalizations after adjusting for several demographic characteristics , comorbidities , and inhospital clinical factors , there was no significant difference in the odds of having a 30day rehospitalization in 20052007 ( or 0.88 , 95% ci 0.74 to 1.04 ) , but there was a reduced odds of being rehospitalized during the subsequent 30 days ( or 0.74 , 95% ci 0.61 to 0.91 ) among patients who survived an ami in 20092011 , in comparison with those discharged in 20012003 ( table 2 ) . after further adjustment for medical procedures and treatments received during hospitalization , there was a significantly reduced odds of being rehospitalized during the subsequent 30 days ( or 0.78 , 95% ci 0.65 to 0.93 ) in 20092011 compared with those discharged from the hospital in 20012003 ( table 2 ) . female sex , having previously diagnosed heart failure and chronic kidney disease , and the development of inhospital cardiogenic shock and heart failure were significantly associated with an increased odds of being rehospitalized for any reason within 30 days after hospital discharge . on the other hand , patients who received various coronary diagnostic and interventional procedures ( cardiac catheterization and pci and/or cabg ) had a reduced odds for being rehospitalized within 30 days after hospital discharge compared with those who did not undergo these procedures ( table 3 ) . association between various prognostic factors and 30day allcause rehospitalizations among patients who survived an ami : worcester heart attack study , 20012011 ( n=4810 ) acei indicates angiotensinconverting inhibitor ; ami , acute myocardial infarction ; arb , angiotensin type ii receptor blocker ; cabg , coronary artery bypass grafting ; or , odds ratios ; pci , percutaneous coronary intervention . overall , the average age of this population was 68.9 years , 41.8% were women , 92.4% were white , and 54.6% were married . during the most recent years under study , patients who survived their ami were more likely to be younger and have a history of hypertension , peripheral vascular disease , diabetes , or chronic kidney disease than were patients who were hospitalized during earlier study periods ( table 1 ) . in addition , the likelihood of developing cardiogenic shock and stroke during hospitalization remained relatively low ( 3.5% and 1.4% overall , respectively ) , whereas the incidence rates of inhospital heart failure and atrial fibrillation were considerably higher ( 34.4% and 18.8% overall , respectively ) during the years under study ( table 1 ) . association between time period of hospitalization and 30day allcause rehospitalizations among patients who survived an ami : worcester heart attack study , 20012011 ( n=4810 ) acei indicates angiotensinconverting inhibitors ; ami , acute myocardial infarction ; arbs , angiotensin type ii receptor blockers ; cabg , coronary artery bypass grafting ; or , odds ratio ; pci , percutaneous coronary intervention . rates of rehospitalizations within 30 days after hospital discharge among patients who survived an acute myocardial infarction : worcester heart attack study , 20012011 ( n=4810 ) . the average postdischarge 30day rehospitalization rates due to non cvdrelated reasons decreased ( p for trend < 0.001 ) during the years under study , while no significant changing trends in 30day rehospitalization rates due to cvd ( excluding ami ) or ami were observed ( figure 2 ) . in examining changing trends in 30day rehospitalizations after adjusting for several demographic characteristics , comorbidities , and inhospital clinical factors , there was no significant difference in the odds of having a 30day rehospitalization in 20052007 ( or 0.88 , 95% ci 0.74 to 1.04 ) , but there was a reduced odds of being rehospitalized during the subsequent 30 days ( or 0.74 , 95% ci 0.61 to 0.91 ) among patients who survived an ami in 20092011 , in comparison with those discharged in 20012003 ( table 2 ) . after further adjustment for medical procedures and treatments received during hospitalization , there was a significantly reduced odds of being rehospitalized during the subsequent 30 days ( or 0.78 , 95% ci 0.65 to 0.93 ) in 20092011 compared with those discharged from the hospital in 20012003 ( table 2 ) . female sex , having previously diagnosed heart failure and chronic kidney disease , and the development of inhospital cardiogenic shock and heart failure were significantly associated with an increased odds of being rehospitalized for any reason within 30 days after hospital discharge . on the other hand , patients who received various coronary diagnostic and interventional procedures ( cardiac catheterization and pci and/or cabg ) had a reduced odds for being rehospitalized within 30 days after hospital discharge compared with those who did not undergo these procedures ( table 3 ) . association between various prognostic factors and 30day allcause rehospitalizations among patients who survived an ami : worcester heart attack study , 20012011 ( n=4810 ) acei indicates angiotensinconverting inhibitor ; ami , acute myocardial infarction ; arb , angiotensin type ii receptor blocker ; cabg , coronary artery bypass grafting ; or , odds ratios ; pci , percutaneous coronary intervention . the results of this large observational study suggest that , among greater worcester residents who survived their hospitalization for an ami at the major medical centers in central massachusetts between 2001 and 2011 , nearly 1 in 5 patients remained at risk for being rehospitalized within 30 days and 36% of all 30day rehospitalizations occurred during the first week after hospital discharge during the years under study . our findings suggest a decline in the odds of being rehospitalized during the first 30 days after hospital discharge in the most recent years under study , though this odds was slightly attenuated after further adjustment for hospital treatment practices . reducing hospital readmissions is a national priority to improve the quality of patient care and lower health care spending.5 , 7 , 21 this is because excess hospital readmissions indicate potentially poor health care quality or inadequate coordination of postdischarge care , represent a significant burden to both patients and the health care system , and are costly.5 , 7 , 21 several prior studies have examined the frequency of 30day rehospitalizations after ami.4 , 8 , 9 between 2007 and 2009 , nearly 1 in every 5 medicare feeforservice patients discharged from all acute care hospitals in the united states after an ami was readmitted within 30 days after hospital discharge.8 in a recent study that used an allpayer administrative data set from california , which consisted of 107 256 hospitalizations for ami among adults younger than 65 years between 2007 and 2009 , the 30day rehospitalization rate was 15%.9 a retrospective cohort study conducted in 3 hospitals in olmsted county , mn , during 19872010 found that the 30day readmission rates among adult patients who survived their hospitalization for a first ami were 23% during 19871992 , 22% during 19932004 , and 19% during the most recent period under study ( 20052010).4 in our study , we found similar results in that nearly onefifth of adult greater worcester residents who survived their hospitalization for an ami on a biennial basis between 2001 and 2011 were readmitted to the hospital within 30 days after hospital discharge . in addition , our study observed an encouraging decline in the odds of having a 30day rehospitalization during the most recent years ( 20092011 ) under study after adjustment for several potentially confounding variables of prognostic importance . a recent study in olmsted county , mn , examined factors associated with 30day rehospitalizations after an incident ami.4 the investigators found that certain comorbid conditions , a longer hospital stay , and complications of coronary angiography and revascularization or reperfusion were associated with an increased risk of being rehospitalized.4 our study observed that patients with a history of previously diagnosed heart failure and chronic kidney disease and the development of inhospital cardiogenic shock and heart failure were significantly associated with an increased odds of being rehospitalized during the first month after hospital discharge for ami . despite the potential for confounding by indication given the nonrandomized nature of the present investigation , our multivariable regression analyses adjusting for the use of various hospital treatment practices showed that the use of invasive coronary interventions was associated with a reduced odds of being rehospitalized among patients hospitalized with ami during the decadelong period under study . a recent study examining 30day rehospitalizations after an acute coronary syndrome among 5219 patients enrolled in the australian and new zealand populations of the global registry of acute coronary events ( grace ) between 1999 and 2007 also observed similar results in that coronary revascularization during the acute hospital stay was associated with a reduced odds of being rehospitalized during the next month.24 the strengths of the present study include its large population of residents of all ages and both sexes from a major central massachusetts metropolitan area who were hospitalized with a confirmed ami and examination of relatively contemporary decadelong trends in 30day rehospitalization rates among hospital survivors of an ami . finally , although our study observed an encouraging decline in 30day rehospitalizations during the most recent years ( 20092011 ) under study , future studies remain warranted to continue monitoring changes in 30day rehospitalization rates after the implementation of financial penalties to hospitals due to excess readmissions in 2012.7 reducing hospital readmissions is a national priority to improve the quality of patient care and lower health care spending.5 , 7 , 21 this is because excess hospital readmissions indicate potentially poor health care quality or inadequate coordination of postdischarge care , represent a significant burden to both patients and the health care system , and are costly.5 , 7 , 21 several prior studies have examined the frequency of 30day rehospitalizations after ami.4 , 8 , 9 between 2007 and 2009 , nearly 1 in every 5 medicare feeforservice patients discharged from all acute care hospitals in the united states after an ami was readmitted within 30 days after hospital discharge.8 in a recent study that used an allpayer administrative data set from california , which consisted of 107 256 hospitalizations for ami among adults younger than 65 years between 2007 and 2009 , the 30day rehospitalization rate was 15%.9 a retrospective cohort study conducted in 3 hospitals in olmsted county , mn , during 19872010 found that the 30day readmission rates among adult patients who survived their hospitalization for a first ami were 23% during 19871992 , 22% during 19932004 , and 19% during the most recent period under study ( 20052010).4 in our study , we found similar results in that nearly onefifth of adult greater worcester residents who survived their hospitalization for an ami on a biennial basis between 2001 and 2011 were readmitted to the hospital within 30 days after hospital discharge . in addition , our study observed an encouraging decline in the odds of having a 30day rehospitalization during the most recent years ( 20092011 ) under study after adjustment for several potentially confounding variables of prognostic importance . a recent study in olmsted county , mn , examined factors associated with 30day rehospitalizations after an incident ami.4 the investigators found that certain comorbid conditions , a longer hospital stay , and complications of coronary angiography and revascularization or reperfusion were associated with an increased risk of being rehospitalized.4 our study observed that patients with a history of previously diagnosed heart failure and chronic kidney disease and the development of inhospital cardiogenic shock and heart failure were significantly associated with an increased odds of being rehospitalized during the first month after hospital discharge for ami . despite the potential for confounding by indication given the nonrandomized nature of the present investigation , our multivariable regression analyses adjusting for the use of various hospital treatment practices showed that the use of invasive coronary interventions was associated with a reduced odds of being rehospitalized among patients hospitalized with ami during the decadelong period under study . a recent study examining 30day rehospitalizations after an acute coronary syndrome among 5219 patients enrolled in the australian and new zealand populations of the global registry of acute coronary events ( grace ) between 1999 and 2007 also observed similar results in that coronary revascularization during the acute hospital stay the strengths of the present study include its large population of residents of all ages and both sexes from a major central massachusetts metropolitan area who were hospitalized with a confirmed ami and examination of relatively contemporary decadelong trends in 30day rehospitalization rates among hospital survivors of an ami . finally , although our study observed an encouraging decline in 30day rehospitalizations during the most recent years ( 20092011 ) under study , future studies remain warranted to continue monitoring changes in 30day rehospitalization rates after the implementation of financial penalties to hospitals due to excess readmissions in 2012.7 the results of this large observational investigation provide insights into trends and causes of 30day rehospitalizations , and factors associated with an increased risk of 30day rehospitalizations , among patients who survived hospitalization for an ami between 2001 and 2011 . the likelihood of subsequent rehospitalizations during the following month remained frequent ; however , we observed an encouraging decline in the 30day rehospitalization rate during the most recent years under study .

even though nystagmus mostly indicates a lesion involving the peripheral vestibular labyrinth , brainstem , or cerebellum , it may occur in lesions involving the cerebral hemisphere , , . nystagmus may be observed in association with seizures or epileptiform discharges originating from the cortical areas involved in the generation of eye movements , . it has been postulated that the epileptic nystagmus stemming from the saccadic areas occurs only in the contraversive hemifield without crossing the midline and has a decreasing slow - phase velocity ( spv ) . in contrast , the epileptic nystagmus from the smooth pursuit regions has been proposed to have a linear spv and corrective quick phases crossing the midline , . even though this distinction seems reasonable in view of the anatomical correlates , , an individual patient may show co - activation of both areas or propagation of the epileptic discharges from one to the other , making the distinction difficult . we also recently encountered a patient who showed epileptic nystagmus with features originating from both the saccade and smooth pursuit areas . even though there have been numerous reports on epileptic nystagmus since the first report in a patient with focal seizure , a systematic review on the features of epileptic nystagmus and the correlation of those with the areas of ictal discharges on eeg has not been available . to address this issue , we describe a patient with epileptic nystagmus and analyzed the characteristics of epileptic nystagmus in the previously reported cases with a systematic review . our patient had continuous eeg monitoring with simultaneous recording of eye movements in the absence of visual fixation using a video - frenzel goggle system ( slmed , seoul , korea ) . brain mri was conducted with a 3.0-t scanner ( achieva , philips , oh , usa ) . ictal and interictal single photon emission computed tomography ( spect , infinia hawkeye 4 spect ct , ge healthcare , waukesha , wi , usa ) were performed using a radiotracer ( tc - hmpao , ceretec , amersham , chalfont , uk ) . the keywords used for searching included epileptic nystagmus , ictal nystagmus , and non - convulsive status epilepticus ( ncse ) . we included all the patients described in systematic reviews , clinical studies , and case reports published in english ( fig . 1 ) . the diagnosis of epileptic nystagmus was based on the following : 1 ) clearly documented seizure activity on the ictal eeg , 2 ) occurrence of nystagmus in accordance with the epileptic discharges , and 3 ) documentation of the nystagmus using oculography , video clip , or periorbital electrodes . from these reports , we analyzed the type of seizure , ictal onset zone and maximal ictal frequency on eeg . we also determined the direction and midline crossing of the nystagmus and the presence of preceding gaze deviation and its direction . we also analyzed findings of oculography even though it was available only in a small number of patients . based on these findings , we attempted any correlation among the epileptic foci and other features of epileptic nystagmus using linear - to - linear correlation . our patient had continuous eeg monitoring with simultaneous recording of eye movements in the absence of visual fixation using a video - frenzel goggle system ( slmed , seoul , korea ) . brain mri was conducted with a 3.0-t scanner ( achieva , philips , oh , usa ) . ictal and interictal single photon emission computed tomography ( spect , infinia hawkeye 4 spect ct , ge healthcare , waukesha , wi , usa ) were performed using a radiotracer ( tc - hmpao , ceretec , amersham , chalfont , uk ) . the keywords used for searching included epileptic nystagmus , ictal nystagmus , and non - convulsive status epilepticus ( ncse ) . we included all the patients described in systematic reviews , clinical studies , and case reports published in english ( fig . 1 ) . the diagnosis of epileptic nystagmus was based on the following : 1 ) clearly documented seizure activity on the ictal eeg , 2 ) occurrence of nystagmus in accordance with the epileptic discharges , and 3 ) documentation of the nystagmus using oculography , video clip , or periorbital electrodes . from these reports , we analyzed the type of seizure , ictal onset zone and maximal ictal frequency on eeg . we also determined the direction and midline crossing of the nystagmus and the presence of preceding gaze deviation and its direction . we also analyzed findings of oculography even though it was available only in a small number of patients . based on these findings , we attempted any correlation among the epileptic foci and other features of epileptic nystagmus using linear - to - linear correlation . she had suffered from hypertension and diabetes mellitus for seven years and alzheimer 's disease for a few years . she had also been admitted to the hospital 4 months ago because of myocardial infarction and cerebral infarction involving the territory of the right anterior cerebral artery . because of global aphasia and dementia , smooth pursuits and saccades could not be fully evaluated , but the reflexive saccades appeared to be intact . however , she intermittently ( 10 per day ) lapsed into a drowsy and lethargic state with both eyes closed and did not respond to noxious stimuli . this altered mental status lasted no more than 1 min , and she recovered to her previous alert state . during the attacks , she showed rightward deviation of both eyes followed by vigorous right - beating nystagmus without any convulsive movements or abnormal postures . eeg revealed 19- to 22-hz polyspikes that were maximal over the left temporo - occipital region , and eye movement recording showed right - beating nystagmus at 34 hz , which did not cross the midline ( video ) . magnetic resonance imaging showed an edematous lesion without gadolinium enhancement on the left temporo - parieto - occipital junction , which was considered a seizure - induced change ( fig . single photon emission computed tomography during the ictus showed hyperperfusion in the left temporo - parieto - occipital junction ( fig . 2b ) . with a suspicion of seizure from a small infarction or alzheimer 's disease itself , fosphenytoin ( 450 mg per day ) and levetiracetam ( 2000 mg per day ) were initiated . her neurological status gradually improved , and follow - up video - eeg one week later showed no epileptiform discharges but regional slowing over the left temporal area . she was discharged two weeks after the admission still with aphasia but without any seizures . through the literature review , we were able to additionally reviewed the reported clinical features of 36 patients ( 21 women , age range : 075 years , median age : 32 years , mean age sd = 35.7 24.1 , table 1 ) . except for four patients [ one ( patient 17 ) with pendular nystagmus , another ( patient 35 ) with upbeat nystagmus , another ( patient 30 ) with generalized ictal discharges on eeg , and the remaining one ( patient 36 ) with alternating nystagmus ] , all the other 33 ( 33/37 , 89.2% ) patients showed nystagmus beating away from the side of ictal discharges on eeg ( table 1 ) . the preceding gaze deviation was described in 25 patients , and 19 of them showed contraversive deviation , while only two had ipsiversive deviation . since oculography was performed in only six patients , the patterns of spv could not be determined in most patients , but four ( patients 4 , 5 , 10 , and 15 , table 1 ) of the six patients showed a linear spv , while two ( patients 11 and 14 ) had a decreasing spv . eight patients , including one with incomplete description of the seizure semiology , had more than one type of seizure in association with the epileptic nystagmus . however , the seizures were mostly focal ( 25/29 , 86.2% ) with ( n = 17 ) or without ( n = 8) loss of consciousness ( loc ) . the ictal discharges originated from the occipital ( n = 17 ) , parietal ( n = 9 ) , temporo - occipital ( n = 5 ) , frontal ( n = 4 ) , and temporal ( n = 3 ) areas , and two patients had multiple epileptic foci ( patients 19 and 20 , table 1 ) . generalized ictal discharges were found only in 2 patients ( patients 17 and 30 , table 1 ) . underlying etiologies included cerebral infarction in 7 patients , cerebral hemorrhage in 3 , infection in 2 , cortical dysplasia in 2 , and head trauma , cerebral metastasis , uremic encephalopathy , hypoglycemic encephalopathy , melas ( mitochondrial myopathy , encephalopathy , lactic acidosis , and stroke ) , sturge weber syndrome , arachnoid cyst , physostigmine intoxication , and birth asphyxia in one patient each . except for one patient ( patient 13 ) without description of the frequency of ictal discharges , almost half ( 16/36 , 44.4% ) of the previously reported patients showed ictal discharges less than 10 hz . even in patients who were alert , epileptic nystagmus occurred with ictal discharges of less than 10 hz ( patients 1 , 8 , 11 , 15 , 16 , 29 , and 34 , table 1 ) . the presence of preceding gaze deviation and midline crossing of the nystagmus did not correlate with the ictal onset zone ( p = 0.509 and p = 0.385 , respectively , linear - to - linear correlation ) . the presence of midline crossing of the nystagmus did not show a correlation with the seizure type either ( p 1.000 , linear - to - linear correlation ) . however , after excluding patients who had more than one type of seizure , all patients with focal seizure and loc showed epileptic nystagmus with preceding gaze deviation , either contraversive ( n = 12 ) or ipsiversive ( n = 2 ) . by contrast , the focal seizures without loc ( n = 3 ) did not have accompanying preceded by a gaze deviation . the gaze deviation was mostly observed in seizures which accompanied loc ( p = 0.027 , linear - to - linear correlation ) . of the 6 patients who had both eeg and oculography , two ( patients 11 and 14 , table 1 ) showed the characteristics of nystagmus from involvement of the saccadic areas , while the other two ( patients 4 and 15 , table 1 ) had the characteristic nystagmus associated with smooth pursuit areas . by contrast , two patients ( patients 5 and 10 , table 1 ) showed epileptic nystagmus with a linear slow phase but without crossing the midline , which can not be classified into one type based on the previous distinction . all 6 patients had the ictal foci in the posterior part ( temporo - occipital , parietal , or occipital areas ) of the cerebral hemisphere and the frequency of ictal discharges on eeg ranged from 2 to 16 hz . she had suffered from hypertension and diabetes mellitus for seven years and alzheimer 's disease for a few years . she had also been admitted to the hospital 4 months ago because of myocardial infarction and cerebral infarction involving the territory of the right anterior cerebral artery . because of global aphasia and dementia , smooth pursuits and saccades could not be fully evaluated , but the reflexive saccades appeared to be intact . however , she intermittently ( 10 per day ) lapsed into a drowsy and lethargic state with both eyes closed and did not respond to noxious stimuli . this altered mental status lasted no more than 1 min , and she recovered to her previous alert state . during the attacks , she showed rightward deviation of both eyes followed by vigorous right - beating nystagmus without any convulsive movements or abnormal postures . eeg revealed 19- to 22-hz polyspikes that were maximal over the left temporo - occipital region , and eye movement recording showed right - beating nystagmus at 34 hz , which did not cross the midline ( video ) . magnetic resonance imaging showed an edematous lesion without gadolinium enhancement on the left temporo - parieto - occipital junction , which was considered a seizure - induced change ( fig . single photon emission computed tomography during the ictus showed hyperperfusion in the left temporo - parieto - occipital junction ( fig . 2b ) . with a suspicion of seizure from a small infarction or alzheimer 's disease itself , fosphenytoin ( 450 mg per day ) and levetiracetam ( 2000 mg per day ) were initiated . her neurological status gradually improved , and follow - up video - eeg one week later showed no epileptiform discharges but regional slowing over the left temporal area . she was discharged two weeks after the admission still with aphasia but without any seizures . through the literature review , we were able to additionally reviewed the reported clinical features of 36 patients ( 21 women , age range : 075 years , median age : 32 years , mean age sd = 35.7 24.1 , table 1 ) . except for four patients [ one ( patient 17 ) with pendular nystagmus , another ( patient 35 ) with upbeat nystagmus , another ( patient 30 ) with generalized ictal discharges on eeg , and the remaining one ( patient 36 ) with alternating nystagmus ] , all the other 33 ( 33/37 , 89.2% ) patients showed nystagmus beating away from the side of ictal discharges on eeg ( table 1 ) . the preceding gaze deviation was described in 25 patients , and 19 of them showed contraversive deviation , while only two had ipsiversive deviation . since oculography was performed in only six patients , the patterns of spv could not be determined in most patients , but four ( patients 4 , 5 , 10 , and 15 , table 1 ) of the six patients showed a linear spv , while two ( patients 11 and 14 ) had a decreasing spv . eight patients , including one with incomplete description of the seizure semiology , had more than one type of seizure in association with the epileptic nystagmus . however , the seizures were mostly focal ( 25/29 , 86.2% ) with ( n = 17 ) or without ( n = 8) loss of consciousness ( loc ) . the ictal discharges originated from the occipital ( n = 17 ) , parietal ( n = 9 ) , temporo - occipital ( n = 5 ) , frontal ( n = 4 ) , and temporal ( n = 3 ) areas , and two patients had multiple epileptic foci ( patients 19 and 20 , table 1 ) . generalized ictal discharges were found only in 2 patients ( patients 17 and 30 , table 1 ) . underlying etiologies included cerebral infarction in 7 patients , cerebral hemorrhage in 3 , infection in 2 , cortical dysplasia in 2 , and head trauma , cerebral metastasis , uremic encephalopathy , hypoglycemic encephalopathy , melas ( mitochondrial myopathy , encephalopathy , lactic acidosis , and stroke ) , sturge weber syndrome , arachnoid cyst , physostigmine intoxication , and birth asphyxia in one patient each . except for one patient ( patient 13 ) without description of the frequency of ictal discharges , almost half ( 16/36 , 44.4% ) of the previously reported patients showed ictal discharges less than 10 hz . even in patients who were alert , epileptic nystagmus occurred with ictal discharges of less than 10 hz ( patients 1 , 8 , 11 , 15 , 16 , 29 , and 34 , table 1 ) . the presence of preceding gaze deviation and midline crossing of the nystagmus did not correlate with the ictal onset zone ( p = 0.509 and p = 0.385 , respectively , linear - to - linear correlation ) . the presence of midline crossing of the nystagmus did not show a correlation with the seizure type either ( p 1.000 , linear - to - linear correlation ) . however , after excluding patients who had more than one type of seizure , all patients with focal seizure and loc showed epileptic nystagmus with preceding gaze deviation , either contraversive ( n = 12 ) or ipsiversive ( n = 2 ) . by contrast , the focal seizures without loc ( n = 3 ) did not have accompanying preceded by a gaze deviation . the gaze deviation was mostly observed in seizures which accompanied loc ( p = 0.027 , linear - to - linear correlation ) . of the 6 patients who had both eeg and oculography , two ( patients 11 and 14 , table 1 ) showed the characteristics of nystagmus from involvement of the saccadic areas , while the other two ( patients 4 and 15 , table 1 ) had the characteristic nystagmus associated with smooth pursuit areas . by contrast , two patients ( patients 5 and 10 , table 1 ) showed epileptic nystagmus with a linear slow phase but without crossing the midline , which can not be classified into one type based on the previous distinction . all 6 patients had the ictal foci in the posterior part ( temporo - occipital , parietal , or occipital areas ) of the cerebral hemisphere and the frequency of ictal discharges on eeg ranged from 2 to 16 hz . the findings in our patient and systematic review may be summarized as follows : 1 ) in almost all of the patients , the nystagmus beat away from the side of ictal discharges ; 2 ) seizures were mostly symptomatic ; 3 ) seizures mostly originated from the posterior part of the brain ; and 4 ) the characteristics of epileptic nystagmus ( presence of preceding gaze deviation and midline crossing ) and patterns of spv were poorly documented and did not correlate with the ictal onset zone . irritative lesions in the cerebral cortex are known to cause deviation of both eyes to the contralesional side , which was demonstrated by electrical stimulation . introduction of eeg and oculography allowed an accurate analysis of eye movements and localization of the corresponding cortical areas responsible for generating such eye movements during the epileptic events . epileptic nystagmus has been ascribed to disruption of cortical controls over saccades or smooth pursuit , . the cortical regions involved in the generation of saccades include the frontal , parietal , and supplementary eye fields . it has been proposed that epileptic nystagmus from the cortical saccadic areas result in contralesional ocular deviation , has decreasing spv , and does not cross the midline toward the ipsilesional hemifield . seizures from the middle temporal ( mt ) and medial superior temporal ( mst ) areas , which are involved in the generation of smooth pursuit are also known to generate epileptic nystagmus . when the regions responsible for smooth pursuit are stimulated , the eyes drift ipsiversively to the eccentric position , and reflexive resetting quick phases beat to the contralateral side crossing the midline . accordingly , contrary to those commencing from the saccadic areas , epileptic nystagmus from the areas involved in the generation of smooth pursuit is known to cause ipsilesional ocular deviation , has linear spv , and crosses the midline toward the contralesional hemifield . even though the pattern of spv and integrity of saccades or smooth pursuits could not be fully evaluated in our patient due to aphasia and underlying alzheimer 's disease , the epileptic nystagmus occurred only in the contralateral right hemifield without crossing the midline , which corresponds to the characteristics of epileptic nystagmus from the saccadic areas . however , our patient also showed interictal spontaneous nystagmus , probably pursuit - paretic , beating to the lesion side ( video ) , and the ictal onset zone on the spect and eeg corresponded to the lateral occipital cortex ( brodmann area 19 ) and the adjacent ventrocaudal aspect of brodmann area 39 , which are known as the human homologues of mt and mst areas . as in our patient , previous reports on epileptic nystagmus frequently encountered the features from both the saccadic and smooth pursuit areas . this may be explained by propagation of the ictal discharges from one to the adjacent cortices . alteration in the level of consciousness during the seizures also indicates propagation of the ictal discharges . as various cortical regions responsible for ocular motility it seems less likely that the epileptic discharges would stimulate either the saccadic or the smooth pursuit regions only . especially in focal seizures with loc or generalized seizures , therefore , the dichotomization of epileptic nystagmus based on the disruption of cortical control over smooth pursuit or saccades may not be applicable to most patients with epileptic nystagmus in clinical practice . although we found a correlation between the characteristics of epileptic nystagmus , the presence of accompanying gaze deviation , and seizure type , it should be interpreted with caution . contraversive gaze deviation is known to be followed by epileptic nystagmus from the saccadic areas . however , other characteristics such as midline crossing and pattern of spv did not correlate with the seizure type . moreover , patients who had more than one type of seizure ( n = 8) exhibited the same semiology of preceding contraversive gaze deviation during the focal seizure without loc ( patients 8 , 11 , 24 , and 25 ) . epileptic nystagmus appears infrequent in that it was reported in only 10% of 42 patients with occipital lobe epilepsy . in an epilepsy monitoring unit , epileptic nystagmus was observed in only 0.5% of 1838 patients who had been monitored for 46 months . the infrequency of epileptic nystagmus has been ascribed to the rarity of the conditions that meet the following : 1 ) involvement of the regions responsible for epileptic nystagmus and 2 ) high - frequency epileptic discharges sufficient to generate epileptic nystagmus ( more than 10 hz ) . however , the ictal frequency required to generate nystagmus was suggested to be lower in patients with decreased mental status or in those with severe neurological impairment . yet , systematic analysis of previous literature showed that epileptic nystagmus can occur with an ictal frequency less than 10 hz , regardless of the neurological status . epileptic nystagmus was observed even seizures absence seizure with 3-hz spikes ( patient 17 , table 1 ) . this implies that factors other than the ictal discharge frequency contribute to the rarity of epileptic nystagmus . the incidence of epileptic nystagmus may have been underestimated since it may escape recognition when abnormal postures or movements are not observed . since most epileptic nystagmus occurs in focal seizures or ncse , the ancillary movements may be as subtle as eye blinking or head rotation . furthermore , propagation of ictal discharges to the frontal areas may suppress the generation of epileptic nystagmus . as stated previously , most epileptic nystagmus has been observed with lesions involving the posterior part of the cerebral hemisphere . the dorsolateral prefrontal cortex is known to inhibit the superior colliculus and may act as a saccadic suppressor , . accordingly , involvement of the frontal lobe by ictal discharges may have suppressed the generation of epileptic nystagmus . detailed characteristics of epileptic nystagmus have rarely been sought in previous reports . even though the localizing value of epileptic nystagmus seems limited in previous reports , the fast phase of epileptic nystagmus was almost always directed away from an epileptic focus that mostly arose from the posterior part of the cerebral hemisphere . the following are the supplementary data related to this articlevideocontinuous video - eeg monitoring of the patient shows subtle spontaneous left - beating nystagmus during the interictal phase . during the ictus , eyelid blinking starts in the left eye and rightward drift of the eyes and right - beating nystagmus follow . electroencephalography shows a build - up of high - frequency polyspike discharges at 1922 hz that arise from the left temporo - occipital region and propagate into the adjacent parietal and parasagittal channels ( channels marked in red ) with a sparing of the channels overlying the frontal areas . continuous video - eeg monitoring of the patient shows subtle spontaneous left - beating nystagmus during the interictal phase . during the ictus , eyelid blinking starts in the left eye and rightward drift of the eyes and right - beating nystagmus follow . electroencephalography shows a build - up of high - frequency polyspike discharges at 1922 hz that arise from the left temporo - occipital region and propagate into the adjacent parietal and parasagittal channels ( channels marked in red ) with a sparing of the channels overlying the frontal areas . kim conducted the design and conceptualization of the study , interpretation of the data , and drafting and revising the manuscript . the patient gave permission for still and moving images of his eyes to be shown in this publication .

purposewe aimed to define the characteristics of epileptic nystagmus and correlate those with other clinical findings in a large number of patients.methodswe report a patient with epileptic nystagmus and additionally reviewed the reported clinical features of 36 more patients through a systematic literature search . we analyzed the characteristics of epileptic nystagmus and attempted correlations of those with alertness of the patients and epileptic foci on eeg.resultsall 33 patients with unilateral horizontal nystagmus showed nystagmus beating away from the side of ictal discharges . epileptic nystagmus was preceded by gaze deviation in 21 patients , with contraversive in 19 and ipsiversive in 2 . seizures associated with epileptic nystagmus were mostly focal ( 25/29 , 86.2% ) with or without loss of awareness . ictal discharges originated from the occipital ( n = 16 ) , parietal ( n = 9 ) , temporo - occipital ( n = 6 ) , frontal ( n = 4 ) , and temporal ( n = 3 ) areas , and two patients had multiple epileptic foci . seizures were usually symptomatic ( 24/37 , 64.9% ) . the presence of preceding gaze deviation and midline crossing of the nystagmus did not correlate with the ictal onset zone or alertness of the patients . recording of epileptic nystagmus was available only in 6 patients , and the epileptic nystagmus could be localized to the saccadic areas in two and to the smooth pursuit areas in another two . two patients showed the features of epileptic nystagmus from both areas.conclusioneven though the localizing value of epileptic nystagmus seems limited in previous reports , the fast phase of epileptic nystagmus was almost always directed away from the epileptic focus that mostly arose from the posterior part of the cerebral hemisphere .

Introduction Methods Laboratory work-ups in our patient Literature review Results Case report Literature review Discussion Conclusion Author contributions Disclosure Conflict of interest

it has been postulated that the epileptic nystagmus stemming from the saccadic areas occurs only in the contraversive hemifield without crossing the midline and has a decreasing slow - phase velocity ( spv ) . in contrast , the epileptic nystagmus from the smooth pursuit regions has been proposed to have a linear spv and corrective quick phases crossing the midline , . even though this distinction seems reasonable in view of the anatomical correlates , , an individual patient may show co - activation of both areas or propagation of the epileptic discharges from one to the other , making the distinction difficult . we also recently encountered a patient who showed epileptic nystagmus with features originating from both the saccade and smooth pursuit areas . even though there have been numerous reports on epileptic nystagmus since the first report in a patient with focal seizure , a systematic review on the features of epileptic nystagmus and the correlation of those with the areas of ictal discharges on eeg has not been available . to address this issue , we describe a patient with epileptic nystagmus and analyzed the characteristics of epileptic nystagmus in the previously reported cases with a systematic review . the keywords used for searching included epileptic nystagmus , ictal nystagmus , and non - convulsive status epilepticus ( ncse ) . we included all the patients described in systematic reviews , clinical studies , and case reports published in english ( fig . the diagnosis of epileptic nystagmus was based on the following : 1 ) clearly documented seizure activity on the ictal eeg , 2 ) occurrence of nystagmus in accordance with the epileptic discharges , and 3 ) documentation of the nystagmus using oculography , video clip , or periorbital electrodes . from these reports , we analyzed the type of seizure , ictal onset zone and maximal ictal frequency on eeg . we also determined the direction and midline crossing of the nystagmus and the presence of preceding gaze deviation and its direction . we also analyzed findings of oculography even though it was available only in a small number of patients . based on these findings , we attempted any correlation among the epileptic foci and other features of epileptic nystagmus using linear - to - linear correlation . the keywords used for searching included epileptic nystagmus , ictal nystagmus , and non - convulsive status epilepticus ( ncse ) . the diagnosis of epileptic nystagmus was based on the following : 1 ) clearly documented seizure activity on the ictal eeg , 2 ) occurrence of nystagmus in accordance with the epileptic discharges , and 3 ) documentation of the nystagmus using oculography , video clip , or periorbital electrodes . from these reports , we analyzed the type of seizure , ictal onset zone and maximal ictal frequency on eeg . we also determined the direction and midline crossing of the nystagmus and the presence of preceding gaze deviation and its direction . we also analyzed findings of oculography even though it was available only in a small number of patients . based on these findings , we attempted any correlation among the epileptic foci and other features of epileptic nystagmus using linear - to - linear correlation . eeg revealed 19- to 22-hz polyspikes that were maximal over the left temporo - occipital region , and eye movement recording showed right - beating nystagmus at 34 hz , which did not cross the midline ( video ) . magnetic resonance imaging showed an edematous lesion without gadolinium enhancement on the left temporo - parieto - occipital junction , which was considered a seizure - induced change ( fig . single photon emission computed tomography during the ictus showed hyperperfusion in the left temporo - parieto - occipital junction ( fig . through the literature review , we were able to additionally reviewed the reported clinical features of 36 patients ( 21 women , age range : 075 years , median age : 32 years , mean age sd = 35.7 24.1 , table 1 ) . except for four patients [ one ( patient 17 ) with pendular nystagmus , another ( patient 35 ) with upbeat nystagmus , another ( patient 30 ) with generalized ictal discharges on eeg , and the remaining one ( patient 36 ) with alternating nystagmus ] , all the other 33 ( 33/37 , 89.2% ) patients showed nystagmus beating away from the side of ictal discharges on eeg ( table 1 ) . the preceding gaze deviation was described in 25 patients , and 19 of them showed contraversive deviation , while only two had ipsiversive deviation . since oculography was performed in only six patients , the patterns of spv could not be determined in most patients , but four ( patients 4 , 5 , 10 , and 15 , table 1 ) of the six patients showed a linear spv , while two ( patients 11 and 14 ) had a decreasing spv . eight patients , including one with incomplete description of the seizure semiology , had more than one type of seizure in association with the epileptic nystagmus . however , the seizures were mostly focal ( 25/29 , 86.2% ) with ( n = 17 ) or without ( n = 8) loss of consciousness ( loc ) . the ictal discharges originated from the occipital ( n = 17 ) , parietal ( n = 9 ) , temporo - occipital ( n = 5 ) , frontal ( n = 4 ) , and temporal ( n = 3 ) areas , and two patients had multiple epileptic foci ( patients 19 and 20 , table 1 ) . generalized ictal discharges were found only in 2 patients ( patients 17 and 30 , table 1 ) . underlying etiologies included cerebral infarction in 7 patients , cerebral hemorrhage in 3 , infection in 2 , cortical dysplasia in 2 , and head trauma , cerebral metastasis , uremic encephalopathy , hypoglycemic encephalopathy , melas ( mitochondrial myopathy , encephalopathy , lactic acidosis , and stroke ) , sturge weber syndrome , arachnoid cyst , physostigmine intoxication , and birth asphyxia in one patient each . except for one patient ( patient 13 ) without description of the frequency of ictal discharges , almost half ( 16/36 , 44.4% ) of the previously reported patients showed ictal discharges less than 10 hz . even in patients who were alert , epileptic nystagmus occurred with ictal discharges of less than 10 hz ( patients 1 , 8 , 11 , 15 , 16 , 29 , and 34 , table 1 ) . the presence of preceding gaze deviation and midline crossing of the nystagmus did not correlate with the ictal onset zone ( p = 0.509 and p = 0.385 , respectively , linear - to - linear correlation ) . the presence of midline crossing of the nystagmus did not show a correlation with the seizure type either ( p 1.000 , linear - to - linear correlation ) . however , after excluding patients who had more than one type of seizure , all patients with focal seizure and loc showed epileptic nystagmus with preceding gaze deviation , either contraversive ( n = 12 ) or ipsiversive ( n = 2 ) . by contrast , the focal seizures without loc ( n = 3 ) did not have accompanying preceded by a gaze deviation . of the 6 patients who had both eeg and oculography , two ( patients 11 and 14 , table 1 ) showed the characteristics of nystagmus from involvement of the saccadic areas , while the other two ( patients 4 and 15 , table 1 ) had the characteristic nystagmus associated with smooth pursuit areas . all 6 patients had the ictal foci in the posterior part ( temporo - occipital , parietal , or occipital areas ) of the cerebral hemisphere and the frequency of ictal discharges on eeg ranged from 2 to 16 hz . she had also been admitted to the hospital 4 months ago because of myocardial infarction and cerebral infarction involving the territory of the right anterior cerebral artery . eeg revealed 19- to 22-hz polyspikes that were maximal over the left temporo - occipital region , and eye movement recording showed right - beating nystagmus at 34 hz , which did not cross the midline ( video ) . through the literature review , we were able to additionally reviewed the reported clinical features of 36 patients ( 21 women , age range : 075 years , median age : 32 years , mean age sd = 35.7 24.1 , table 1 ) . except for four patients [ one ( patient 17 ) with pendular nystagmus , another ( patient 35 ) with upbeat nystagmus , another ( patient 30 ) with generalized ictal discharges on eeg , and the remaining one ( patient 36 ) with alternating nystagmus ] , all the other 33 ( 33/37 , 89.2% ) patients showed nystagmus beating away from the side of ictal discharges on eeg ( table 1 ) . the preceding gaze deviation was described in 25 patients , and 19 of them showed contraversive deviation , while only two had ipsiversive deviation . since oculography was performed in only six patients , the patterns of spv could not be determined in most patients , but four ( patients 4 , 5 , 10 , and 15 , table 1 ) of the six patients showed a linear spv , while two ( patients 11 and 14 ) had a decreasing spv . eight patients , including one with incomplete description of the seizure semiology , had more than one type of seizure in association with the epileptic nystagmus . however , the seizures were mostly focal ( 25/29 , 86.2% ) with ( n = 17 ) or without ( n = 8) loss of consciousness ( loc ) . the ictal discharges originated from the occipital ( n = 17 ) , parietal ( n = 9 ) , temporo - occipital ( n = 5 ) , frontal ( n = 4 ) , and temporal ( n = 3 ) areas , and two patients had multiple epileptic foci ( patients 19 and 20 , table 1 ) . generalized ictal discharges were found only in 2 patients ( patients 17 and 30 , table 1 ) . underlying etiologies included cerebral infarction in 7 patients , cerebral hemorrhage in 3 , infection in 2 , cortical dysplasia in 2 , and head trauma , cerebral metastasis , uremic encephalopathy , hypoglycemic encephalopathy , melas ( mitochondrial myopathy , encephalopathy , lactic acidosis , and stroke ) , sturge weber syndrome , arachnoid cyst , physostigmine intoxication , and birth asphyxia in one patient each . except for one patient ( patient 13 ) without description of the frequency of ictal discharges , almost half ( 16/36 , 44.4% ) of the previously reported patients showed ictal discharges less than 10 hz . even in patients who were alert , epileptic nystagmus occurred with ictal discharges of less than 10 hz ( patients 1 , 8 , 11 , 15 , 16 , 29 , and 34 , table 1 ) . the presence of preceding gaze deviation and midline crossing of the nystagmus did not correlate with the ictal onset zone ( p = 0.509 and p = 0.385 , respectively , linear - to - linear correlation ) . the presence of midline crossing of the nystagmus did not show a correlation with the seizure type either ( p 1.000 , linear - to - linear correlation ) . however , after excluding patients who had more than one type of seizure , all patients with focal seizure and loc showed epileptic nystagmus with preceding gaze deviation , either contraversive ( n = 12 ) or ipsiversive ( n = 2 ) . by contrast , the focal seizures without loc ( n = 3 ) did not have accompanying preceded by a gaze deviation . of the 6 patients who had both eeg and oculography , two ( patients 11 and 14 , table 1 ) showed the characteristics of nystagmus from involvement of the saccadic areas , while the other two ( patients 4 and 15 , table 1 ) had the characteristic nystagmus associated with smooth pursuit areas . all 6 patients had the ictal foci in the posterior part ( temporo - occipital , parietal , or occipital areas ) of the cerebral hemisphere and the frequency of ictal discharges on eeg ranged from 2 to 16 hz . the findings in our patient and systematic review may be summarized as follows : 1 ) in almost all of the patients , the nystagmus beat away from the side of ictal discharges ; 2 ) seizures were mostly symptomatic ; 3 ) seizures mostly originated from the posterior part of the brain ; and 4 ) the characteristics of epileptic nystagmus ( presence of preceding gaze deviation and midline crossing ) and patterns of spv were poorly documented and did not correlate with the ictal onset zone . irritative lesions in the cerebral cortex are known to cause deviation of both eyes to the contralesional side , which was demonstrated by electrical stimulation . it has been proposed that epileptic nystagmus from the cortical saccadic areas result in contralesional ocular deviation , has decreasing spv , and does not cross the midline toward the ipsilesional hemifield . seizures from the middle temporal ( mt ) and medial superior temporal ( mst ) areas , which are involved in the generation of smooth pursuit are also known to generate epileptic nystagmus . when the regions responsible for smooth pursuit are stimulated , the eyes drift ipsiversively to the eccentric position , and reflexive resetting quick phases beat to the contralateral side crossing the midline . accordingly , contrary to those commencing from the saccadic areas , epileptic nystagmus from the areas involved in the generation of smooth pursuit is known to cause ipsilesional ocular deviation , has linear spv , and crosses the midline toward the contralesional hemifield . even though the pattern of spv and integrity of saccades or smooth pursuits could not be fully evaluated in our patient due to aphasia and underlying alzheimer 's disease , the epileptic nystagmus occurred only in the contralateral right hemifield without crossing the midline , which corresponds to the characteristics of epileptic nystagmus from the saccadic areas . however , our patient also showed interictal spontaneous nystagmus , probably pursuit - paretic , beating to the lesion side ( video ) , and the ictal onset zone on the spect and eeg corresponded to the lateral occipital cortex ( brodmann area 19 ) and the adjacent ventrocaudal aspect of brodmann area 39 , which are known as the human homologues of mt and mst areas . as in our patient , previous reports on epileptic nystagmus frequently encountered the features from both the saccadic and smooth pursuit areas . this may be explained by propagation of the ictal discharges from one to the adjacent cortices . alteration in the level of consciousness during the seizures also indicates propagation of the ictal discharges . as various cortical regions responsible for ocular motility it seems less likely that the epileptic discharges would stimulate either the saccadic or the smooth pursuit regions only . especially in focal seizures with loc or generalized seizures , therefore , the dichotomization of epileptic nystagmus based on the disruption of cortical control over smooth pursuit or saccades may not be applicable to most patients with epileptic nystagmus in clinical practice . although we found a correlation between the characteristics of epileptic nystagmus , the presence of accompanying gaze deviation , and seizure type , it should be interpreted with caution . contraversive gaze deviation is known to be followed by epileptic nystagmus from the saccadic areas . however , other characteristics such as midline crossing and pattern of spv did not correlate with the seizure type . moreover , patients who had more than one type of seizure ( n = 8) exhibited the same semiology of preceding contraversive gaze deviation during the focal seizure without loc ( patients 8 , 11 , 24 , and 25 ) . the infrequency of epileptic nystagmus has been ascribed to the rarity of the conditions that meet the following : 1 ) involvement of the regions responsible for epileptic nystagmus and 2 ) high - frequency epileptic discharges sufficient to generate epileptic nystagmus ( more than 10 hz ) . however , the ictal frequency required to generate nystagmus was suggested to be lower in patients with decreased mental status or in those with severe neurological impairment . this implies that factors other than the ictal discharge frequency contribute to the rarity of epileptic nystagmus . furthermore , propagation of ictal discharges to the frontal areas may suppress the generation of epileptic nystagmus . as stated previously , most epileptic nystagmus has been observed with lesions involving the posterior part of the cerebral hemisphere . accordingly , involvement of the frontal lobe by ictal discharges may have suppressed the generation of epileptic nystagmus . detailed characteristics of epileptic nystagmus have rarely been sought in previous reports . even though the localizing value of epileptic nystagmus seems limited in previous reports , the fast phase of epileptic nystagmus was almost always directed away from an epileptic focus that mostly arose from the posterior part of the cerebral hemisphere . electroencephalography shows a build - up of high - frequency polyspike discharges at 1922 hz that arise from the left temporo - occipital region and propagate into the adjacent parietal and parasagittal channels ( channels marked in red ) with a sparing of the channels overlying the frontal areas . electroencephalography shows a build - up of high - frequency polyspike discharges at 1922 hz that arise from the left temporo - occipital region and propagate into the adjacent parietal and parasagittal channels ( channels marked in red ) with a sparing of the channels overlying the frontal areas . kim conducted the design and conceptualization of the study , interpretation of the data , and drafting and revising the manuscript .

language can be counted as the first cognitive enhancement , and the invention of writing also dramatically improved our cognitive ability to remember , to learn , and to communicate. today , with the aid of recent phenomenal development of biomedical technology , we are witnessing the emergence of more direct biological cognitive enhancementschemical , physical , or electromagnetic intrusions into our physical brain. brain enhancement has been considered as one of the most important intersections of law and neuroscience . the idea of directly enhancing the brain with drugs , brain stimulation , or neurosurgery might seem frightening . but more than frightening , the use of these enhancement technologies raises crucial neuroethical and legal issues . for example , fairness can be a serious problem , if an effective enhancement technology is very costly and thus , available only to the rich . also , individuals could be coerced , explicitly or implicitly , to undergo cognitive enhancements for various reasons , such as criminal rehabilitation , medical treatment , or even academic success at school . despite these potential risks , professor hank greely has argued that safe and effective cognitive enhancement will benefit both individuals and society , and we should require better research on , and better regulation of , cognitive enhancement instead of banning the use of all cognitive enhancements : biomedicine will be creating more and more products and procedures that can be used for cognitive enhancement . some of them will be used in ways that will , on balance , improve human life and society . at the same time i am confident , though , that a knee - jerk rejection of all direct brain enhancement will be at least a missed opportunity and at worst an opening for a damaging underground and uncontrolled world of enhancement . in order to maximize the benefits and minimize the harms of these new technologies , we will need to look at particular enhancements rationally and to adopt , ban or regulate them carefully . one of the most eye - catching and readily accessible direct brain enhancement technologies is transcranial direct current stimulation ( tdcs ) . tdcs is a type of non - invasive neuromodulation , which delivers weak direct current ( dc ) ( 12 ma ) to the brain using small saline - soaked electrodes . this weak current is insufficient to cause neurons to fire , but it can alter the excitability of neurons by shifting their membrane potentials in a de- or hyper polarizing direction ; thus making them more or less likely to fire. anodal dc stimulation is known to increase neuronal excitability , while cathodal stimulation decreases it . application of electric currents to the human brain for therapeutic purposes is not a new idea . in ancient times , people placed an electric fish over the scalp to treat headache or epilepsy . later in 18th century , advances in the science of electrophysiology by galvani and volta inspired the use of direct currents for the treatment of mental disorders . nonetheless , erratic results and the advent of electroconvulsive therapy led to the decrease of interest in dc brain stimulation . however , reappraisal in the last decade has shed new light on the effects of dc stimulation on the human brain . a number of neurophysiological studies have demonstrated the efficacy of tdcs for the treatment of patients suffering from neuropsychiatric diseases , such as stroke , chronic pain , and depression . moreover , recent studies have reported that the use of tdcs on specific brain regions can improve cognitive functions , such as attention span , working and long - term memory , language , and mathematical ability of healthy subjects . for example , de vries and colleagues showed that anodal tdcs over broca 's area a region in the frontal lobe of the hemisphere of the brain improves artificial grammar learning . numerical learning was enhanced by anodal tdcs of the parietal cortex , and this effect was stable for at least 6 months after training. also , subjects who received anodal tdcs on the right anterior temporal lobe while naming pictures of famous people showed improved retrieval of proper names . in one study funded by the us defense advanced research projects agency , subjects who received anodal tdcs on right inferior frontal and right parietal cortex showed learning and performance improvement in a video game designed to train military personnel to accurately identify obscured and concealed objects in a complex environment . these interesting experiment results began to draw the media 's attention a new york times article in october 2013 described tdcs devices as jump starter kits for the mind. also , the number of tdcs articles published per year has been growing rapidly , and currently at least 224 clinical studies on tdcs are going on around the world . on top of the fact that it can be an effective tool for both treatment and cognitive enhancement , tdcs has two other especially appealing features most of the known side effects of tdcs are minor adverse effects , such as headache , or slight tingling or itching under the electrodes . at least nine tdcs devices for personal use can be purchased through websites as a complete unit with prices between approximately \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}200 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}400 . but for less than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}40 for parts and thanks to these special features of tdcs , in recent years , a group of people who built or purchased the tdcs device for home use has appeared . researchers have expressed concerns about this so - called do - it - yourself ( diy ) use of tdcs . they have argued that even if there are no serious side effects , potential risks of misusing this device should not be ignored . for example , lay people who are diy users may not have sufficient knowledge on the structure of brain to place the electrodes accurately , causing unintended effects . moreover , reversing anodal and cathodal electrodes placement could lead to impairment of the brain by switching areas affected by excitatory and inhibitory stimulation . it is also possible that tdcs interacts with other treatment that diy users are undergoing , such as psychoactive medication , in detrimental ways . lastly , using tdcs to enhance some of the cognitive functions may adversely affect other functions of the brain , and potential long - term ( side ) effects of tdcs on the brain have not been fully explored . the fda 's own description of its regulatory regime for medical devices reads : if a product is labeled , promoted or used in a manner that meets the following definition in section 201(h ) of the federal food , drug , and cosmetic ( fd&c ) act , it will be regulated by the food and drug administration ( fda ) as a medical device and is subject to premarketing and postmarketing regulatory controls . an instrument , apparatus , implement , machine , contrivance , implant , in vitro reagent , or other similar or related article , including a component part , or accessory which is : ( 1 ) recognized in the official national formulary , or the united states pharmacopoeia , or any supplement to them , ( 2 ) intended for use in the diagnosis of disease or other conditions , or in the cure , mitigation , treatment , or prevention of disease , in man or other animals , or ( 3 ) intended to affect the structure or any function of the body of man or other animals , and which does not achieve its primary intended purposes through chemical action within or on the body of man or other animals and which is not dependent upon being metabolized for the achievement of any of its primary intended purposes . fda requires all medical products manufacturers to register their facilities , list their devices with fda , and follow general control requirements. medical devices are classified in three categories low ( class i ) , moderate ( class ii ) , and high - risk ( class iii ) devices according to the risks that the devices present . devices with low risk are considered as exempt from fda 's pre - market review and can be legally marketed upon registration . in terms of moderate- and high - risk devices , there are two administrative paths that manufacturers must take to bring the devices to markets . the first path is pre - market approval ( pma ) , which consists of conducting clinical studies , submitting pre - market approval ( pma ) application and requires evidence providing reasonable assurance that the device is safe and effective. usually novel and high - risk devices are subject to this process , and it is known to be very lengthy and expensive . the other path involves submitting a 510(k ) notification demonstrating that the devices is substantially equivalent to a device already on the market ( a predicate device) and results in fda clearance . this path , which tends to be less expensive and time - consuming than pma , is available for moderate - risk medical devices not exempt from pre - market review . once approved or cleared for marketing , manufacturers must comply with regulation on manufacturing , labeling , surveillance , device tracking , and adverse event reporting. some neurostimulation devices are already classified and regulated as medical devices by fda . for example , in 2011 , repetitive transcranial magnetic stimulation ( rtms ) , another non - invasive neuromodulation that generates electric currents using electromagnetic coils , was classified as a class ii medical device for treatment of major depressive disorder . also , cranial electrotherapy stimulation , whose mechanism is similar to tdcs except that it applies alternating current , not dc , to the brain , has been regulated as a medical device ( class iii ) for treatment of insomnia , depression , or anxiety since 1979 . fda guidance explicitly provides that there is no regulation for the therapeutic tdcs. a few clinics offer tdcs treatment to their patients with various medical conditions , such as depression and chronic pain , but these clinics apply tdcs as off - label use of the iontophoresis device a dc stimulator for introducing ions of soluble salts or other drugs into the body for medical purposes that fda classified as a medical device ( class ii / iii ) in 2004 . in addition , devices marketed for cognitive enhancement are not covered by any existing legislation in the usa . in their recent article , nick fitz and peter reiner stated that this regulatory vacuum is understandable given that the myriad applications of tdcs are fairly new. however , since tdcs devices are already on the market and being sold to the public , they argued that now we should begin the discussion on how to develop a regulatory policy for the diy use of tdcs . for example , foc.us , a fancy tdcs device marketed as a gamer headset to boost attention , completely sold out its first batch of 3000 units less than a month after its release in may 2013 . fitz and reiner urged all stakeholders regulators , scientists and the diy community to share in crafting policy proposals that ensure public safety while supporting diy innovation. they emphasized the importance of communication between policy makers and diy users to develop the ethos of responsible use based on the idea that people should have access to a diversity of opportunities created by enhancement technologies. responding to fitz and reiner 's call for a policy debate , maslen and colleagues proposed a regime that regulates cognitive enhancement devices such as tdcs by extending the existing legislation for medical devices . in line with the managed technological optimism that fitz and reiner advocate , they suggested the incorporation of a low - risk exemption for any cognitive enhancement devices falling below a given level of risk . in june 2013 , tdcs device kit , inc . , a tdcs device manufacturer based in california , voluntarily recalled its products after an inspection by california department of public health ( cdph ) . cdph determined that these products were not manufactured in compliance with good manufacturing practices for medical devices and that the devices lacked adequate labeling for directions for use and warnings against dangerous uses . this was a meaningful step taken by the government authorities , but it only had power in one state as there is no regulation on tdcs under the current fda regime for medical devices . despite the recent surge of attention to the potential risks and regulations of tdcs , there has been no attempt to systemically study what is really happening in the diy tdcs user community . discussions of issues related to the diy use of tdcs are mostly based on speculation , which may lead to importantly incorrect understanding of the diy community and its use of tdcs . this study is the first exploratory research on who diy tdcs users are and what is happening in this community . specifically , this study aims to answer following four questions on the diy use of tdcs through an online survey , a content analysis of web postings , and interviews with the diy users . what are the general demographic characteristics of diy users?why and how do they use this device?what are the perceived effects or side effects of this device?what are their concerns , if any , about the diy use of this device ? what are their concerns , if any , about the diy use of this device ? in particular , its survey was not of random users but of a set of self - selected diy tdcs users who responded to an invitation to participant the author posted on the two major diy tdcs internet web sites . also , only a small number of people interviewed , chosen from among those who , based on the websites , appeared to be influential in the diy tdcs user community . the unregulated , unregistered , and otherwise unreachable nature of the community of tdcs users made these the only feasible approaches , but they do undercut , to some extent , the confidence that can be placed in the findings . nonetheless , even if viewed as only preliminary data , these results provide some basic information on diy tdcs users and their attitudes and practices of diy tdcs which will help us to lay the groundwork for designing sound future policy and official guidelines on tdcs . section 1 describes the three research methodologies recruited in this study , and the findings on the diy tdcs user community are presented in section 2 . section 3 explores potential legal and regulatory implications of these findings considering possible regulatory options for the diy use of tdcs . finally , section 4 summarizes the paper 's major findings and briefly discusses future directions for research . there has been no official report on the size of the diy tdcs user population in the usa , in any other country , or worldwide . users do not need a prescription , do not need to register their use , and do not even need to purchase tdcs units . however , two major diy tdcs internet websites , www.reddit.com/r/tdcs and www.diytdcs.com , where diy users usually visit and share information about tdcs are potential resources for exploring diy tdcs users . the site , www.reddit.com/r/tdcs/ ( hereinafter subreddit tdcs ) , is an open forum for tdcs users and www.diytdcs.com ( hereinafter diytdcs ) is a personal blog by a famous lay expert on diy tdcs . the research subjects of this study are the registered users or visitors of these two websites ( 1 ) who built or purchased their personal tdcs devices and ( 2 ) who have used them for treatment or as a cognitive enhancer . the research methodology of this study has three parts : ( 1 ) an online questionnaire survey , ( 2 ) a content analysis of web postings on the diy use of tdcs , and ( 3 ) in - depth interviews . the survey was composed of a minimum of 21 to a maximum of 29 questions depending on the respondents answers to some questions on basic demographic information of diy users , their aims of practicing diy tdcs , current practices of tdcs ( including types of devices , stimulation protocols , and frequency of use ) , effects and side effects of tdcs , and their concerns about the diy tdcs ( see appendix a for the questionnaire ) . the survey was posted on subreddit tdcs from december 27 , 2013 to march 10 , 2014 and on diytdcs from december 29 , 2013 to march 10 , 2014 . the responses of this online survey do not form a random sample , and there is a potential bias because these respondents are self - selected to participate in this survey . however , since this study is the first preliminary attempt to learn about the diy tdcs user community , responses from this convenience sample should provide previously unknown but useful facts about this community . the second research methodology is a content analysis of web postings on subreddit tdcs . in this well - known and very active online forum , diy users ask and answer questions on the diy use of tdcs device , post interesting recent tdcs studies , and share their experiences of positive and adverse effects of tdcs devices . these web postings are an important source of information to answer some of the research questions . subreddit tdcs was established in april 2011 ; since then , there has been a significant increase in the number of postings ( fig . this study took a census of all 825 postings , and the comments to these postings were selectively included in the analysis in cases where the comments showed meaningful discussions on the issues raised in the postings . number of posting per quarter in www.reddit.com/r/t/dcs ( 20112013 ) . since there is no previously established theory or research study on the diy tdcs user community or subreddit tdcs , the codes were derived from an emergent process . through a preliminary examination of the postings , five major codes to categorize the main theme of each posting were developed : ( 1 ) building and operation of the tdcs device , ( 2 ) marketed devices , ( 3 ) effects / side effects of the device , ( 4 ) references ( external links ) to research papers , newspaper / magazine articles , or blog posts on tdcs , and ( 5 ) others . when there was more than one theme in a posting , this study coded the posting according to its primary purpose . for example , if a user began his or her posting with the side effect he or she has experienced , but then later asked about how to improve his or her diy circuit to prevent this side effect , this posting would be classified under the code , building and operation of the tdcs device. in addition to these major codes , two additional emergent codes the commenters reasons for using the tdcs device and their warnings about safety were prepared to describe recurrent topics that answer some of the research questions of this study but do not constitute the main theme of the postings . for example , diy tdcs users usually mentioned their purpose for using the tdcs device while asking questions about building and operation of the device ( see appendix b for detailed classification of the codes ) . after survey data analysis and content analysis of web postings were completed , five semistructured interviews were conducted to collect more detailed information on the user experience and users opinions on the official guidelines and potential government regulation of the tdcs device . first , this study conducted a brief analysis on the user ids and postings at subreddit tdcs to identify power users who could provide a general overview and informative facts on the diy tdcs user community . the analysis revealed five users who have made more than 200 postings and comments at subreddit tdcs ( appendix c ) . four of these five power users participated in the interviews : the moderator of subreddit tdcs , the webmaster of diytdcs , a user who has uploaded popular self - experimentation videos on his youtube channel , and a user who has been using tdcs for treatment of bipolar type ii and other medical conditions . the interviewees were also asked to give their thoughts on the major findings of the survey and content analysis as an active user or an observer who is familiar with the trend in the community . second , a physician who has been treating about 350 patients with tdcs for the last seven years was also recruited for the interview to provide a medical perspective on the diy use of tdcs . the interview with webmaster of diytdcs was conducted through email , and the moderator of subreddit tdcs and the physician was interviewed over skype . the phone and skype interviews were conducted for about 30 minutes to an hour and audiotaped with the consent of the interviewees . although the number of samples is very small , given the exploratory nature of this study , these interviews should provide a meaningful preliminary sketch of the diy tdcs community and current uses of tdcs along with the findings of the survey and content analysis of the web postings . in this section , the results of the survey , content analysis of web postings , and in - depth interviews will be presented and analysed under the four main research topics of this study : general demographic characteristics of diy users ; their reasons for and current uses of tdcs ; effects and side effects of tdcs ; and their concerns about the diy use of tdcs . there is no official estimate of the size of the diy tdcs user population . as of the end of 2013 , subreddit tdcs had about 2700 subscribers . according to the moderator of the website , there were 4000 individual hits on this forum per month in 2012 . the number has been gradually increasing , and this forum is now getting 6000 individual hits per month . although it is very hard to track whether a particular person is really using tdcs , the moderator estimated that 6000 might be the upper bound of the diy tdcs user population and the lower bound might be the number of subscribers to this website , about 3000 , assuming that an active , regular tdcs user will visit the website at least once a month . one of the power users who has posted self - experiment videos said that 800 people were subscribing his youtube channel , and another famous youtube channel on tdcs has 2700 subscribers . this power user estimated that the size of the diy tdcs user population is probably a few thousand , but not more than 10,000 . however , the webmaster of diytdcs , who is also one of the most active participants in subreddit tdcs since the establishment of that forum , questioned this estimate of the size of the diy tdcs community in the media and academic studies : i 'm not sure how large the community is just now . it seems people pop in for a period and then move on , though there are also committed diy tdcs users who are self - treating daily . my sense is that tdcs is a fringe interest that wo n't attain popularity unless some sort of yet to be seen benefit emerges . only half ( 62 ; 51% ) of the respondents reported that they are continuous and regular tdcs users and among these continuous and regular users , 61% of them have been using tdcs less than six months ( appendix d ) . the majority of the respondents who claimed that they are continuous and regular users began to use tdcs pretty recently . thus , although , based on evidence of internet uses , the diy tdcs user population seems to have been growing for the last few years , these rough indicators might suggest that the dedicated tdcs user population has been overstated . there seems to be a small core of dedicated diy tdcs users that enable a sustained presence at the tdcs subreddit . but apart from that a lot of people come in for a quick look and then wander off. whatever the community 's actual size , the results on the demographic characteristics of the surveyed community are analysed below . first , the survey respondents are overwhelmingly male114 respondents ( 94% ) are male , 5 respondents ( 4% ) are female . second , most of the respondents are in their 20s and 30s ( 86 ; 71% ) , but there are a significant number of respondents in their 50s and over ( 15 ; 12% ) ( fig . the physician who has been treating patients with tdcs reported that there are some elderly patients who want to restore their diminishing cognitive abilities . he argued that tdcs is an appealing brain stimulation technology not only for young self - experimenters or bio hackers but also for seniors suffering from deterioration of mental functioning . also , it is noteworthy that there are some minors using tdcs ( 7 ; 6% ) . most of the respondents ( 90 ; 74% ) are from north america the usa and canada , although the rest of them are widely spread around the world . in addition , the majority of the respondents have four - year undergraduate degree or masters degree ( 62 ; 51% ) , though a significant number of respondents only have a high - school degree or below ( 29 ; 24% ) ( appendix e ) . relatedly , most of the respondents are students ( 25 ; 21% ) or professionals ( 45 ; 37% ) ( appendix f ) . the reported annual income level of the respondents also seems to fall in line with their occupations the respondents either have reported income of less than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}20,000(26 ; 21% ) or reported income of more than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}90,000 ( 26 ; 21% ) ( appendix g ) . however , making generalizations on respondents socio - economic status based on these data can be problematic given that the respondents are from all over the world . this part will delineate and analyse the data on diy users aims of using tdcs device , as well as their current use of tdcs such as types of tdcs devices , stimulation protocols , and frequency of use . according to the survey results , respondents first learned about tdcs through academic research studies on tdcs ( 43 ; 35.5% ) , tdcs websites ( 43 ; 35.5% ) , and newspaper or magazine articles on tdcs ( 30 ; 25% ) ( appendix h ) . this indicates that information on tdcs is mainly disseminated and shared through online media and forums , assuming that many of respondents may not have direct access to academic journals , and recent intriguing studies on the various effects of tdcs have been vigorously covered by the media . 3 presents the number and the percentage of subreddit tdcs postings classified under the five codes . this shows that this website functions as a forum for discussing how to build and use tdcs and sharing new scientific discoveries on the effects of tdcs featured in media and various online forums . postings at subreddit tdcs . a total of 13 ( 11% ) respondents use tdcs for treatment of medical conditions and 71 ( 59% ) respondents use it for cognitive enhancement . a total of 29 ( 24% ) respondents reported that they are using tdcs for both cognitive enhancement and treatment . the most common types of cognitive enhancement purpose for which the respondents use tdcs were attention ( 72 ) , learning ( motor , verbal , or numerical learning , etc . ) ( 68 ) , and working memory ( 67 ) ( fig . 4 ) . types of cognitive enhancement purpose for which respondents use tdcs . in addition , the most common medical condition for which the respondents use tdcs was depression ( fig . interviewees of this study also reported that tdcs is very effective for the treatment of depression , especially for treatment - resistant depression . according to the physician who has been treating patients with tdcs , most of his patients came to the clinic because they do not respond to psychoactive medications . based on his extensive clinical experiences , he claimed that tdcs is significantly beneficial for the treatment - refractory patient suffering from chronic depressionit takes only about five treatments before the effects of tdcs become noticeable. one of the power users who has bipolar type ii disorder also reported that the effect of tdcs is immediate and he can even tell when the machine is shutting off. types of medical condition for which respondents use tdcs . furthermore , this survey also asked , in cases where the respondents are using tdcs for treatment , whether these respondents are also taking other conventional medications . this question is intended to assess the risk of diy tdcs , assuming that there could be unknown adverse compounding effects of tdcs and other extant medications . the pharmacological status of the brain can have meaningful effect on the outcome of tdcs , and the variety of psychoactive agents that home users may employ is legion. in this survey , 18 respondents44% of respondents who are using tdcs for treatment of medical conditions reported that they are currently taking other medications for the same medical conditions . thus , a significant percentage of respondents who are using tdcs for treatment are exposed to the potential detrimental interactions with other medications . the survey result showed that among the 121 respondents , 47 ( 39% ) respondents built their own device and 58 ( 48% ) respondents bought out of the box tdcs devices . some of the respondents ( 11 ; 9% ) reported that they both built and bought their devices . in terms of home - built tdcs devices , the analysis of subreddit tdcs postings revealed that some open source tdcs circuit designs proposed by diy users had been available at this forum and improved through active discussions among users . openstim tdcs , by the moderator of subreddit tdcs , and another designed by a user named shawn nock , are the two most well - known open source tdcs circuits . the moderator of subreddit tdcs initially had wanted to buy one of the devices used at clinics or research labs but the prices of these devices were over his budget . after he found out that building a tdcs device does not require complicated knowledge of electrical engineering , he decided to design his own circuit , which is affordable ( costs less than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}50 ) and , he believes , safe , and to share it with other diy users . later , it has been evolved with the inputs from other users in the community in terms of what kind of features would be useful. in addition to these open source circuits , some users also compiled and posted basic schematics , tools and electrical components needed to build the tdcs device , and online or offline shops that one could get those tools and components , as well as their prices . thus , at subreddit tdcs , instructions on how to build an easy - to - use tdcs device at a very low cost are easily accessible to anyone who is interested in trying self - experimentation with tdcs . regarding the out of the box tdcs devices , currently about 10 devices are on the market . the survey result showed that the most prevalent device in this community is the foc.us headset . 6 ) types of out of the box tdcs device that respondents are using . however , both power users and the physician had unfavorable opinions on the foc.us headset , mostly because its placement of electrodes , which is called montage , is fixed . the physician argued that the montage of foc.us device is different from montages used in any of the clinical studies , and he did not think it has the same effects as the ordinary two electrodes system using iontophoresis devices that researchers have used in finding evidence of effectiveness . the webmaster of diytdcs also stated that unless some sort of tangible evidence of obvious benefit emerges , foc.us will turn out to be a fad and will most likely fade away. the fact that these commercial devices , such as foc.us , are gaining popularity among the diy users has caused a demographic shift in this diy tdcs community . the moderator of subreddit tdcs recalled that two years ago when the website was first established people were talking about designing electronics , designing techniques , and designing experiments. however , there is now a population of people who are basically end users and are not programmers , hackers or electronics people. this is also reflected in the recent sharp increase in the number of postings on these commercial devices at subreddit tdcs ( fig . the moderator argued that this has good and bad sides. it is good in the sense that many of these out of the box products are quite sophisticated. for example , the foc.us device can deliver custom waveforms , which is difficult with diy devices . if commercial devices become more popular , they can spur people 's interest in developing tdcs methods that can take advantage of the more advanced functionality . on the other hand , the moderator said we can not assume that these end users are knowledgeable enough to make informed decisions about their safety in regard to issues like whether a specific type of circuit is safe to use in these devices , and thus , three factors determine the safety of a tdcs protocol : dose of current , size of electrodes , and duration of stimulation . the first two factors determine current density ( current dose divided by electrode size ) , and the total current dosage is then measured by multiplying current density by the duration of stimulation . researchers have suggested that a stimulation protocol using 2535 cm electrodes with currents of 12 milliamperes ( ma ) for up to 2040 minutes is considered safe . the survey results showed that most of the respondents are following this safety guideline , though there are some exceptions . ( figs 810 ) for example , 43 respondents are using electrodes smaller than 10 cm . however , it seems that most of them are foc.us users : the size of electrodes of foc.us headset is about 46 cm , and there are 37 foc.us users among the respondents . when asked how they try to find out about these three factors that determine the safety of a stimulation protocol , most respondents answered that they find relevant information from academic research studies on tdcs or website postings , such as postings in subreddit tdcs ( appendix i ) . how often one can safely use tdcs per day or per week for an extended period of time is a question that does not have a clear answer yet . most of the tdcs studies applied tdcs to the subjects less than five to six times per week for a relatively short term ( ie few weeks ) . thus , these studies do not provide proper guidelines for people who are using tdcs at home more often for a longer period of time . a few postings on the frequency of use at subreddit tdcs led to users replying based on their own experiences and acknowledging the absence of proven guidelines by researchers . a total of 52 out of 56 respondents who are using tdcs regularly reported that they have stimulation sessions fewer than seven times per week ( on average less than once a day ) . this part presents , the data on the respondents self - reports about the effects and side effects of tdcs . in this exploratory study , it is not feasible to measure and test the actual effects of tdcs , such as to what extent tdcs has affected users cognitive abilities or medical conditions . the self - reports on the effects of tdcs posted at subreddit tdcs show that diy users themselves are having problems in assessing the effects of tdcs while using it at home . although the physician who has been treating his patients with tdcs described the effects of tdcs on chronic pain and depression as too good to be true , a power user who used tdcs mainly for cognitive enhancement reported that the effects of tdcs are extremely subtle. for example , there can be placebo effects and in cases where users take cognitive tests after or while simulating their brain ( ie lumosity , dual n - back game , or cambridge brain challenge ) , learning by repetition can also affect the test results . in addition , types of side effects which diy users are experiencing and the degree of these side effects will be analysed . a total of 54 ( 44% ) out of 121 responding users rated the effects of tdcs at four or five on the scale of one ( totally unsuccessful ) to five ( extremely successful ) ( appendix j ) . the quotes below are two examples of self - reports on the effects of tdcs posted at subreddit tdcs . the first quote is describing the effects of tdcs on cognitive abilities : a series of memory tests were administered by an electrical engineer who supervised the experiment , initially without tdcs . these tests demonstrated standard working memory results with a measured span of 58 components retained after a series of 15 terms were listed aloud to remember . with tdcs the data showed very promising results . apart from the subjective experience of heightened focus and a calmness that can be likened to being in the zone , the results of the memory test indicated a measurable increase in working memory span . for each subsequent test , all 15 words were remembered in correct order and recalled in correct order after tdcs was administered . [ t]he subjective experience combined with data reflecting very promising cognitive enhancements leads me to conclude tdcs overall effectiveness in increasing my learning potential during and post stimulation . the second quote describes the perceived effects of tdcs on a medical condition depression : from a subjective perspective , this is awesome . i actually went out jogging . mostly walking , actually , because there is n't enough electricity in the world to make me not be a fat ass who 's running for the first time since 2010 , but still way out of my usual . i initially questioned if this was a hypomanic episode or just what the release of 15 years of depression feels like , but i judge it to likely be hypomania . does n't matter , i feel like frankenstein 's monster . i was dead , if only in my mind , and then with a little bit of lightning , boom . subjectively perceived effects of tdcs , survey respondents were asked whether they are planning to use tdcs in the future . a total of 111 out of 121 respondents ( 92% ) replied that they would continue using tdcs in the future . moreover , 56 ( 46% ) of the respondents rated their willingness to recommend tdcs to family members or friends , who have the same aim(s ) or conditions(s ) as they do , at four or five on the scale of one ( will not recommend ) to five ( strongly recommend ) ( appendix k ) . a total of 56 out of 121 responding users ( 46% ) reported that they have experienced side effects while using tdcs . these respondents have experienced headache , discomforting changes such as pain , tingling , itching or burning under the electrodes , fatigue , nervousness , visual perceptual changes , acute mood changes , difficulties in concentrating , nausea , and sleeping disturbance ( appendix l ) . however , the degree of side effects was not severe the average severity of most types of side effects were rated at about one , on the scale of one ( not severe at all ) to five ( extremely severe ) , except discomforting changes , such as pain , tingling , itching or burning under the electrodes , where the average severity was about 2.3 ( appendix m ) . power users and the physician reported that the side effects of tdcs are almost negligible , and this result corresponds to previous empirical results showing that there are no severe short - term adverse or side effects . what concerns did diy users have , if any , about the use of tdcs are and what they think about the possibility of government regulation on tdcs or guidelines from government agencies , researchers , or physicians ? a total of 97 out of 121 survey respondents ( 80% ) reported that they have concerns about the diy use of tdcs . most of these respondents are worried about long - term potential impairment on the brain and the use of inaccurate position of electrodes ( fig . the most interesting finding in the analysis of web postings at subreddit tdcs was the users provided safety warnings . in subreddit tdcs , 100 safety warnings were found in the comments to the postings , and 77 out of these 100 warnings were the comments to the postings on building and operation of tdcs ( table 1 ) . if you have no idea what a 4 milliamp is , i suggest you stay away from this . a 9v battery can kill you!remember safety first before you try this.don't up the current , that is the wrong way around.seriously , if you are n't comfortable with your knowledge of currents and what effects different amperages can have on your body , do not attempt this. if you have no idea what a 4 milliamp is , i suggest you stay away from this remember safety first before you try this. do n't up the current , that is the wrong way around. seriously , if you are n't comfortable with your knowledge of currents and what effects different amperages can have on your body , do not attempt this. sometimes diy users also ask more specific questions on their own diy circuit at subreddit tdcs . they post pictures or detailed descriptions of their home - built circuit and ask whether it is safe to do self - experiment with the circuit . then , other users actively correct them if there are issues on the circuit that can cause danger . the general safety warnings and questions and comments about the safety of a specific diy circuit design show the existence of a self - regulating system within this online forum . the moderator of subedit tdcs stated that there is a lot of emphasis on individual responsibility a bio hacker 's mentality , but there is also a very much of a culture that values giving people information about dangerous things. the webmaster of diytdcs also acknowledged that there is definitely a self - regulating system in subreddit tdcs : those who do know about electronics easily recognize people who should n't be experimenting with self - built devices. other power users also agreed that there were a certain number of users in the community who have corrected and guided other users not to make dangerous mistakes : we are self - regulating community . everybody in reddit has to express some type of knowledge as he / she writes a posting and nobody in the community will let up on you understanding the dangers of messing around with electronic current . the reason that it happens is because i believe at least in the beginning most of the tdcs community on reddit were people who had a background in engineering such as building circuits . over the course of last three years , i have yet seen anybody post anything that tdcs has done some real damage . the majority of survey respondents ( 97 out of 121 respondents ) thought that it would be helpful to have an official guideline from government agency or experts ( researchers or physicians ) for the diy use of tdcs . all of the power user interviewees claimed that the part that most needs guidelines from the government or experts is placement of electrodes . they reported that getting reliable repeated placement of anodes and cathodes , and what montages should be used to treat or improve which things are the most confusing parts of practicing diy tdcs . moreover , the power users expressed concern that even though diy users locate the electrodes in the right place according to a specific montage , whether a person is left - handed or right - handed might affect the outcome because left - handed people 's brains may be organized differently from those of right - handed people . it is also possible that subtle differences in the neuroanatomy of individuals may alter the effects of the device. the physician also warned that the area under the anode that is being stimulated could be changed depending on where you locate the cathodes . some of the survey respondents also provided more detailed comments on the potential official guidelines or government regulation on tdcs . official guidelines would only be helpful if they were more than do n't do it . i think in many cases our society focuses too much on safety rather than encouraging curiosity.i believe that government regulation would be a detriment and stifle any new developments.tdcs has great potential . i 'd like guidelines to come out that are backed by proper and thorough research into the effects.while i think more information and studies being published from academic and medical sources would be great , i hope that the industry does not undergo any governmental regulation , and remains a practice individuals can perform on themselves and buy the related devices without legal issues.the use of these technologies will evolve and outpace the medically established research merely based on the network effect that the internet creates around anything it touches . we need to create a sort of social network for experimenters to share and teach each other . if they are smart , they will study and not interfere except to add useful insight from time to time from their own research.about official guidelines i'd welcome any helpful information , but such guidelines , in my experience tend to be over - cautious . i ca n't imagine any official agency ever advising the public to do what i do . my own experience has been very satisfactory , but i know that there are a lot of unknowns and that i 'm accepting an unknown measure of risk when i run an electrical current through my cranium . nobody should do that until they 've researched the matter thoroughly and assessed the risks for themselves . what i would like is an advisory agency for psychologists and psychiatrists to help them deliver tdcs treatment where it 's appropriate . i think it can be very beneficial , and not many professionals seem to be aware of it.i have great concerns with some of the equipment people are using to attempt this on their own brains . i 'm an electrical engineer so when i look at some of the devices people have designed it 's terrifying what these people are hooking up to themselves . it would be very , very useful for an official guideline to be available since some of these people are using simple voltage dividers with no understanding how current behaves when they apply these things to their heads. official guidelines would only be helpful if they were more than do n't do it . i think in many cases our society focuses too much on safety rather than encouraging curiosity. i believe that government regulation would be a detriment and stifle any new developments. tdcs has great potential . i 'd like guidelines to come out that are backed by proper and thorough research into the effects. while i think more information and studies being published from academic and medical sources would be great , i hope that the industry does not undergo any governmental regulation , and remains a practice individuals can perform on themselves and buy the related devices without legal issues. the use of these technologies will evolve and outpace the medically established research merely based on the network effect that the internet creates around anything it touches . we need to create a sort of social network for experimenters to share and teach each other . if they are smart , they will study and not interfere except to add useful insight from time to time from their own research. about official guidelines i'd welcome any helpful information , but such guidelines , in my experience tend to be over - cautious . i ca n't imagine any official agency ever advising the public to do what i do . my own experience has been very satisfactory , but i know that there are a lot of unknowns and that i 'm accepting an unknown measure of risk when i run an electrical current through my cranium . nobody should do that until they 've researched the matter thoroughly and assessed the risks for themselves . what i would like is an advisory agency for psychologists and psychiatrists to help them deliver tdcs treatment where it 's appropriate . i think it can be very beneficial , and not many professionals seem to be aware of it. i have great concerns with some of the equipment people are using to attempt this on their own brains . i 'm an electrical engineer so when i look at some of the devices people have designed it 's terrifying what these people are hooking up to themselves . it would be very , very useful for an official guideline to be available since some of these people are using simple voltage dividers with no understanding how current behaves when they apply these things to their heads. these comments suggest that although diy users acknowledge the need for guidelines for the diy tdcs ; many are opposed to and worried about blanket government regulations on tdcs devices , which would prevent them from continuing self - experiments . these blanket regulations could backfire and draw further attention to diy tdcs , and lead to the opening of uncontrollable underground markets for tdcs . according to the interviewees , one of the most serious concerns about government regulations seems to be the potential increase in the cost of practicing tdcs . the moderator of subreddit tdcs said that the community has a strong preference for devices that are extremely low cost . it is very difficult to convince people to have a current meter on the home - built device because it costs \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}5 more. criticizing the current us health care system that discourages physicians from reducing the costs of medical treatments , the physician also argued that other available options , comparable to tdcs , for treatment refractory patients are too expensive . for example , it costs \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}20,000\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}30,000 per year for the treatment of chronic depression using rtms , which is a classified medical device by fda , and insurance often does not cover the treatment . in contrast , his clinic offers a tdcs treatment program at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}2500 , which includes four years of at - home supervision , as well as the initial evaluation , training , and a device although it still could be considered expensive for some patients . in other words , the costs could be the main reason why people with specific treatment refractory medical conditions , such as chronic pain or depression , opt to build or purchase a tdcs device and self - treat them . these people might view a strict regulatory oversight as a threat to deprive them of the only affordable last resort to keep their medical condition under control . as a last note , below quote from the interview with the moderator of subreddit tdcs suggests an interesting angle for a future regulatory regime regarding tdcs : if the goal of regulating tdcs device is to make sure that when people are getting tdcs device , it is safe and effective , i think labeling and regulating it as a medical device is not practical and that 's because they are so easy to make . the medical device testing is onerous and expensive and not something that many of the diy users are going to like to go through so , there are always going to be a supply of unregulated devices and you can not stop people from making this device . it would be better to adopt a wide - spread and easily understandable standard system for tdcs that is based on research on safety of tdcs and have a group to test tdcs devices to make sure that the devices meet the standard you would think twice about buying a car that has one star crash rating , and i would like to see something like that applies to tdcs devices as well . the findings of this study presented in the last section may provide preliminary empirical indicators to determine whether we need regulations and guidelines for diy tdcs and , if we do , how to design them to ensure a safe and responsible use of tdcs . this section will summarize some of the main findings and explore the tentative regulatory implications of these findings . first , unlike the implicit assumption made in media coverage on tdcs , the diy community does not comprise just risk takers with eccentric interests , such as young bio hackers or self - experimenters . the survey results showed some distinctive characteristics of the respondents clear male dominance and slight skew to ( a ) higher than average education level , ( b ) students and professionals , and ( c ) very low and high income . also , it should be noted that there was a wide distribution of ages among the respondents , which shows the potential of tdcs as an appealing technology satisfying various aims across generations . although the survey respondents did not necessarily form a representative sample , this demographic profile of diy tdcs users provides preliminary information on who will be the target subjects of potential regulations or official guidelines on the use of tdcs . second , according to the findings of the study , diy users interest in tdcs seems transient only half of the survey respondents are continuous and regular users , and the majority of these respondents ( 61% ) began to use tdcs less than six months ago . one of the interviewees also pointed out that there seem to be only a small number of committed diy users in the community . from this data , it can be inferred that there may be some hype around the estimation on the current size of the diy population . however , it is also true that findings on the perceived effects of tdcs appear to support more affirmative predictions on the future of the diy use of tdcs . a total of 54 ( 44% ) survey respondents reported that their use of tdcs was quite successful and satisfactory , and 111 respondents ( 92% ) replied that they would continue using tdcs in the future . also , 56 respondents ( 46% ) are willing to recommend tdcs to family members or friends , who have the same aim(s ) or condition(s ) as they do . interviewees expressed conflicting views on the future of the diy use of tdcs . some argued that unless more robust evidence on the effects of tdcs emerges in contrast , others who strongly believed in the potential of tdcs as treatment and cognitive enhancement predicted substantial increase in the diy tdcs user population . these somewhat contradictory findings suggest that there are a lot of ambiguities and mistaken assumptions about the use of tdcs . in general , it can be said that at this stage , diy use of tdcs is not currently widespread , that it does not seem to pose an imminent risk or danger to the public , and that there seems to be only a remote possibility of a dramatic increase of diy use of tdcs in the near future . nonetheless , more systematic and detailed investigations on the current state and prospect of diy tdcs are required so that regulatory agencies can carefully examine the need for and the appropriate timing of potential regulatory interventions . as discussed above , there is no regulation on therapeutic use of tdcs and devices intended for cognitive enhancement are not covered by any current regulations in the usa considering possible regulatory options , maslen et al . have expressed concerns about establishing a new separate regulatory regime for cognitive enhancement devices different from the current regime for medical devices . one of the reasons for their concern is that it is hard to categorically distinguish cognitive enhancement devices from medical devices . most of the current enhancing technologies , including tdcs , were originally developed for therapeutic purposes , such as treatments for illness or age - related mental deterioration , but the uses of these technologies have been subsequently expanded for enhancements . in other words , [ t]he very same device may be used both for therapeutic and enhancement purposes , in some cases using similar parameters. the types and levels of risks that are involved with therapeutic and enhancement use of a device also may not be meaningfully different . in addition , some scholars have argued that there is no philosophical difference between treatment and enhancement[b]oth therapy and enhancement aim to improve a human being 's biology and/or psychology. this study provides preliminary empirical support to the claim that the practical distinction between medical devices and enhancements can be muddy . the survey results showed the mixed and overlapping use of tdcs13 ( 11% ) respondents use tdcs for treatment of medical conditions , 71 ( 59% ) respondents for cognitive enhancement , and 29 ( 24% ) respondents for both cognitive enhancement and treatment . this suggests that if a regulatory agency decides to regulate tdcs either as a medical device or as a cognitive enhancement only , it will turn out to be an essentially underinclusive policy . according to the survey results , diy users claim to conform very well to safety guidelines for the current density ( current dose divided by electrode size ) , and the total current dosage ( current density multiplied by the duration of stimulation ) set by previous clinical studies . the analysis on the web postings in subreddit tdcs demonstrated that diy users are not only concerned about the safety of their own stimulation protocols but also cared about other users practice of tdcs . the general safety warnings by the users and the feedback process to verify the safety of individual diy circuit show the existence of an active self - regulating system in this online forum . according to previous literatures on self - regulation , self - regulation and state regulation can be intertwined and complement each other. the self - regulating system in the diy tdcs community suggests that it may be possible to build a partnership between the diy community and regulatory agencies to oversee the use of tdcs . in addition , the fact that many users consult website postings to get information on the three main elements ( dose of current , size of electrodes , and duration of stimulation ) of stimulation protocol reflects the users reliance on web postings to self - experiment with tdcs . thus , incorporating this bottom - up self - regulating system into a top - down state regulatory framework can increase the efficiency of the framework by self - motivating diy users to conform to the regulation and allowing the framework to more flexibly respond to unforeseen circumstances . fitz and reiner claimed that regulatory approach taken to diy by synthetic biologists ( diy bio ) could be a good example of a potential regulatory option for diy tdcs . synthetic biology is the design and construction of new biological parts , devices , and systems , and the re - design of existing , natural biological systems for useful purposes. in this case , the national science advisory board for biosecurity chose open communication and education through public events and conferences instead of traditional regulatory practice to develop a culture of responsibility by diy bio practitioners . fitz and reiner acknowledged that compared to diy bio , the costs of practicing diy tdcs are relatively low or sometimes even negligible , and thus , the risks associated with diy tdcs are greater than those of diy bio . however , they argued that the new regulatory approach for diy bio can be a practical option at least for the interim period until a regulatory agency comes up with a comprehensive regime for tdcs . the existence of the self - regulating system demonstrated in this study may provide support to fitz and reiner 's argument for the new regulatory approach . with voluntary participation and oversight by diy tdcs users , open communication and education can be a more viable and effective scheme for developing the culture of safe and responsible use . however , it is noteworthy that recently , there has been a demographic shift within the diy tdcs user community . the interviews with the power users of subreddit tdcs revealed that when this online forum was first established two years ago , most people in this forum had a background in engineering or did their homework on the effects and risks of tdcs by researching previous clinical studies on tdcs before their self - experiments . the self - regulating body was comprised of these people , and they were extremely helpful in giving real advices. however , the recent release of out of the box tdcs devices , such as foc.us , has increased interest in tdcs among the general public , and now there is a growing population of end users who do not know how tdcs circuits work or do not care to thoroughly investigate the effects and risks of tdcs . the increase in the size of the diy tdcs user community particularly the influx of nave end users may undermine the vitality of the self - regulating system in subreddit tdcs . if the regulatory agency wants to incorporate the self - regulating system into any future regulatory regime for tdcs , it should take into account the potential impacts of this demographic shift in the community on the self - regulating system . a total of 97 out of 121 survey respondents ( 80% ) reported that they had concerns about the diy use of tdcs . these respondents are mostly worried about long - term impairment on the brain and the use of inaccurate placement of electrodes . also , the same number of respondents answered that it would be helpful to have official guidelines from government agency or experts ( researchers or physicians ) for the diy use of tdcs . in other words , it can be said that diy users are waiting for the open communication and education by the authorities proposed in fitz and reiner 's article to ensure the safety of their self - experiments with tdcs . in addition , consistent with the survey results , all of the interviewed power user concurred that the issue where guidelines from the government or experts are most needed is placement of electrodes . as discussed above , locating the electrodes on the scalp to target a specific underlying brain region can be not only confusing but also very risky for lay users . therefore , in designing a guideline or education program for diy use of tdcs , the regulatory agency or expert groups may want to focus more on how to guide the placement of electrodes rather than other issues such as size of current or size of electrodes , for which there are already relatively clear safety standards . in contrast to their desire for official guidelines , the survey respondents took a very negative stance toward potential government regulation . some of the survey respondents answers showed that diy users would practice tdcs regardless of the government regulations , to wit : levying onerous requirements on diy users may drive the work further underground to the benefit of none. the significant financial advantages of diy tdcs compared to other treatments for chronic medical conditions seem to make this scenario more plausible . this raises a question whether it is legally or practically possible to enforce regulations on tdcs devices . however , for the fda to intervene and take enforcement actions , there need to be prohibited acts , such as the introduction of adulterated or misbranded drugs or devices into interstate commerce . building and using a tdcs device only for oneself does not seem to constitute any of the prohibited acts listed in the act . moreover , even if the fda may legally be able to outlaw the use of home - built tdcs devices through the broad interpretation or amendment of the act , how could it enforce such a ban , considering the ease and low cost of making such devices and the difficulty of detecting them , especially considering the limited resources that the fda has . in addition , regulations of marketing and sales of out of the box tdcs devices that would make device manufacturers subject to rigorous supervision , would likely lead to cost increases for the manufacturers that could increase the appeal of home - made tdcs devices . for example , arguing that enhancement devices in european union should be regulated through extending the existing legislations for medical devices , maslen et al . transparent , detailed , evidence - based information pertaining to the mechanisms , risks and effects that might be construed as benefits of the devices. providing such detailed information is currently not compulsory. this demanding requirement will inflict additional costs on the manufacturers and eventually , the consumers . however , given the ease of building tdcs devices and the diy community 's strong preference for low - cost devices , increasing the costs of devices may lead to uncontrollable self - manufacturer of the devices , causing users to hide out of reach of the fda . thus , further investigation is needed to analyse whether , in practice , the fda intervention and supervision can effectively ensure the safety and responsible use of tdcs devices . direct brain enhancement is not an imaginary story that exists only in the remote future . it was originally developed for treatment for medical conditions , such as depression , but clinical studies have also reported that it can improve various cognitive abilities among healthy people . this study is the first empirical attempt to investigate the diy tdcs user community . a questionnaire survey of diy users , interviews with some active power users , and a content analysis of web postings on tdcs showed distinctive demographic characteristics of the diy users , ambiguities and mistaken assumptions around the current state and future prospects of the diy use of tdcs , mixed use of tdcs for both treatment and cognitive enhancement , the existence of an active self - regulating system in the community , and users demands for official guidelines and their concerns about government regulations on tdcs . even with the limitation of a small non - random sample , the findings of this study provide a preliminary but useful empirical illustration of the diy tdcs user community , and increase our current understanding of the practice of diy tdcs however , more detailed subsequent studies are required to determine the need for and the timing of government interventions and to assess the strength and weakness of regulatory options proposed by researchers . furthermore , although this study just focused on one type of human biological cognitive enhancement there are numerous other enhancements , such as drugs , biologics , and dietary supplements , which could be used to improve the function of human bodies or brains . as discussed above , there have been growing discussions on whether and how to regulate these enhancements , including off - label uses of fda - approved drugs and devices for enhancement purposes . however , empirical understanding on the use of these enhancements is incomplete , as in the case of tdcs . owing to advances in biomedical science and increasing interest in human biological enhancements thus , for more meaningful and informed discussions of potential regulation , future studies on the actual practices of various human biological enhancements are required to clarify critical issues and factual assumptions regarding the use of these enhancements .

among currently available technologies , transcranial direct current stimulation ( tdcs ) is one of the most promising neuroenhancements because it is relatively effective , safe , and affordable . recently , lay people have begun to build or purchase the tdcs device to use it at home for treatment or as a cognitive enhancer . the tdcs device is currently not covered by the existing regulatory framework , but there are still significant potential risks of misusing this device , and its long - term effects on the brain have not been fully explored . thus , researchers have argued the need for regulations or official guidelines for the personal use of tdcs . however , until now , no systematic research on the do - it - yourself ( diy ) tdcs user community has been done . the present study explores the basic demographic characteristics of diy tdcs users as well as why and how they are using this device through a questionnaire survey , in - depth interviews , and a content analysis of web postings on the use of tdcs . this preliminary but valuable picture of the diy tdcs user community will shed light on future studies and policy analysis to craft sound regulations and official guidelines for the use of tdcs .

INTRODUCTION RESEARCH METHODOLOGIES FINDINGS TENTATIVE REGULATORY IMPLICATIONS OF THE FINDINGS CONCLUSION: SUMMARY AND FUTURE DIRECTIONS

brain enhancement has been considered as one of the most important intersections of law and neuroscience . despite these potential risks , professor hank greely has argued that safe and effective cognitive enhancement will benefit both individuals and society , and we should require better research on , and better regulation of , cognitive enhancement instead of banning the use of all cognitive enhancements : biomedicine will be creating more and more products and procedures that can be used for cognitive enhancement . one of the most eye - catching and readily accessible direct brain enhancement technologies is transcranial direct current stimulation ( tdcs ) . moreover , recent studies have reported that the use of tdcs on specific brain regions can improve cognitive functions , such as attention span , working and long - term memory , language , and mathematical ability of healthy subjects . researchers have expressed concerns about this so - called do - it - yourself ( diy ) use of tdcs . they have argued that even if there are no serious side effects , potential risks of misusing this device should not be ignored . lastly , using tdcs to enhance some of the cognitive functions may adversely affect other functions of the brain , and potential long - term ( side ) effects of tdcs on the brain have not been fully explored . an instrument , apparatus , implement , machine , contrivance , implant , in vitro reagent , or other similar or related article , including a component part , or accessory which is : ( 1 ) recognized in the official national formulary , or the united states pharmacopoeia , or any supplement to them , ( 2 ) intended for use in the diagnosis of disease or other conditions , or in the cure , mitigation , treatment , or prevention of disease , in man or other animals , or ( 3 ) intended to affect the structure or any function of the body of man or other animals , and which does not achieve its primary intended purposes through chemical action within or on the body of man or other animals and which is not dependent upon being metabolized for the achievement of any of its primary intended purposes . for example , in 2011 , repetitive transcranial magnetic stimulation ( rtms ) , another non - invasive neuromodulation that generates electric currents using electromagnetic coils , was classified as a class ii medical device for treatment of major depressive disorder . also , cranial electrotherapy stimulation , whose mechanism is similar to tdcs except that it applies alternating current , not dc , to the brain , has been regulated as a medical device ( class iii ) for treatment of insomnia , depression , or anxiety since 1979 . a few clinics offer tdcs treatment to their patients with various medical conditions , such as depression and chronic pain , but these clinics apply tdcs as off - label use of the iontophoresis device a dc stimulator for introducing ions of soluble salts or other drugs into the body for medical purposes that fda classified as a medical device ( class ii / iii ) in 2004 . however , since tdcs devices are already on the market and being sold to the public , they argued that now we should begin the discussion on how to develop a regulatory policy for the diy use of tdcs . despite the recent surge of attention to the potential risks and regulations of tdcs , there has been no attempt to systemically study what is really happening in the diy tdcs user community . discussions of issues related to the diy use of tdcs are mostly based on speculation , which may lead to importantly incorrect understanding of the diy community and its use of tdcs . specifically , this study aims to answer following four questions on the diy use of tdcs through an online survey , a content analysis of web postings , and interviews with the diy users . what are the general demographic characteristics of diy users?why and how do they use this device?what are the perceived effects or side effects of this device?what are their concerns , if any , about the diy use of this device ? also , only a small number of people interviewed , chosen from among those who , based on the websites , appeared to be influential in the diy tdcs user community . the unregulated , unregistered , and otherwise unreachable nature of the community of tdcs users made these the only feasible approaches , but they do undercut , to some extent , the confidence that can be placed in the findings . nonetheless , even if viewed as only preliminary data , these results provide some basic information on diy tdcs users and their attitudes and practices of diy tdcs which will help us to lay the groundwork for designing sound future policy and official guidelines on tdcs . section 1 describes the three research methodologies recruited in this study , and the findings on the diy tdcs user community are presented in section 2 . section 3 explores potential legal and regulatory implications of these findings considering possible regulatory options for the diy use of tdcs . there has been no official report on the size of the diy tdcs user population in the usa , in any other country , or worldwide . the site , www.reddit.com/r/tdcs/ ( hereinafter subreddit tdcs ) , is an open forum for tdcs users and www.diytdcs.com ( hereinafter diytdcs ) is a personal blog by a famous lay expert on diy tdcs . the research subjects of this study are the registered users or visitors of these two websites ( 1 ) who built or purchased their personal tdcs devices and ( 2 ) who have used them for treatment or as a cognitive enhancer . the research methodology of this study has three parts : ( 1 ) an online questionnaire survey , ( 2 ) a content analysis of web postings on the diy use of tdcs , and ( 3 ) in - depth interviews . the survey was composed of a minimum of 21 to a maximum of 29 questions depending on the respondents answers to some questions on basic demographic information of diy users , their aims of practicing diy tdcs , current practices of tdcs ( including types of devices , stimulation protocols , and frequency of use ) , effects and side effects of tdcs , and their concerns about the diy tdcs ( see appendix a for the questionnaire ) . however , since this study is the first preliminary attempt to learn about the diy tdcs user community , responses from this convenience sample should provide previously unknown but useful facts about this community . the second research methodology is a content analysis of web postings on subreddit tdcs . in this well - known and very active online forum , diy users ask and answer questions on the diy use of tdcs device , post interesting recent tdcs studies , and share their experiences of positive and adverse effects of tdcs devices . since there is no previously established theory or research study on the diy tdcs user community or subreddit tdcs , the codes were derived from an emergent process . through a preliminary examination of the postings , five major codes to categorize the main theme of each posting were developed : ( 1 ) building and operation of the tdcs device , ( 2 ) marketed devices , ( 3 ) effects / side effects of the device , ( 4 ) references ( external links ) to research papers , newspaper / magazine articles , or blog posts on tdcs , and ( 5 ) others . for example , diy tdcs users usually mentioned their purpose for using the tdcs device while asking questions about building and operation of the device ( see appendix b for detailed classification of the codes ) . after survey data analysis and content analysis of web postings were completed , five semistructured interviews were conducted to collect more detailed information on the user experience and users opinions on the official guidelines and potential government regulation of the tdcs device . first , this study conducted a brief analysis on the user ids and postings at subreddit tdcs to identify power users who could provide a general overview and informative facts on the diy tdcs user community . four of these five power users participated in the interviews : the moderator of subreddit tdcs , the webmaster of diytdcs , a user who has uploaded popular self - experimentation videos on his youtube channel , and a user who has been using tdcs for treatment of bipolar type ii and other medical conditions . second , a physician who has been treating about 350 patients with tdcs for the last seven years was also recruited for the interview to provide a medical perspective on the diy use of tdcs . although the number of samples is very small , given the exploratory nature of this study , these interviews should provide a meaningful preliminary sketch of the diy tdcs community and current uses of tdcs along with the findings of the survey and content analysis of the web postings . in this section , the results of the survey , content analysis of web postings , and in - depth interviews will be presented and analysed under the four main research topics of this study : general demographic characteristics of diy users ; their reasons for and current uses of tdcs ; effects and side effects of tdcs ; and their concerns about the diy use of tdcs . there is no official estimate of the size of the diy tdcs user population . although it is very hard to track whether a particular person is really using tdcs , the moderator estimated that 6000 might be the upper bound of the diy tdcs user population and the lower bound might be the number of subscribers to this website , about 3000 , assuming that an active , regular tdcs user will visit the website at least once a month . this power user estimated that the size of the diy tdcs user population is probably a few thousand , but not more than 10,000 . however , the webmaster of diytdcs , who is also one of the most active participants in subreddit tdcs since the establishment of that forum , questioned this estimate of the size of the diy tdcs community in the media and academic studies : i 'm not sure how large the community is just now . thus , although , based on evidence of internet uses , the diy tdcs user population seems to have been growing for the last few years , these rough indicators might suggest that the dedicated tdcs user population has been overstated . second , most of the respondents are in their 20s and 30s ( 86 ; 71% ) , but there are a significant number of respondents in their 50s and over ( 15 ; 12% ) ( fig . this part will delineate and analyse the data on diy users aims of using tdcs device , as well as their current use of tdcs such as types of tdcs devices , stimulation protocols , and frequency of use . this indicates that information on tdcs is mainly disseminated and shared through online media and forums , assuming that many of respondents may not have direct access to academic journals , and recent intriguing studies on the various effects of tdcs have been vigorously covered by the media . this shows that this website functions as a forum for discussing how to build and use tdcs and sharing new scientific discoveries on the effects of tdcs featured in media and various online forums . the pharmacological status of the brain can have meaningful effect on the outcome of tdcs , and the variety of psychoactive agents that home users may employ is legion. in this survey , 18 respondents44% of respondents who are using tdcs for treatment of medical conditions reported that they are currently taking other medications for the same medical conditions . after he found out that building a tdcs device does not require complicated knowledge of electrical engineering , he decided to design his own circuit , which is affordable ( costs less than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}50 ) and , he believes , safe , and to share it with other diy users . in addition to these open source circuits , some users also compiled and posted basic schematics , tools and electrical components needed to build the tdcs device , and online or offline shops that one could get those tools and components , as well as their prices . the physician argued that the montage of foc.us device is different from montages used in any of the clinical studies , and he did not think it has the same effects as the ordinary two electrodes system using iontophoresis devices that researchers have used in finding evidence of effectiveness . on the other hand , the moderator said we can not assume that these end users are knowledgeable enough to make informed decisions about their safety in regard to issues like whether a specific type of circuit is safe to use in these devices , and thus , three factors determine the safety of a tdcs protocol : dose of current , size of electrodes , and duration of stimulation . thus , these studies do not provide proper guidelines for people who are using tdcs at home more often for a longer period of time . the self - reports on the effects of tdcs posted at subreddit tdcs show that diy users themselves are having problems in assessing the effects of tdcs while using it at home . what concerns did diy users have , if any , about the use of tdcs are and what they think about the possibility of government regulation on tdcs or guidelines from government agencies , researchers , or physicians ? most of these respondents are worried about long - term potential impairment on the brain and the use of inaccurate position of electrodes ( fig . the most interesting finding in the analysis of web postings at subreddit tdcs was the users provided safety warnings . i 'd like guidelines to come out that are backed by proper and thorough research into the effects.while i think more information and studies being published from academic and medical sources would be great , i hope that the industry does not undergo any governmental regulation , and remains a practice individuals can perform on themselves and buy the related devices without legal issues.the use of these technologies will evolve and outpace the medically established research merely based on the network effect that the internet creates around anything it touches . if they are smart , they will study and not interfere except to add useful insight from time to time from their own research.about official guidelines i'd welcome any helpful information , but such guidelines , in my experience tend to be over - cautious . these comments suggest that although diy users acknowledge the need for guidelines for the diy tdcs ; many are opposed to and worried about blanket government regulations on tdcs devices , which would prevent them from continuing self - experiments . as a last note , below quote from the interview with the moderator of subreddit tdcs suggests an interesting angle for a future regulatory regime regarding tdcs : if the goal of regulating tdcs device is to make sure that when people are getting tdcs device , it is safe and effective , i think labeling and regulating it as a medical device is not practical and that 's because they are so easy to make . the medical device testing is onerous and expensive and not something that many of the diy users are going to like to go through so , there are always going to be a supply of unregulated devices and you can not stop people from making this device . it would be better to adopt a wide - spread and easily understandable standard system for tdcs that is based on research on safety of tdcs and have a group to test tdcs devices to make sure that the devices meet the standard you would think twice about buying a car that has one star crash rating , and i would like to see something like that applies to tdcs devices as well . the findings of this study presented in the last section may provide preliminary empirical indicators to determine whether we need regulations and guidelines for diy tdcs and , if we do , how to design them to ensure a safe and responsible use of tdcs . although the survey respondents did not necessarily form a representative sample , this demographic profile of diy tdcs users provides preliminary information on who will be the target subjects of potential regulations or official guidelines on the use of tdcs . however , it is also true that findings on the perceived effects of tdcs appear to support more affirmative predictions on the future of the diy use of tdcs . interviewees expressed conflicting views on the future of the diy use of tdcs . some argued that unless more robust evidence on the effects of tdcs emerges in contrast , others who strongly believed in the potential of tdcs as treatment and cognitive enhancement predicted substantial increase in the diy tdcs user population . nonetheless , more systematic and detailed investigations on the current state and prospect of diy tdcs are required so that regulatory agencies can carefully examine the need for and the appropriate timing of potential regulatory interventions . as discussed above , there is no regulation on therapeutic use of tdcs and devices intended for cognitive enhancement are not covered by any current regulations in the usa considering possible regulatory options , maslen et al . according to the survey results , diy users claim to conform very well to safety guidelines for the current density ( current dose divided by electrode size ) , and the total current dosage ( current density multiplied by the duration of stimulation ) set by previous clinical studies . the self - regulating system in the diy tdcs community suggests that it may be possible to build a partnership between the diy community and regulatory agencies to oversee the use of tdcs . fitz and reiner acknowledged that compared to diy bio , the costs of practicing diy tdcs are relatively low or sometimes even negligible , and thus , the risks associated with diy tdcs are greater than those of diy bio . however , it is noteworthy that recently , there has been a demographic shift within the diy tdcs user community . however , the recent release of out of the box tdcs devices , such as foc.us , has increased interest in tdcs among the general public , and now there is a growing population of end users who do not know how tdcs circuits work or do not care to thoroughly investigate the effects and risks of tdcs . the increase in the size of the diy tdcs user community particularly the influx of nave end users may undermine the vitality of the self - regulating system in subreddit tdcs . these respondents are mostly worried about long - term impairment on the brain and the use of inaccurate placement of electrodes . also , the same number of respondents answered that it would be helpful to have official guidelines from government agency or experts ( researchers or physicians ) for the diy use of tdcs . thus , further investigation is needed to analyse whether , in practice , the fda intervention and supervision can effectively ensure the safety and responsible use of tdcs devices . a questionnaire survey of diy users , interviews with some active power users , and a content analysis of web postings on tdcs showed distinctive demographic characteristics of the diy users , ambiguities and mistaken assumptions around the current state and future prospects of the diy use of tdcs , mixed use of tdcs for both treatment and cognitive enhancement , the existence of an active self - regulating system in the community , and users demands for official guidelines and their concerns about government regulations on tdcs . even with the limitation of a small non - random sample , the findings of this study provide a preliminary but useful empirical illustration of the diy tdcs user community , and increase our current understanding of the practice of diy tdcs however , more detailed subsequent studies are required to determine the need for and the timing of government interventions and to assess the strength and weakness of regulatory options proposed by researchers . however , empirical understanding on the use of these enhancements is incomplete , as in the case of tdcs . owing to advances in biomedical science and increasing interest in human biological enhancements thus , for more meaningful and informed discussions of potential regulation , future studies on the actual practices of various human biological enhancements are required to clarify critical issues and factual assumptions regarding the use of these enhancements .

dna in all eukaryotic organisms is bound by nucleosomes , forming a physiological chromatin context in which all molecular processes involving dna operate . the integrity of the chromatin template is constantly compromised by fundamental biological processes , such as dna replication , repair , and transcription , following which the normal chromatin structure is restored with the help of histone chaperone proteins ( gurard - levin et al . , 2014 , ransom et al . , 2010 ) . advances in recent years have shed light on some of the molecular players involved in chromatin assembly and maintenance , separating ( on the molecular level ) dna replication - dependent and -independent pathways ( burgess and zhang , 2013 ) . histone proteins themselves are central to these processes : the expression and incorporation of canonical histones is tightly coupled to dna replication ; in contrast , histone variants can be incorporated into chromatin independent of the cell cycle ( maze et al . , 2014 ) . while it has become increasingly clear that the activity of histone chaperone proteins is of critical importance during dna replication and repair ( adam et al . , 2013 , hoek and stillman , 2003 , polo et al . , 2006 , ransom et al . , 2010 ) , studies that have attempted to dissect the importance of histone replacement in the interphase nucleus have revealed only a limited contribution of histone chaperones and/or variants to transcriptional regulation ( banaszynski et al . , 2013 , goldberg et al . , 2010 , hdl and basler , 2009 , sakai et al . , 2009 ) . these studies , however , have been complicated by the use of proliferative cell systems that could ( at least partially ) restore chromatin integrity through replication - coupled chromatin assembly ( banaszynski et al . , 2013 , hdl and basler , 2009 , sakai et al . , 2009 , wyrick et al . , 1999 ) . thus , the extent to which basic physiological processes , such as transcription , are dependent on or regulated by histone replacement remains unclear . to overcome the limitations of these previous studies , we took advantage of the unique system presented by mammalian oogenesis . over an extended time span and in the absence of dna replication , postnatal mammalian oocytes execute the oogenesis - specific developmental program , involving widespread transcriptional changes and de novo dna methylation , ultimately acquiring the competencies required for fertilization and embryogenesis ( de la fuente , 2006 , li and albertini , 2013 , tomizawa et al . , 2012 ) . to address the importance of histone turnover during this process , we have generated a mouse oocyte - specific knockout of the histone chaperone hira . as opposed to caf1 , which is implicated in the replication - coupled deposition of canonical histones h3.1 and h3.2 , hira has been shown to deposit the h3.3 variant during replication - independent chromatin assembly ( ray - gallet et al . , 2002 , our findings show that depletion of hira in primordial oocytes causes a severe developmental defect associated with extensive oocyte death . on the molecular level this causes significantly increased dna accessibility , accumulation of dna damage , and chromosome segregation defects . the lack of normal chromatin structure has a striking impact on transcriptional regulation : lack of normal h3.3/h4 replacement prevents the oocytes from maintaining full dynamic range of gene expression , and it leads to compromised transcriptional transitions normally associated with the oocyte development . furthermore , we show that histone replacement is necessary for silencing of spurious transcription and , in the context of the oocyte , also for the efficient deposition of de novo dna methylation . our results thus uniquely demonstrate the critical relationship between continuous histone replacement , the structural integrity of the chromatin template , the normal regulation of transcription , and the establishment of dna methylation , in the context of an in vivo developmental system . in the mouse , female germ cells arrest in prophase of the first meiotic division at embryonic day ( e)13.5 . the female gonocytes subsequently resume growth in postnatal ovaries where they undergo oogenesis and progress through meiosis ( de la fuente , 2006 ) . this process involves the following : ( 1 ) pronounced transcriptional changes as the developing oocytes progress from primordial to large antral follicle stages ; ( 2 ) the deposition of de novo dna methylation starting in the secondary follicle stage ; ( 3 ) the silencing of transcription in the germinal vesicle ( gv ) stage ; and , finally , ( 4 ) the condensation of chromosomes in the mii stage , resulting in an oocyte that is competent to undergo fertilization and support early embryonic development ( figure 1a ) . we first addressed the capacity of developing oocytes to incorporate histones by microinjection of mrna for tagged versions of histones . in agreement with the absence of replication during oocyte maturation , growing oocytes did not show any incorporation of canonical histones h3.1 and h3.2 ( figure 1b ; akiyama et al . , 2011 ) . in contrast , microinjected flag - tagged h3.3 and h2a.x , previously shown to be enriched in the developing oocyte ( akiyama et al . , 2011 , 2010 ) , were readily incorporated into the chromatin of growing oocytes ( figure 1b ) . we thus conclude that h3.3 is the only h3 subtype incorporated during postnatal oocyte development . in further support of this observation , oocytes isolated from our h3.3b - egfp knockin mice ( figure s1 g ) confirmed that both growing and gv oocytes contain high levels of nuclear h3.3 ( figure 1e ) . however , we also detected a high level of chromatin incorporation for flag - tagged h2a.x , h4 , and h3.3 upon injection of tagged histone mrna into transcriptionally inactive gv - stage oocytes ( figure 1c ) , demonstrating that the chromatin in the mature gv oocyte is still dynamic even after global transcription has ceased . previous studies have shown that the majority of h3.3 incorporation is critically dependent on the presence of histone chaperone hira ( goldberg et al . , 2010 , pchelintsev et al . , 2013 ) . in agreement with the observed h3.3 incorporation , hira is expressed and shows nuclear localization during postnatal oocyte development ( figure 1d ) . to investigate whether hira is specifically required for the observed h3.3 incorporation during oogenesis , we took advantage of gdf9- and zp3-driven expression of cre recombinase and deleted hira in the primordial and primary stages of follicle development , respectively ( figures 1a and s1a ) . we confirmed that both gdf9-cre- and zp3-cre - deleted oocytes undergo genetic recombination ( figure s1b ; data not shown ) and are depleted of hira mrna , protein and complex ( figures 1d and s1c s1f ) . the absence of hira severely decreased h3.3 content and completely abolished the incorporation of microinjected tagged h3.3 in both growing and gv - stage oocytes ( figures 1b , 1c , and 1e ) . the observed impairment of h3.3 incorporation occurred despite the presence of an alternative atrx / daxx chaperone complex that has been well characterized to incorporate h3.3 ( burgess and zhang , 2013 , maze et al . , , the presence and the localization of this complex in the gv oocyte were not affected by hira deletion ( figure s2a ) , and we could observe h3.3b - egfp loci overlapping with atrx staining in hira gdf9-cre , h3.3b - egfp gv oocytes at higher laser detection intensity ( figure s2c ) . the severe reduction of h3.3 incorporation in hira gdf9-cre oocytes thus demonstrates that the atrx / daxx chaperone complex can not compensate for the lack of hira , as was also observed in mouse embryonic stem cells ( escs ) ( goldberg et al . , 2010 ) . the lack of hira and h3.3 incorporation also was not compensated for by incorporation of the canonical histones h3.1 or h3.2 ( figure 1b ) , and it coincided with the lack of detectable incorporation of histone h4 ( figure 1c ) . taken together , these findings demonstrate that the histone variant h3.3 , rather than h3.1 or h3.2 , is actively incorporated into chromatin during oocyte development , and confirm that hira is responsible for the incorporation of the majority of h3.3 in conjunction with histone h4 ( figures 1b1e and s2b ) . of note , although hira - deficient oocytes failed to incorporate tagged h3 and h4 histones , the incorporation of tagged h2a.x was readily detectable ( figures 1b and 1c ) , confirming that distinct molecular pathways are implicated in the deposition of h3/h4 and h2a / h2b histones ( burgess and zhang , 2013 ) . assessment of females with oocyte - specific hira deletion revealed that loss of hira led to a severe ovarian phenotype . significantly lower numbers of mii oocytes could be recovered following superovulation ( figure 2a ) ; zp3-cre - driven deletion of hira led to a more than 50% reduction in the number of ovulated mii oocytes , and this effect was even more dramatic upon earlier gdf9-cre - driven deletion ( figure 2a ) . furthermore , gv oocytes ( figure 1a ) isolated from both hira zp3-cre and hira gdf9-cre ovaries showed dramatically reduced competency to complete the gv - to - mii transition during in vitro maturation ( figure s2e ) . hira gdf9-cre ovaries were significantly smaller ( figures 2b and s2f ) ; and , although we could recover some gv oocytes from 3-week - old females , the ovaries of older mice contained only very few oocytes , which were predominantly at early developmental stages ( figures 2c and s2 g ) . further analysis of the superovulated hira gdf9-cre mii oocytes revealed frequent defects in chromosome alignment and segregation and instances of aberrant polar body extrusion ( figure 2d ) . in line with a severe ovarian phenotype and in agreement with recently published reports ( inoue and zhang , 2014 , lin et al . 2005 ) , the surviving hira - depleted oocytes were unable to either reprogram the paternal pronucleus or support early embryonic development after fertilization . in agreement with the observed enrichment of h3.3 and hira in the paternal pronucleus of control zygotes ( figures s3a s3c ) , fertilized hira gdf9-cre oocytes could not fully de - condense the male pronucleus ( figure s3d ) , incorporate h3.3 ( figure s3e ) , or support normal zygotic development ( figure s3f ) . failure to incorporate h3.3 occurred despite the efficient removal of protamines ( figure s4a ) , indicating that protamine removal and chromatinization of the paternal genome are mechanistically distinct ( inoue and zhang , 2014 , lin et al . , 2014 ) . although the histone load of the paternal pronucleus was severely reduced upon maternal hira deletion , we could detect a residual amount of histones on the paternal dna by immunofluourescent staining ( figure s4b ) . however , it remains to be addressed whether the residual histones were inherited from sperm or whether they were incorporated in a hira - independent manner . in agreement with the severe chromatinization defect , the partially decondensed paternal pronucleus of zygotes in which hira had been maternally deleted failed to undergo dna demethylation ( figure s4c ) and could not provide a normal template for zygotic replication or transcription ( figures s4d and s4e ; lin et al . , 2014 ) . apart from the failure to support normal reprogramming of the paternal pronucleus following fertilization , hira - depleted oocytes showed severe maternal defects . the atp content was significantly reduced in hira - depleted mii oocytes ( figure s2h ) , and , upon parthenogenetic activation , only a fraction of these oocytes reached the two - cell stage and none progressed further , contrary to the control oocytes that efficiently supported development of parthenogenetic blastocysts ( figure 2e ) . considering the pronounced oocyte phenotype , we set out to understand the molecular mechanisms by which hira regulates oocyte development . we hypothesized that , in the absence of h3/h4 incorporation , the chromatin structure in hira - depleted oocytes would be severely affected . in agreement with this , the histone content of hira - depleted gv - stage oocytes was severely reduced ( figures 3a and s2b ) , and the resulting chromatin displayed altered structure and increased dnase i sensitivity ( figures 3b and 3c ) . moreover , in accordance with the predicted role of histones in protecting genetic material from environmental stress and mutagenesis ( ljungman and hanawalt , 1992 ) , hira - depleted oocytes showed clear signs of dna damage both by increased levels of -h2ax ( figure 3d ) and upregulation of dna damage - responsive genes ( figure 3e ) . the importance of hira and continuous h3.3 replacement has been previously assessed in the context of proliferating mouse pluripotent escs ( banaszynski et al . these studies surprisingly revealed that hira deletion led to only minor transcriptional changes ( banaszynski et al . the severity of the phenotype observed following the deletion of hira in oocytes , we next asked whether the reduced h3/h4 incorporation and increased dna accessibility ( figures 1b1e , 3a , and 3c ) affected the transcriptional program of the hira - depleted oocytes . surprisingly , although the lack of maternal hira severely affected the amount of transcription emanating from the paternal pronucleus following fertilization ( figure s4e ; lin et al . , 2014 ) , hira - depleted and control growing oocytes showed similar global levels of transcription , as judged by eu incorporation ( figure 4a ) . to address the role of hira in transcription during oocyte development in depth , we carried out single - cell rna sequencing ( scrna - seq ) on four hira gdf9-cre and four hira control oocytes . we chose to analyze ovulated mii oocytes that had successfully completed their first meiotic division to avoid inconsistencies due to differences in staging or developmental progression . of note , consistent with the identical patterns of eu incorporation in the hira gdf9-cre and control growing oocytes ( figure 4a ) , normalization to the ercc rna spike - ins included in our scrna - seq library preparations revealed no significant differences in the total quantity of poly - adenylated rna between hira gdf9-cre and control mii oocytes ( figure 4b ) . in agreement with the observed severe developmental phenotype , the initial data analysis revealed pronounced transcriptional differences upon deletion of hira ( figures 4c and s6a ) . expression analysis identified 2,101 differentially expressed genes ( false discovery rate [ fdr ] < 0.1 ) between hira gdf9-cre and control oocytes ( 1,274 upregulated in hira gdf9-cre oocytes relative to controls ; 827 downregulated ) , representing over 15% of all annotated transcripts detected ( table s1 ) . gene ontology ( go ) analysis of differentially upregulated genes showed enrichment for cell membrane , biological adhesion molecules , and cell junctions ( figure 4d ) . in contrast , go analysis on differentially downregulated genes confirmed enrichment for processes important in late - stage oocytes , including chromosome , nucleolus , mitochondria , and pronucleus ( figure 4e ) . surprisingly , we detected significantly more annotated transcripts ( 7.7% increase , p value < 0.01 ) in hira gdf9-cre oocytes relative to controls ( figure 4f ) . the increased number of transcripts was not evenly distributed across all expression levels ( figure 4 g ) ; rather , deletion of hira resulted in relatively fewer transcripts with very low ( first decile ) or very high ( ninth and tenth deciles ) expression ( figure 4 g ) . furthermore , differentially down- and upregulated genes showed higher and lower than expected expression , respectively ( figure 4h ) ; and , while the genes normally not expressed or expressed at very low levels ( bottom 20% ) were upregulated upon hira deletion ( figure 4i ; gene set enrichment analysis [ gsea ] , fdr < 0.001 ; data not shown ) , genes with very high expression in normal oocytes ( top 20% ) were specifically downregulated in hira gdf9-cre oocytes ( figure 4i ; gsea , fdr < this also was evident by the greatly reduced variance in expression levels of annotated transcripts within the hira - depleted oocytes ( figure 4j ; p value < 0.001 ) , suggesting that hira is necessary to maintain the full dynamic range of gene expression . thus , contrary to previous findings in proliferating pluripotent escs , hira appears to be critical for normal transcriptional regulation during oocyte development . the major transcriptional transition during oogenesis occurs between primordial stage oocytes ( when gdf9-driven cre is first expressed ) and primary stage oocytes ( when zp3-driven cre is first expressed ) , during which the major wave of gene activation is observed ( pan et al . , 2005 ) . to test whether the observed transcriptional defects are due to hira gdf9-cre oocytes failing to execute this transcriptional activation or to other dominant secondary effects , we additionally carried out scrna - seq on four hira zp3-cre and three hira control oocytes ( figure s6 ; table s3 ) . remarkably , a nearly identical transcriptional phenotype was observed in hira zp3-cre and hira gdf9-cre oocytes : differentially down- and upregulated genes showed higher and lower than average expression in the control oocytes , respectively ( figure s6e ) , and genes normally not expressed or expressed at very low levels ( bottom 20% ) were upregulated upon hira deletion ( figure s6f ) , while genes with very high expression in normal oocytes ( top 20% ) were specifically downregulated in hira gdf9-cre oocytes ( figure s6f ) . similar to the effect observed in hira gdf9-cre oocytes , the variance in expression levels of annotated transcripts within the hira zp3-cre oocytes was greatly reduced ( figure s6 g ; p value < 0.001 ) . the developing oocyte is a unique experimental system , as it allows us to assess the importance of continuing histone h3.3/h4 replacement not only for steady - state transcription , but also for affecting transcriptional transitions associated with development , such as activation or repression of dynamically regulated genes . to address this , we used k - means clustering of published expression data ( pan et al . , 2005 ; geo : gse3351 ) to identify eight clusters of genes that show distinct expression patterns over the course of oocyte development ( figures s5a and s5b ) , and we compared these to the expression profiles in hira gdf9-cre and control oocytes . remarkably , genes downregulated upon hira depletion ( hira gdf9-cre ) were specifically enriched for genes whose transcription is activated either between the earliest stages of oocyte development ( figure 5a , cluster 4 , primordial and primary follicle stages ; gsea , fdr < 0.001 ) or between early and late oogenesis ( figure 5a , cluster 5 ; gsea , fdr < 0.001 ) . in contrast , genes upregulated upon hira deletion were most significantly enriched for clusters of genes that all uniquely show downregulation of expression between early and late oogenesis ( figure 5a , cluster 2 ; fdr = 0.017 ) or between primordial and primary follicle stages ( figure s5b , clusters 6 and 8) . of note , similar phenotypes were observed upon zp3-cre - driven deletion of hira ( figure s6h ) . to confirm that these observations were not an artifact of the reduced dynamic range observed in the hira - depleted mii oocytes , we carried out a similar gsea analysis on only median expressed genes ( i.e. , genes in the second , third , or fourth expression quintile ) . cluster 2 ( genes progressively downregulated during oogenesis ) remained enriched among upregulated genes in hira gdf9-cre oocytes ( gsea , fdr = 0.08 ; data not shown ) ; similarly , cluster 4 ( genes upregulated in early oogenesis ) still showed enrichment ( although this was less pronounced ) among genes downregulated in the absence of hira ( gsea , fdr = 0.26 ; data not shown ) . to further test the direct link between normal transcriptional activation and hira , we carried out differential expression analysis between hira gdf9-cre and hira zp3-cre mii oocytes . given the same observed reduction of the dynamic range of transcription in both systems ( figures 4 g , 4j , and s6 g ) , we hypothesized that , if normal transcriptional activation was dependent on hira , genes that are specifically upregulated between primordial ( when gdf9-driven cre is first expressed ) and primary ( when zp3-driven cre is first expressed ) stage oocytes should show higher expression in hira zp3-cre mii oocytes . indeed , this hypothesis was confirmed by the gsea analysis ( figure s6i ; gsea , fdr < 0.001 ) . taken together , our data bring to light the critical role of hira ( and h3.3/h4 turnover ) for affecting changes to the transcriptional state . histone h3.3/h4 replacement is required for normal transcriptional activation of genes associated with developmental progression of oocytes , and , surprisingly , it is also necessary for genes to become or to remain transcriptionally silent . nucleosomes have been hypothesized to constitute a barrier for transcription ( teves et al . , 2014 ) . considering the failure of hira - deleted oocytes to silence genes , we asked whether the increased dna accessibility in the absence of continuous h3.3/h4 replacement may lead to aberrant transcription from regions not normally transcribed within the genome . to address this possibility , we used a previously described approach that allows for the identification of unannotated transcripts originating entirely from intronic or intergenic regions ( trapnell et al . , 2010 ) . consistent with our previous mapping , we observed an increase ( 7.3% , p value < 0.05 ) in the number of annotated transcripts detected in hira gdf9-cre oocytes ( figure 5b ) . remarkably , a much larger increase was observed in the total number of transcribed fragments ( 37.7% , p value < 0.05 ; figure 5b ) , with the largest increases observed in unannotated transcripts originating entirely from intronic ( 112.6% increase , p value < 0.05 ) and intergenic ( 92.0% increase , p value < 0.05 ) regions ( figure 5c ) of the hira - deleted oocytes . of note , while expression of unannotated intergenic transcripts was clearly detectable ( 90% transcripts with > 1 fragments per kilobase of transcript per million mapped reads [ fpkm ] ) and often at high level ( 8% transcripts with > 10 fpkm ) in individual knockout samples , expression of a given unannotated intergenic transcript was only ever observed in one biological replicate , reflecting stochastic events consistent with spurious transcription ( table s2 ) . also of note , a nearly identical phenotype was observed upon zp3-cre - driven deletion of hira ( figures s6d , s6j , and s6k ; table s4 ) . progression through oocyte development is accompanied by the accumulation of dna methylation . during this process , the largely hypomethylated genome of primordial oocytes acquires dna methylation through the activity of the de novo dna methyltransferase dnmt3a and its co - factor dnmt3l ( kaneda et al . , 2004 , smallwood and kelsey , 2012 , smallwood et al . , 2011 ) . acquisition of dna methylation in oocytes is known to be positively correlated with transcription , which could relate to the recruitment of dnmt3a/3l to gene bodies through binding of its pwwp domain to h3k36me3 , a histone modification associated with transcriptional elongation ( chotalia et al . , 2009 , dhayalan et al . although h3k36me3-driven dna methylation in gene bodies has recently been attributed to the enzymatic activity of dnmt3b in mouse escs ( baubec et al . , 2015 , yang et al . , 2014 ) , dnmt3a is responsible for the accumulation of oocyte dna methylation as dnmt3b is absent in growing oocytes ( kaneda et al . , 2004 , shirane et al . , 2013 ) . given the widespread transcriptional changes observed in the hira knockout oocytes , we set out to examine the potential impact of hira deletion on de novo dna methylation in the oocyte . although the total amount of transcription was not altered upon hira deletion ( figures 4a and 4b ) , the observed reduced histone load was connected with lower levels of global h3k36me3 in hira - depleted gv oocytes ( figures 3a and s7a ) . interestingly , initial immunofluorescence ( if ) detection of 5mc indicated that hira gdf9-cre gv oocytes also contain lower levels of dna methylation ( figure s7b ) . further quantitative assessment by liquid chromatography - mass spectrometry ( lc - ms ) revealed a dramatic reduction of 5mc in hira - deleted oocytes , with a more pronounced effect following earlier gdf9-cre - driven deletion ( figure 6a ) . importantly , the reduction of dna methylation occurred despite the transcriptional upregulation of dnmt3a and without statistically significant changes in the expression of other dna methyltransferases ( figures s5c , s7c , and s7d ) . to further understand the relationship among hira deletion , transcriptional changes , and global changes in dna methylation , we profiled dna methylation genome - wide by bisulphite sequencing . because of the very limited availability of hira - deleted oocytes , we performed single - cell bisulphite sequencing ( scbs - seq ) on 14 individual hira gdf9-cre mii oocytes and 14 individual hira control mii oocytes , followed by in silico merging of the individual datasets , as previously described ( smallwood et al . , 2014 ) . we first confirmed that our scbs - seq dataset represented an accurate depiction of oocyte methylation , as we observed a high correlation ( r = 0.96 ) between our merged scbs - seq control oocytes and an independent whole - genome bisulphite sequencing ( wgbs ) dataset of pooled oocytes ( shirane et al . , 2013 ; dna data bank of japan : dra000570 ; figures 6c and s7e ) . in agreement with the lc - ms measurements and if data , our wgbs analysis confirmed the dramatic reduction in dna methylation in both cpg ( 42.7% decrease ) and chh ( 73.2% decrease ) contexts ( figures 6b and s7f ) . further analysis revealed that only 3.8% of the 3-kb genomic windows classed as methylated in wild - type oocytes ( > 80% methylation ) remained methylated to the same extent in hira - depleted oocytes , with the majority of methylated ( > 80% ) regions showing less than 50% dna methylation in hira - depleted oocytes ( figure 6d ) . at the whole - genome level , 40.2% of 3-kb genomic windows displayed a statistically significant loss of dna methylation in hira gdf9-cre oocytes ( figure 6e ; chi - square test , p < 0.001 ) . this extensive reduction of dna methylation was observed across all types of genomic regions , including genic regions , cpg islands , maternally methylated imprint control regions , and repetitive elements ( figures 6f , 6 g , s7 g , and s7h ) . in agreement with previously published observations ( kobayashi et al . , 2012 , 2015 ) , we observed a positive correlation between transcription and the level of dna methylation in control oocytes ( figure 6h ) . although the methylome of hira gdf9-cre oocytes was severely reduced in comparison to control oocytes , they too retained a positive correlation between transcription and dna methylation across most of the transcriptional range ( figure 6h ) ; however , this correlation was abolished for highly expressed genes ( figure 6h ) . consequently , the effect on dna methylation in hira - deleted oocytes was more pronounced at highly expressed genes compared to median expressed genes ( figure s7i ) . surprisingly , beyond the observed relationship between transcription level and dna methylation described above , on average , dna methylation was reduced both at genes whose expression was unaffected by hira deletion and also at genes differentially expressed between control and hira gdf9-cre oocytes , regardless of whether they were up- or downregulated ( figure 6i ) . cumulatively , these observations suggest that , although de novo methylation is still targeted to transcribed regions in hira - depleted oocytes , this process appears to be inefficient , clearly indicating that transcription is not sufficient to ensure normal levels of de novo dna methylation in this system . considering the high levels of dnmt3a and dnmt3l in hira gdf9-cre oocytes ( figures s7c and s7d ) , we reasoned that the observed effect was likely caused either by inefficient recruitment or reduced enzymatic activity on chromatin of the dnmt3a/3l complex . we first asked whether chromatin association of dnmt3a is grossly altered following hira depletion in hira gdf9-cre oocytes . in agreement with our rna - seq and qpcr data , hira knockout oocytes contained higher levels of dnmt3a ( figures s7c , s7d , and s5c ) . importantly , dnmt3a remained associated with chromatin following triton pre - extraction of soluble proteins , confirming that altered chromatin structure in hira - deleted oocytes does not completely abolish recruitment of this enzyme ( figure s7j ) . in this context , the pwwp domain of dnmt3a has previously been shown to interact in vitro with h3k36me3 ( dhayalan et al . , 2010 ) . although the recognition of this histone modification by dnmt3b ( and not by dnmt3a ) has recently been linked to gene body dna methylation in mouse escs ( baubec et al . , 2015 ) , it is possible that reduced h3k36me3 levels observed in hira - depleted gv oocytes contribute to the observed loss of dna methylation in this system . in addition , using in vitro biochemical assays , it has recently been shown that the enzymatic activity of dnmt3a is specifically enhanced by binding to the n - terminal tail of histone h3 lacking methylation at lysine 4 ( h3k4me0 ) ( guo et al . , 2015 ) . despite the lack of h3.3/h4 replacement , growing hira gdf9-cre oocytes remained highly transcriptionally active ( figure 4a ) . it is , thus , conceivable that passage of the transcriptional machinery would result in histone h3 depletion and increased dna accessibility , both of which we observed ( figures 3a and 3c ) . although our developmental system precludes direct analysis of locus - specific histone turnover due to severely limited material , a very strong correlation had been observed previously between transcription levels and the degree of histone ( and specifically h3.3 ) replacement , a relationship that is conserved among various model organisms including mouse ( deal et al . , 2010 , kraushaar et al . , 2013 , dna methylation was most affected at genes with the highest level of transcription ( figures 6h and s7i ) . we also note that , while the combination of both compromised dnmt3a/3l recruitment and reduced enzymatic activity would explain the reduction of dna methylation in transcribed regions , reduced histone load is likely to underpin the methylation defect observed in other non - transcribed parts of the genome ( figure 6f ; guo et al . , 2015 ) . to address the biological significance of continuous histone replacement in a physiological context , we have generated a genetic deletion of the histone chaperone hira in the early stages of mouse oogenesis . developing mouse oocytes represent a unique experimental system as postnatal oocytes undergo developmental transitions , including major transcriptional changes and widespread de novo dna methylation , in the absence of dna replication . our results show that the chromatin of developing oocytes is highly dynamic , with histone turnover being observed also in the transcriptionally inert gv - stage oocytes ( figures 1c and 4a ) . we demonstrate that constant histone replacement is necessary for the maintenance of normal chromatin homeostasis in vivo . depletion of hira during early oocyte development leads to a severe reduction of histone load , compromised developmental progression , and progressive oocyte loss ( figures 2 , 3a , and s2 g ) . we note that , albeit pronounced , our observed phenotype is milder in comparison to the recently reported oocyte death in h3.3 knockout mice ( tang et al . , 2015 ) . we attribute this difference to the presence of an alternative atrx / daxx h3.3 chaperone complex , which , although present in our system , can not functionally replace hira - driven h3.3 deposition ( figures s2a s2c ) . our experiments document that , while lacking the normal ability to incorporate h3.3 and h4 , hira - deleted growing oocytes remain transcriptionally active , which results in chromatin with severely reduced histone content , increased dnase i sensitivity , and signs of dna damage ( figures 3 and s2b ) . reminiscent of the phenotype associated with hira depletion in yeast ( blackwell et al . , 2004 , greenall et al . , 2006 ) and h3.3 depletion in mice ( bush et al . , 2013 , lin et al . , 2013 ) and drosophila ( sakai et al . , 2009 ) , the compromised chromatin structure leads to chromosome segregation defects and aberrant first polar body extrusion ( figure 2d ) . however , the observed chromosome segregation defects in the hira - depleted oocytes are not linked with aberrant cenpa incorporation , as previously suggested in somatic cells ( figure s2c ; bush et al . , 2013 ) . non - replicative hira - depleted oocytes uniquely reveal the importance of histone replacement on transcriptional regulation in the absence of replication - coupled chromatin assembly . although transcription can continue from histone - depleted chromatin , our study shows that the lack of histone replacement has a major impact on the dynamic range of gene expression . in the context of histone - depleted chromatin additionally , the lack of normal histone occupancy leads to increased spurious transcription from otherwise not - transcribed regions of the genome , suggesting an evolutionarily conserved role for the hira histone chaperone complex ( anderson et al . , 2009 ) . our results additionally reveal an unexpected connection among continuous h3.3 replacement , transcription , and de novo dna methylation in developing oocytes . following hira depletion , more accessible chromatin with reduced histone load leads to significantly reduced dna methylation ( figures 6a6 g ) . we note that , although in other systems dna methylation changes result in pronounced transcriptional outcomes ( yang et al . , 2014 ) , reduced dna methylation is not likely to contribute to the observed transcriptional changes in hira - depleted oocytes , as dnmt3l knockout oocytes lacking dna methylation do not display any transcriptional phenotype ( kobayashi et al . , 2012 ) . our data suggest that the observed methylation phenotype can not be attributed to downregulation of the dnmt3a/3l complex , and , although we can not exclude locus - specific effects on dnmt3a/3l recruitment , the observed reduced dna methylation is likely due to altered enzymatic activity of dnmt3a in the absence of normal levels of chromatin - bound h3 ( figures 1c , 1e , and s2b ) . in this context , the n - terminal part of h3 has been shown in vitro to be required for allosteric activation of dnmt3a catalytic activity ( guo et al . , 2015 ) . our study thus provides the first support for this effect in an in vivo setting . the observed effect is more pronounced in highly expressed genes ( figure s7i ) , in agreement with the expected role of transcription in inducing nucleosome depletion in hira - depleted oocytes . furthermore , cpg island methylation is greatly reduced in hira - depleted oocytes ( figure 6 g ) and methylation loss is more pronounced at regions with high cpg density ( figure s7k ) . as the dnmt3a enzyme has been shown to operate in a non - processive manner ( dhayalan et al . , 2010 ) , stimulatory effect of the unmodified ( h3k4me0 ) h3 tail might be necessary to ensure a high level of methylation at these regions . we also considered that loss of hira early during oocyte development ( gdf9-cre - driven deletion ) might lead to a variety of secondary effects that could contribute to the observed phenotypes . in this context it is important to note that deletion of hira at a later time point during oogenesis ( zp3-cre system ) fully recapitulated the transcriptional and dna methylation defects observed in hira gdf9-cre oocytes ( figures 6a and s6 ) , providing further support for our findings and indicating that the observed effects are not solely attributable to compromised development of the oocytes . although we can not exclude that some of the effects could be secondary to alterations in gene expression , the most parsimonious view is that the abnormal chromatin resulting from impaired h3.3/h4 deposition is responsible for the majority of the phenotypes we observed . in addition , while it is important to consider that the observed transcriptional and methylation effects could be connected with the specific absence of the h3.3 variant upon hira depletion in oocytes ( santenard et al . , 2010 ) , it is likely that the lack of normal chromatin structure due to the inability to continuously replace h3/h4 is the main cause underlying the described phenotype . consistent with this view , the pronounced transcriptional phenotype observed in hira - depleted oocytes is in stark contrast to the only limited transcriptional changes previously described in hira knockout escs ( banaszynski et al . , 2013 , goldberg et al . , 2010 ) . in that case , replication - dependent deposition of canonical h3 by the caf1 complex was likely to compensate in the absence of hira - mediated h3.3 deposition in rapidly dividing escs ( banaszynski et al . indeed , h3.3 depletion does not lead to pronounced changes in nucleosome occupancy in proliferating escs ( banaszynski et al . , in contrast , our findings document that post - replicative cells in vivo require continuous histone replacement in order to maintain an intact chromatin structure , which in turn is required for normal regulation of transcription and for de novo dna methylation in the context of developing oocytes ( figure 7 ) . interestingly , the critical role of continuous histone replacement also has been demonstrated recently in post - mitotic neurons , where the lack of h3.3 incorporation had an impact on both transcription and physiological function of neurons ( maze et al collectively , our studies thus provide new insights into the histone dynamics in vivo and highlight the importance of studying chromatin in a physiological context and in non - proliferating post - mitotic cells . oocyte - specific hira depletion was achieved by crossing hira mice with gdf9-icre or with zp3-cre mice , respectively . the h3.3b - egfp knockin strain was generated in the mrc csc transgenic facility and protamine 1-egfp strain was generated by dr . all animal experiments were carried out under a uk home office project license in a home office - designated facility . detailed information is available in the supplemental experimental procedures . in vitro fertilization ( ivf ) , in vitro maturation ( ivm ) , parthenogenesis , mrna microinjection , tunel assay , if , and h&e staining were carried out as described previously ( hajkova et al . , 2010 , the scrna - seq was performed on hira , hira gdf9-cre , and hira zp3-cre mii oocytes using the smarter ultra low input rna kit ( clontech laboratories ) with ercc rna spike - in mix 1 ( life technologies ) . detailed information is available in the supplemental experimental procedures . an scbs - seq described previously ( smallwood et al . , 2014 ) was used to profile the dna methylation landscape of hira and hira gdf9-cre oocytes . ultra - sensitive lc - ms was used to determine overall 5-methylcytosine level . for detailed information ,

summarythe integrity of chromatin , which provides a dynamic template for all dna - related processes in eukaryotes , is maintained through replication - dependent and -independent assembly pathways . to address the role of histone deposition in the absence of dna replication , we deleted the h3.3 chaperone hira in developing mouse oocytes . we show that chromatin of non - replicative developing oocytes is dynamic and that lack of continuous h3.3/h4 deposition alters chromatin structure , resulting in increased dnase i sensitivity , the accumulation of dna damage , and a severe fertility phenotype . on the molecular level , abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression , the appearance of spurious transcripts , and inefficient de novo dna methylation . our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non - replicative system in vivo .

Introduction Results Discussion Experimental Procedures Author Contributions

the integrity of the chromatin template is constantly compromised by fundamental biological processes , such as dna replication , repair , and transcription , following which the normal chromatin structure is restored with the help of histone chaperone proteins ( gurard - levin et al . advances in recent years have shed light on some of the molecular players involved in chromatin assembly and maintenance , separating ( on the molecular level ) dna replication - dependent and -independent pathways ( burgess and zhang , 2013 ) . , 2010 ) , studies that have attempted to dissect the importance of histone replacement in the interphase nucleus have revealed only a limited contribution of histone chaperones and/or variants to transcriptional regulation ( banaszynski et al . over an extended time span and in the absence of dna replication , postnatal mammalian oocytes execute the oogenesis - specific developmental program , involving widespread transcriptional changes and de novo dna methylation , ultimately acquiring the competencies required for fertilization and embryogenesis ( de la fuente , 2006 , li and albertini , 2013 , tomizawa et al . to address the importance of histone turnover during this process , we have generated a mouse oocyte - specific knockout of the histone chaperone hira . as opposed to caf1 , which is implicated in the replication - coupled deposition of canonical histones h3.1 and h3.2 , hira has been shown to deposit the h3.3 variant during replication - independent chromatin assembly ( ray - gallet et al . , 2002 , our findings show that depletion of hira in primordial oocytes causes a severe developmental defect associated with extensive oocyte death . on the molecular level this causes significantly increased dna accessibility , accumulation of dna damage , and chromosome segregation defects . the lack of normal chromatin structure has a striking impact on transcriptional regulation : lack of normal h3.3/h4 replacement prevents the oocytes from maintaining full dynamic range of gene expression , and it leads to compromised transcriptional transitions normally associated with the oocyte development . furthermore , we show that histone replacement is necessary for silencing of spurious transcription and , in the context of the oocyte , also for the efficient deposition of de novo dna methylation . our results thus uniquely demonstrate the critical relationship between continuous histone replacement , the structural integrity of the chromatin template , the normal regulation of transcription , and the establishment of dna methylation , in the context of an in vivo developmental system . this process involves the following : ( 1 ) pronounced transcriptional changes as the developing oocytes progress from primordial to large antral follicle stages ; ( 2 ) the deposition of de novo dna methylation starting in the secondary follicle stage ; ( 3 ) the silencing of transcription in the germinal vesicle ( gv ) stage ; and , finally , ( 4 ) the condensation of chromosomes in the mii stage , resulting in an oocyte that is competent to undergo fertilization and support early embryonic development ( figure 1a ) . however , we also detected a high level of chromatin incorporation for flag - tagged h2a.x , h4 , and h3.3 upon injection of tagged histone mrna into transcriptionally inactive gv - stage oocytes ( figure 1c ) , demonstrating that the chromatin in the mature gv oocyte is still dynamic even after global transcription has ceased . previous studies have shown that the majority of h3.3 incorporation is critically dependent on the presence of histone chaperone hira ( goldberg et al . to investigate whether hira is specifically required for the observed h3.3 incorporation during oogenesis , we took advantage of gdf9- and zp3-driven expression of cre recombinase and deleted hira in the primordial and primary stages of follicle development , respectively ( figures 1a and s1a ) . the absence of hira severely decreased h3.3 content and completely abolished the incorporation of microinjected tagged h3.3 in both growing and gv - stage oocytes ( figures 1b , 1c , and 1e ) . , , the presence and the localization of this complex in the gv oocyte were not affected by hira deletion ( figure s2a ) , and we could observe h3.3b - egfp loci overlapping with atrx staining in hira gdf9-cre , h3.3b - egfp gv oocytes at higher laser detection intensity ( figure s2c ) . the lack of hira and h3.3 incorporation also was not compensated for by incorporation of the canonical histones h3.1 or h3.2 ( figure 1b ) , and it coincided with the lack of detectable incorporation of histone h4 ( figure 1c ) . taken together , these findings demonstrate that the histone variant h3.3 , rather than h3.1 or h3.2 , is actively incorporated into chromatin during oocyte development , and confirm that hira is responsible for the incorporation of the majority of h3.3 in conjunction with histone h4 ( figures 1b1e and s2b ) . significantly lower numbers of mii oocytes could be recovered following superovulation ( figure 2a ) ; zp3-cre - driven deletion of hira led to a more than 50% reduction in the number of ovulated mii oocytes , and this effect was even more dramatic upon earlier gdf9-cre - driven deletion ( figure 2a ) . hira gdf9-cre ovaries were significantly smaller ( figures 2b and s2f ) ; and , although we could recover some gv oocytes from 3-week - old females , the ovaries of older mice contained only very few oocytes , which were predominantly at early developmental stages ( figures 2c and s2 g ) . in agreement with the observed enrichment of h3.3 and hira in the paternal pronucleus of control zygotes ( figures s3a s3c ) , fertilized hira gdf9-cre oocytes could not fully de - condense the male pronucleus ( figure s3d ) , incorporate h3.3 ( figure s3e ) , or support normal zygotic development ( figure s3f ) . although the histone load of the paternal pronucleus was severely reduced upon maternal hira deletion , we could detect a residual amount of histones on the paternal dna by immunofluourescent staining ( figure s4b ) . in agreement with the severe chromatinization defect , the partially decondensed paternal pronucleus of zygotes in which hira had been maternally deleted failed to undergo dna demethylation ( figure s4c ) and could not provide a normal template for zygotic replication or transcription ( figures s4d and s4e ; lin et al . we hypothesized that , in the absence of h3/h4 incorporation , the chromatin structure in hira - depleted oocytes would be severely affected . in agreement with this , the histone content of hira - depleted gv - stage oocytes was severely reduced ( figures 3a and s2b ) , and the resulting chromatin displayed altered structure and increased dnase i sensitivity ( figures 3b and 3c ) . moreover , in accordance with the predicted role of histones in protecting genetic material from environmental stress and mutagenesis ( ljungman and hanawalt , 1992 ) , hira - depleted oocytes showed clear signs of dna damage both by increased levels of -h2ax ( figure 3d ) and upregulation of dna damage - responsive genes ( figure 3e ) . the importance of hira and continuous h3.3 replacement has been previously assessed in the context of proliferating mouse pluripotent escs ( banaszynski et al . the severity of the phenotype observed following the deletion of hira in oocytes , we next asked whether the reduced h3/h4 incorporation and increased dna accessibility ( figures 1b1e , 3a , and 3c ) affected the transcriptional program of the hira - depleted oocytes . to address the role of hira in transcription during oocyte development in depth , we carried out single - cell rna sequencing ( scrna - seq ) on four hira gdf9-cre and four hira control oocytes . furthermore , differentially down- and upregulated genes showed higher and lower than expected expression , respectively ( figure 4h ) ; and , while the genes normally not expressed or expressed at very low levels ( bottom 20% ) were upregulated upon hira deletion ( figure 4i ; gene set enrichment analysis [ gsea ] , fdr < 0.001 ; data not shown ) , genes with very high expression in normal oocytes ( top 20% ) were specifically downregulated in hira gdf9-cre oocytes ( figure 4i ; gsea , fdr < this also was evident by the greatly reduced variance in expression levels of annotated transcripts within the hira - depleted oocytes ( figure 4j ; p value < 0.001 ) , suggesting that hira is necessary to maintain the full dynamic range of gene expression . remarkably , a nearly identical transcriptional phenotype was observed in hira zp3-cre and hira gdf9-cre oocytes : differentially down- and upregulated genes showed higher and lower than average expression in the control oocytes , respectively ( figure s6e ) , and genes normally not expressed or expressed at very low levels ( bottom 20% ) were upregulated upon hira deletion ( figure s6f ) , while genes with very high expression in normal oocytes ( top 20% ) were specifically downregulated in hira gdf9-cre oocytes ( figure s6f ) . the developing oocyte is a unique experimental system , as it allows us to assess the importance of continuing histone h3.3/h4 replacement not only for steady - state transcription , but also for affecting transcriptional transitions associated with development , such as activation or repression of dynamically regulated genes . to confirm that these observations were not an artifact of the reduced dynamic range observed in the hira - depleted mii oocytes , we carried out a similar gsea analysis on only median expressed genes ( i.e. cluster 2 ( genes progressively downregulated during oogenesis ) remained enriched among upregulated genes in hira gdf9-cre oocytes ( gsea , fdr = 0.08 ; data not shown ) ; similarly , cluster 4 ( genes upregulated in early oogenesis ) still showed enrichment ( although this was less pronounced ) among genes downregulated in the absence of hira ( gsea , fdr = 0.26 ; data not shown ) . given the same observed reduction of the dynamic range of transcription in both systems ( figures 4 g , 4j , and s6 g ) , we hypothesized that , if normal transcriptional activation was dependent on hira , genes that are specifically upregulated between primordial ( when gdf9-driven cre is first expressed ) and primary ( when zp3-driven cre is first expressed ) stage oocytes should show higher expression in hira zp3-cre mii oocytes . taken together , our data bring to light the critical role of hira ( and h3.3/h4 turnover ) for affecting changes to the transcriptional state . histone h3.3/h4 replacement is required for normal transcriptional activation of genes associated with developmental progression of oocytes , and , surprisingly , it is also necessary for genes to become or to remain transcriptionally silent . considering the failure of hira - deleted oocytes to silence genes , we asked whether the increased dna accessibility in the absence of continuous h3.3/h4 replacement may lead to aberrant transcription from regions not normally transcribed within the genome . progression through oocyte development is accompanied by the accumulation of dna methylation . during this process , the largely hypomethylated genome of primordial oocytes acquires dna methylation through the activity of the de novo dna methyltransferase dnmt3a and its co - factor dnmt3l ( kaneda et al . acquisition of dna methylation in oocytes is known to be positively correlated with transcription , which could relate to the recruitment of dnmt3a/3l to gene bodies through binding of its pwwp domain to h3k36me3 , a histone modification associated with transcriptional elongation ( chotalia et al . , 2014 ) , dnmt3a is responsible for the accumulation of oocyte dna methylation as dnmt3b is absent in growing oocytes ( kaneda et al . given the widespread transcriptional changes observed in the hira knockout oocytes , we set out to examine the potential impact of hira deletion on de novo dna methylation in the oocyte . importantly , the reduction of dna methylation occurred despite the transcriptional upregulation of dnmt3a and without statistically significant changes in the expression of other dna methyltransferases ( figures s5c , s7c , and s7d ) . to further understand the relationship among hira deletion , transcriptional changes , and global changes in dna methylation , we profiled dna methylation genome - wide by bisulphite sequencing . at the whole - genome level , 40.2% of 3-kb genomic windows displayed a statistically significant loss of dna methylation in hira gdf9-cre oocytes ( figure 6e ; chi - square test , p < 0.001 ) . this extensive reduction of dna methylation was observed across all types of genomic regions , including genic regions , cpg islands , maternally methylated imprint control regions , and repetitive elements ( figures 6f , 6 g , s7 g , and s7h ) . , 2012 , 2015 ) , we observed a positive correlation between transcription and the level of dna methylation in control oocytes ( figure 6h ) . cumulatively , these observations suggest that , although de novo methylation is still targeted to transcribed regions in hira - depleted oocytes , this process appears to be inefficient , clearly indicating that transcription is not sufficient to ensure normal levels of de novo dna methylation in this system . although our developmental system precludes direct analysis of locus - specific histone turnover due to severely limited material , a very strong correlation had been observed previously between transcription levels and the degree of histone ( and specifically h3.3 ) replacement , a relationship that is conserved among various model organisms including mouse ( deal et al . we also note that , while the combination of both compromised dnmt3a/3l recruitment and reduced enzymatic activity would explain the reduction of dna methylation in transcribed regions , reduced histone load is likely to underpin the methylation defect observed in other non - transcribed parts of the genome ( figure 6f ; guo et al . to address the biological significance of continuous histone replacement in a physiological context , we have generated a genetic deletion of the histone chaperone hira in the early stages of mouse oogenesis . developing mouse oocytes represent a unique experimental system as postnatal oocytes undergo developmental transitions , including major transcriptional changes and widespread de novo dna methylation , in the absence of dna replication . our results show that the chromatin of developing oocytes is highly dynamic , with histone turnover being observed also in the transcriptionally inert gv - stage oocytes ( figures 1c and 4a ) . we demonstrate that constant histone replacement is necessary for the maintenance of normal chromatin homeostasis in vivo . depletion of hira during early oocyte development leads to a severe reduction of histone load , compromised developmental progression , and progressive oocyte loss ( figures 2 , 3a , and s2 g ) . we attribute this difference to the presence of an alternative atrx / daxx h3.3 chaperone complex , which , although present in our system , can not functionally replace hira - driven h3.3 deposition ( figures s2a s2c ) . our experiments document that , while lacking the normal ability to incorporate h3.3 and h4 , hira - deleted growing oocytes remain transcriptionally active , which results in chromatin with severely reduced histone content , increased dnase i sensitivity , and signs of dna damage ( figures 3 and s2b ) . , 2009 ) , the compromised chromatin structure leads to chromosome segregation defects and aberrant first polar body extrusion ( figure 2d ) . non - replicative hira - depleted oocytes uniquely reveal the importance of histone replacement on transcriptional regulation in the absence of replication - coupled chromatin assembly . although transcription can continue from histone - depleted chromatin , our study shows that the lack of histone replacement has a major impact on the dynamic range of gene expression . in the context of histone - depleted chromatin additionally , the lack of normal histone occupancy leads to increased spurious transcription from otherwise not - transcribed regions of the genome , suggesting an evolutionarily conserved role for the hira histone chaperone complex ( anderson et al . our results additionally reveal an unexpected connection among continuous h3.3 replacement , transcription , and de novo dna methylation in developing oocytes . our data suggest that the observed methylation phenotype can not be attributed to downregulation of the dnmt3a/3l complex , and , although we can not exclude locus - specific effects on dnmt3a/3l recruitment , the observed reduced dna methylation is likely due to altered enzymatic activity of dnmt3a in the absence of normal levels of chromatin - bound h3 ( figures 1c , 1e , and s2b ) . our study thus provides the first support for this effect in an in vivo setting . as the dnmt3a enzyme has been shown to operate in a non - processive manner ( dhayalan et al . although we can not exclude that some of the effects could be secondary to alterations in gene expression , the most parsimonious view is that the abnormal chromatin resulting from impaired h3.3/h4 deposition is responsible for the majority of the phenotypes we observed . in addition , while it is important to consider that the observed transcriptional and methylation effects could be connected with the specific absence of the h3.3 variant upon hira depletion in oocytes ( santenard et al . in that case , replication - dependent deposition of canonical h3 by the caf1 complex was likely to compensate in the absence of hira - mediated h3.3 deposition in rapidly dividing escs ( banaszynski et al . , in contrast , our findings document that post - replicative cells in vivo require continuous histone replacement in order to maintain an intact chromatin structure , which in turn is required for normal regulation of transcription and for de novo dna methylation in the context of developing oocytes ( figure 7 ) . interestingly , the critical role of continuous histone replacement also has been demonstrated recently in post - mitotic neurons , where the lack of h3.3 incorporation had an impact on both transcription and physiological function of neurons ( maze et al collectively , our studies thus provide new insights into the histone dynamics in vivo and highlight the importance of studying chromatin in a physiological context and in non - proliferating post - mitotic cells .

children 's development of emotional self - regulation is important for many aspects of their health and wellbeing , including their ability to tolerate frustration , curb aggressive impulses , delay gratification , and express emotions in socially acceptable ways . children who are able to regulate their own emotions are better able to interact with their peers , whereas poor emotion regulation in preschool - aged children has been related to higher levels of externalizing behaviors [ 68 ] . children 's deficits in the ability to self - regulate their own behavior have been linked to rapid weight gain and obesity in middle childhood . there is a strong body of evidence to support that parents play an important role in children 's development of self - regulation of emotions in the early years [ 7 , 10 , 11 ] . at birth , instead , infants ' emotional arousal is regulated by their own biological needs and how parents respond to those needs . parents comfort infants when they express negative emotions as well as arouse positive emotions in their infants through play and other stimulating interactions . for example , if a one - month - old infant experiences an aversive stimulus , the infant 's crying signals to the parent that he or she is upset . parents ' actions such as calming or soothing the infant serve to regulate the child 's emotions . therefore , young infants rely heavily on their parents to regulate their emotions . as children age , they require less assistance in regulating emotions . for example , a four - year - old might self - sooth in response to an aversive stimulus instead of immediately crying . children 's self - regulation of energy intake refers to children 's ability ( inborn and socialized ) to eat and not eat in response to cues of hunger and satiety . the development of self - regulation of energy intake in childhood is important for many aspects of health and wellbeing . notably , overweight children have been found to have deficits in self - regulation of energy intake compared to normal weight peers [ 14 , 15 ] . researchers have demonstrated that preschool children are capable of self - regulation of energy intake [ 16 , 17 ] . at the same time , large individual differences in children 's self - regulation of energy intake have been found . temple and colleagues found food to be more reinforcing for overweight children than for normal weight children . additionally , fmri studies have demonstrated greater anticipatory and consummatory reward responses in brain regions of overweight adolescents compared to normal weight adolescents . to the extent that individuals are motivated by external cues , they may be less sensitive to internal signals of hunger and less able to self - regulate their energy intake in the presence of external food cues . given that socialized ( non - inborn ) aspects of self - regulation of energy intake develop in the context of parent - child interactions during feeding with parents , parents are likely to play an important role in the development of such individual differences . a small but growing body of research is indicating that this is the case ; individual differences in parents ' feeding practices have been linked to individual differences in children 's self - regulation of energy intake [ 2024 ] . at the same time , there are gaps in our knowledge of how parents influence children 's development of self - regulation of energy intake . there is a large body of research on parental influences around emotion regulation , and this represents an exemplary model system for understanding parenting influences on children 's development of self - regulation of energy intake . thus , the literature on how parents influence children 's emotion regulation can provide useful information for child obesity researchers concerning how parents may contribute to an obesogenic environment by influencing the development of children 's self - regulation of energy intake . the purpose of this paper is to examine the literature on children 's early development of emotion regulation , and in particular , parents ' role in this development , to provide insight into the developmental processes by which parents influence children 's self - regulation of energy intake . throughout the first year of life , infants gradually increase their ability to control their own emotional states [ 25 , 26 ] and they begin to self - regulate emotions before the second half - year of life . individual differences in children 's abilities to regulate their emotions are apparent by this time , and whether they intend to or not , parents substantially influence these individual differences . power indentified some of the ways that parents can help children regulate their emotions . these parental techniques are relevant as infants progress through toddlerhood into their preschool years and beyond . they include ( 1 ) modeling emotion regulation or specific emotion regulation strategies ; ( 2 ) responding to their children 's emotional expression by acknowledging emotions , helping children process their emotions , helping children calm down , or questioning / punishing emotional expression ; ( 3 ) assisting children in the moment by teaching them various emotion regulation strategies ( e.g. , attend to relevant stimuli , seek out appropriate information , analyze the situation , generate , and evaluate alternatives ) and ( 4 ) motivating children through various social and material rewards and punishments . this line of research on parental influences on children 's self - regulation of emotions provides a useful framework that researchers interested in parental influences on children 's self - regulation around food can utilize . for example , children experience both stress and hunger in the presence of their parents . parents ' reactions to children in these situations influence how children react to future experiences ; therefore , how parents interact with their children influences children 's self - regulation of both emotions and eating . the following discussion of each of these parenting processes shows how the literature on parental influences on children 's emotional regulation can inform our understanding of the impact of parents ' behaviors on children 's self - regulation of energy intake . parents may not be aware of it , but they model behaviors around both emotional expressivity and food intake beginning when their children are infants . the literature on self - regulation of emotions indicates that parents provide very important models by which children learn to express emotions and later learn to control emotional expressivity . for example , researchers have found that infants in their first half - year of life mimic the emotions of their parents [ 2 , 6 , 29 ] . when parents display a wide range of positive and negative emotions in appropriate social contexts , their children are more likely to learn which emotions are appropriate to display in which situations ; conversely , when parents display high levels of anger or personal distress , children are less likely to observe and learn appropriate ways to regulate and express their negative emotions . similarly , parents can provide both positive and negative models of self - regulation of energy intake through their own eating practices . for example , researchers have found that parents report that they often eat foods during meals that they would like their child to eat . however , parents ' modeling behaviors may either be conducive or not conducive to children developing good self - regulation of energy intake . after being asked if they want seconds , parents who respond by saying that they are full provide a positive model to their child . however , parents who say they are stuffed but then ask for dessert may provide negative models . to this point , johnson and birch found parental self - report of disinhibited eating was correlated with children 's lessened ability to self - regulate energy intake . it may be that parents who report disinhibited eating are providing negative models of self - regulation of energy intake , thereby setting forth the pathway to poor self - regulation of energy intake in their children . although , to our knowledge , most of the literature on modeling and energy - intake deals with food acceptance , and little - to - no work has been conducted on parent modeling and self - regulation of energy intake with children . some research has been conducted on peer modeling and energy intake in children . notably , salvy et al . found differences in the amount of food consumed by overweight and normal weight children when alone and with peers . overweight children consumed more when in solitude than when in the presence of peers . as discussed by the authors , one possible reason for the lessened amount consumed in the presence of peers for overweight children is that normal weight children were modeling lower energy intake amounts and overweight children modified their intake amounts accordingly when in the presence of normal weight peers . researchers interested in understanding parental contributions to children 's self - regulation of energy intake should further examine how parents model behaviors around their own regulation of energy intake and the influence of these parental behaviors on children . research in the area of emotional expressivity indicates that the specific ways in which parents respond to children 's emotions , beginning in infancy and continuing throughout early childhood , are influential in shaping children 's self - regulation of emotions . parents ' acceptance of positive and negative emotions is important for optimal development of emotional expression and emotional regulation because children whose parents are accepting of their negative and positive emotions feel free to express both types of emotions when distressed and are better able to eventually express these emotions in socially accepted ways [ 7 , 33 , 34 ] . parental responses to their children 's negative emotions should also be contingent on children 's behavior , as well as appropriate given the child 's behavior , age , and needs ( see black and aboud for review of responsive parenting ) [ 3538 ] . in contrast , the parental practice of over - control around emotions occurs when parents respond in punitive , distressed , or minimizing ways to children 's expressions of negative emotions [ 13 , 14 ] . stop that crying or i will give you something to cry about . minimizing responses are those that fail to validate the child 's emotions by belittling them , for example , oh , you 're being a baby . distressed responses are those in which parents respond to their children 's negative emotions by displaying distressed negative emotions themselves ( e.g. , a frustrated or angry vocal tone ) . all of these responses show a lack of acceptance of the child 's emotion and do not help the child learn to deal with that emotion in a productive way . parental over - restriction of emotional expressivity has been linked to children 's lessened capacity to regulate their own emotions and thus to undesirable outcomes for children . research has indicated that negative parent reactions to children 's negative emotions can have one of two possible outcomes . first , they can ultimately result in greater negative emotional expressivity in children since the children become frustrated with having to continually suppress their emotions [ 11 , 40 ] . for example , researchers have found that school - aged children whose parents react in more punitive ways to their negative emotions have deficits in their ability to regulate emotion and express higher levels of externalizing behaviors based on teacher reports [ 68 ] . the second possible outcome is that some children succeed in suppressing their negative emotions , leading to flat emotional expressivity ( suppression of both negative and positive emotions ) , and possibly to later internalizing problems . similarly , parental responses to children 's expressions of hunger and satiety cues are likely to be important influences in shaping their children 's self - regulation of energy intake . parents can respond to children 's cues in ways that encourage optimal self - regulation of energy intake ( e.g. , letting children leave the table when they say they are full , encouraging children to reflect on how full they are before they are served some more , encouraging children to slow down the rate of eating ) or in ways that interfere with self - regulation of energy intake ( e.g. , refusing to believe children 's statements about fullness and encouraging them to eat more ) . just as parental restriction of children 's emotional expressivity has been thought to interfere with children 's ability to self - regulate their own emotions , parental restriction around food has been linked to deficits in self - regulation of energy intake in children . parental restriction around food occurs when parents overly restrict the quantities and types of foods available for the child to eat . findings from a laboratory assessment of satiety in children suggest that children whose parents reported more restrictive and controlling feeding behaviors ate more beyond satiety compared to their peers [ 42 , 43 ] . thus , researchers interested in the development of children 's self - regulation of energy intake should examine over - restriction of foods parents make available to children as well as overcontrol of the amount of food children can eat as a possible mechanism for children developing less than optimal self - regulation of energy intake . additionally , researchers should further look into different types of control that parents use ( i.e. , overt control and covert control ) and the reasons why parents restrict foods ( i.e. , restriction for health and restriction for weight ) . children who experience too much parental control around eating may not learn to identify their own satiety cues and thus may have poor abilities to self - regulate their energy intake . to this point , researchers have found associations between controlling parent feeding practices ( as assessed by survey measures ) and children 's lessened ability to self - regulate energy intake . thus , as parental overcontrol of young children 's emotional expressivity results in children not learning adaptive ways of expressing emotions ( either over- or underregulating their emotions ) , parental over - restrictiveness around food may diminish children 's ability to read their own hunger and satiety cues and respond with appropriate self - regulation of energy intake . some children may thus overeat when parents are not present to monitor their eating , and others may undereat . in addition to affecting the development of their children 's abilities to self - regulate energy intake through controlling their children 's food intake or access to certain foods , parents may also affect their children 's energy intake through the ways in which they respond to children 's emotions surrounding food . this can elicit frustration , sadness , and even anger from children , who , in turn , can also frustrate and anger the parents , altering the emotional context of the meal altogether . this is an important process for researchers interested in self - regulation of energy intake to better understand because parental responses to children 's negative emotions around food have the potential to influence the child 's view of that particular food or the process of eating in general . for example , if a parent responds with anger or frustration to the child 's refusal to eat spinach , this response may serve to increase the emotionally negative quality of spinach for the child . similarly , if having sweets is always associated with emotionally happy occasions like holidays or birthdays , this would increase the emotionally positive quality of sweets . according to gottman et al . , parents who engage in emotion coaching are highly aware of their children 's emotions , they are accepting of their children 's emotions ( positive and negative ) and assist them in the process of understanding their emotions and expressing their emotions in socially appropriate ways . a parent might engage in emotion - coaching by saying , i can see you are angry and it is ok to be angry . you can not scream in here . take a deep breath and use your words to tell me what is wrong . these techniques used by parents have been associated with better self - regulation of emotions in children . these findings have great potential to inform researchers interested in understanding parental influences on children 's ability to self - regulate energy intake . parents can assist children in the process of self - regulation of energy intake by helping them attend to relevant stimuli ( e.g. , appropriate portion size , internal fullness , or hunger cues ) , encouraging them to seek out appropriate information ( e.g. , reading and understanding food labels ) , and generating and evaluating alternatives ( e.g. , considering options when they are hungry for a snack ) . theoretically , parents can engage in intake - coaching by understanding and accepting their children 's cues of hunger and satiety and assisting their children with making appropriate choices about the types and amounts of foods that they consume . for example , if choosing from a children 's menu , parents can present their children with a few acceptable choices instead of the entire menu . additionally , given that portion sizes have increased in recent years , parents can control the amount of food presented to children when out at restaurants by modifying portions on their children 's plates before they begin eating . this can be accomplished through splitting large portioned meals between two children or packaging some food as leftovers before beginning a meal . researchers should investigate whether these parental practices of intake - coaching , a concept similar to scaffolding energy intake , foster better self - regulation of energy intake in children . as noted above , punitive parental responses to children 's expression of negative emotion have negative consequences for children 's emotion regulation . much of the time , parents respond to children 's emotions with no conscious intent to socialize them , such as when they show frustration and distress themselves when their children cry . often , however , parents consciously try to motivate their children to suppress their display of negative emotions by threatening punishment or offering rewards . however , when this is a common practice , children are less likely to learn how to productively deal with their negative emotions [ 11 , 40 ] . similarly , some parents may try to motivate self - regulation of energy intake through external rewards and punishments . experiments have indicated better ability to self - regulate energy intake in children who have been encouraged by caregivers to pay attention to internal cues of hunger and satiety rather than external cues such as rewards . rewards tend to increase the amount of a food consumed , however , evidence suggests that they might also undermine children 's natural intrinsic motivation to eat that particular food ( see cooke and colleagues for a review ) . birch and colleagues found that children who were instructed to finish their meals before receiving a reward consumed more after preload than their same - aged peers who had been instructed to pay attention to internal cues of fullness . their experiment was meant to simulate the real - life condition in which parents require children to clean their plate before engaging in a desired behavior . jimmy , you can go play with your sister once you finish your chicken and broccoli . it has been theorized that contingency - based parent behaviors such as this impair children 's ability to self - regulate their energy intake . punishments are likely to be counterproductive because they are likely to override sensitivity to internal cues of fullness and hunger . for example , some children receive a lollipop as a reward for enduring a shot at the doctor 's office . although this is a mundane example , it is a demonstration of how two things that are not at all related ( emotional / physical pain and candy ) can become related over time . later in life , people who find comfort in food because of childhood experiences , might be more likely to turn to a pint of ice cream to cope with a breakup or a candy bar as a pick - me - up after getting reprimanded by their boss . although parental reports of providing food as comfort have not been associated with overweight status in children [ 50 , 51 ] , high parental use of this technique has the potential of teaching children to rely on food to cope with difficult emotions or stress and may influence energy intake over time and should be further investigated by researchers . additionally , parents may be less likely to admit using food to comfort their children on self - report measures or may not even be aware if they do this often . studies examining parental influences on children 's emotion regulation have examined the role of numerous sources of variation that relate to parent - child interaction patterns relevant to children 's development of healthy patterns of emotional self - regulation . these sources of variation have included variations in parenting ( e.g. , parenting style ) , variations in children ( e.g. , the child 's inborn temperament ) , and variations in the parent - child relationship ( e.g. , child - parent attachment security ) . we next explore implications of these sources of variation for the development of children 's energy intake . parenting styles are relevant to self - regulation of both emotions and eating because , as rhee suggested , they can be looked at as the although parenting styles were first conceptualized by baumrind , scholars further elaborated these styles using dimensions of demandingness and responsiveness . parents with an authoritative style are highly demanding and highly responsive , those with an authoritarian style are highly demanding and low in responsiveness , those with an indulgent style are highly responsive but low in demandingness , and those with an uninvolved style are low in both . the authoritative style presents an optimal context for fostering children 's self - regulation in many situations since authoritative parents are more likely than other parents to model positive and socially appropriate emotional responses to frustrating situations and to provide adaptive emotional coaching . in contrast , authoritarian parents are more likely than other parents to model undercontrolled , angry emotions in frustrating situations and to respond in a punitive fashion to their children 's expression of negative emotions . these parenting styles have also been hypothesized to be relevant for explaining differences in children 's self - regulation of energy intake . rhee suggested that researchers look at feeding behaviors that authoritative parents engage in because these are likely the most effective for children 's health and well - being . authoritative parents might require that their children finish their meatloaf and peas before playing with their toys but give into their children 's needs when he or she expresses that they can not eat any more food ; for example , they might change their request such that their child eat some of the meatloaf and peas rather than all . this would be conducive to children developing good self - regulation around eating because it would encourage them to pay attention to their own internal cues of hunger and fullness . additionally , parents responding to children 's cues of hunger and fullness should empower children to self - regulate energy intake instead of energy intake being regulated solely by parents . in contrast , it is unlikely that authoritarian parents would be responsive to children 's negative emotions around food or children 's requests to eat or not eat due to feelings of hunger or fullness . given what is known in the literature related to emotion regulation , parental lack of responsiveness to children 's cues of hunger and satiety paired with parents being demanding might result in a flattening of these cues and , as a result , be detrimental to self - regulation around eating in children of authoritarian parents . in support of these ideas , findings of a study that examined the relation of the four parenting styles to the overweight status of first - grade children indicated that children with authoritarian mothers were at the highest risk for being obese , whereas children with authoritative mothers were at the least risk . similarly , adolescents with authoritative parents were significantly more likely to consume fruit and to have positive attitudes toward fruit consumption than those with authoritarian parents . it is important for researchers to look further into the relation between parenting style and children 's overweight status to examine whether children 's self - regulation of energy intake does in fact mediate this association . researchers have found some interesting differences across ethnicities in regards to how parenting styles relate to child outcomes . for example , in contrast with the typical research findings that children of authoritative parents have better outcomes , tamis - lemonda et al . found that authoritarian parenting styles were associated with positive child outcomes in low - income african - american families . given that this research was done among only one economic strata of african - american families , researchers do not yet know if these findings are due to differences in culture , income , or both . hughes and colleagues found a similar relationship between feeding styles and weight status ; their studies suggest a negative relationship between authoritarian feeding styles and weight status in low - income , ethnically diverse samples [ 59 , 60 ] . understanding findings across low income samples of children is especially important given that , in the united states , people of low ses have a higher rate of obesity than people who are not low ses . highly permissive parenting ( including both indulgent and uninvolved ) has also been linked with young children 's poor emotion regulation and aggressive behavior , indicating that parents ' sensitive responsiveness alone is not sufficient for children 's development of optimal emotion regulation . permissive parents may be less likely than authoritarian parents to scaffold their children 's development of healthy emotional regulation by modeling appropriate emotional expressivity and providing emotion - coaching . similarly , they may be less likely to provide children with the motivation and guidance needed to help them develop effective self - regulation of energy intake . in support of this idea , rhee and colleagues found that indulgent and uninvolved parents were twice as likely as authoritative parents to have overweight children , though these children were at less risk than those with authoritarian parents . however , hughes and colleagues found in a low - income sample , parents ' use of indulgent feeding styles actually predicted the greatest risk for childhood obesity , even greater than the authoritarian feeding styles . parents who engage in indulgent feeding place few demands on their children 's eating behavior , although the few demands they do make are nondirective and supportive . it is possible that these children lack the scaffolding provided by authoritative and authoritarian parents to help teach self - regulation of energy intake . again , research on whether or not self - regulation of energy intake and low parental scaffolding mediate the relationship between indulgent parenting / feeding and higher child bmi is needed . hughes and colleagues did not find differences in children 's energy intake as a function of parents having an uninvolved style of parenting . the authors reasoned that uninvolved parents should be unlikely to foster self - regulated eating habits in their children ; although not measured in their study , they also speculated that uninvolved parents may provide a generally poor diet for their children . future research on the feeding patterns of uninvolved parents is needed . self - regulation of emotions and energy intake are both likely to be influenced by children 's inborn traits . although temperament is defined as a stable and enduring trait , the behavioral manifestations of infants ' temperament are modified over time as a consequence of repeated interactions with parents and others . thus , some researchers have studied temperament from a more developmental , cyclical perspective to better understand how infants ' inborn temperament influences their development of emotion self - regulation . for example , thompson approached the subject of parental influence on children 's self - regulation of emotion from a bidirectional point of view ; he argued that both intrinsic components ( e.g. , temperament ) and extrinsic components ( e.g. , parental socialization ) are involved in the development of emotional regulation . that is , although it has been demonstrated that temperament can be influenced by environment , the child 's temperament can also influence the way that people react to the child . according to cassidy , when an infant 's strategic response to a mother 's caregiving is considered , two contributions of temperament are acknowledged : ( a ) the response is likely to fall within a range that is constrained by the infant 's temperament and ( b ) infants are not only responsive to their mother 's caregiving behavior but also contribute to shaping its nature infants are born with different levels of emotional reactivity that influence their care - giving environment . people often refer to highly reactive infants as fussy or difficult and infants who are not reactive as easygoing . a fussy baby might elicit more frustration from his or her caregiver than an infant who rarely cries . additionally , a fussy baby might elicit a different response from his or her caregiver upon crying than an infant who rarely cries . according to the dynamic viewpoint of temperament posited by thompson , cassidy , and others , the difference in quantity or quality of caregiver response to the infant 's emotional expressivity has the potential to shape the infant 's subsequent emotional expressivity . in turn , the way the infant comes to express emotions elicits further emotional socialization from the parent . thus , a fussy infant might elicit greater expression of personal distress from a parent , which may increase the infants ' negative emotionality over time , whereas another parent who responds to the infants ' crying with comforting may decrease the infants ' negative emotionality . borrowing from the literature on the development of self - regulation of emotions , it is evident that temperament is likely to be an important factor in the cyclical process of how interactions with parents influence children 's self - regulation of energy intake . for example , parents may be more tempted to use foods to comfort and sooth infants and children who have more difficult temperaments , and the parent - child interactions surrounding foods are likely to be more stressful for infants with highly reactive temperaments compared to infants who are more easily soothed . some research evidence for this exists . agras and colleagues found child temperament to mediate the relationship between parent overweight and child overweight . specifically , children of overweight parents who were rated as highly emotional in the children 's behavior questionnaire were more than twice as likely to be overweight at 9.5 years than children of overweight parents who were not highly emotional . however , specific ways in which temperament influences interactions between children and their parents around food are not well known . therefore , researchers interested in the developmental processes by which parents influence children 's self - regulation of energy intake should take child temperament into account . research about the influence of attachment on children 's interactions with their parents and children 's subsequent self - regulation of emotions is also likely to have important implications for understanding the relation between children 's attachment and self - regulation of food intake . based on their caregivers ' history of success in providing comfort and protection to their infants when they are distressed , infants have expectations as to how their caretaker will react to future expressions of distress and form strategies based on these expectations that they will use later when distressed in order to seek comfort from their caregivers . these individual differences in the strategies that infants use to gain comfort from their caregivers can be seen in the strange situation paradigm , the classic method of assessing security of infant - caregiver attachment . after entering an unfamiliar room with their caregiver , secure infants explore the room freely when their caregiver is present , using the caregiver as a secure base . security of attachment is assessed primarily as a function of infants ' behavior during reunions , after being separated from their caregiver . if securely attached infants become distressed during separations , they immediately seek proximity to the caregiver upon reunion and are calmed easily by their caregiver , and if not distressed , they still show accepting behavior towards their caregiver in reunion episodes and clearly prefer the caregiver over the stranger . in contrast , infants classified as insecure - avoidant show less distress during separation and turn away from the caregiver upon reunion , and those classified as insecure - ambivalent can not be comforted . they show ambivalence toward the caregiver , mixing clinging , and proximity - seeking behavior with displays of anger , such as hitting . cassidy has proposed that individual differences in children 's emotion regulation can be predicted from their attachment histories . numerous studies have found that parents of securely attached infants are more likely than those of insecure infants to respond to them with sensitivity . because of this , cassidy hypothesized that children with secure attachment histories should develop healthier patterns of flexible emotion regulation than those with insecure histories . particularly , since their mothers have been more likely to respond in a sensitive way to their emotional cues , secure infants should be more likely to express both negative and positive emotions in a healthy way by neither suppressing nor heightening their emotional expressivity , but rather , expressing both in appropriate contexts . in contrast , children with insecure - avoidant attachment histories are hypothesized to suppress their expression of negative emotions , becoming emotionally overregulated since their mothers have generally rejected their expression of negative emotions during the course of their early development . results of several studies support the idea that caregivers of insecure - avoidant infants inadvertently socialize them to minimize their emotional expressivity , leading to emotional overregulation . for example , these infants had been found to approach their mothers in the strange situation primarily when calm and contented rather than when distressed [ 70 , 71 ] , even though heart - rate measures indicated that they were actually more physiologically distressed by separations than were secure babies . avoidantly - attached infants have also been found to use self - soothing behaviors such as thumb sucking when distressed more often than other infants do . in toddlerhood , children with avoidant attachment were found to be more likely than other children to show flat affect during stressful situations , indicating suppression of negative emotions . interestingly , however , avoidantly attached preschoolers have been found to more angry and aggressive with peers than secure children in childhood and adolescence . it may be that avoidantly attached children generally try to suppress their negative emotion , but continual suppression of negative emotion may lead to frustration and , ultimately , to poor emotional regulation later in development . at the other end of the spectrum , children with insecure - ambivalent attachment histories are expected to become emotionally underregulated since their mothers have been likely to respond inconsistently , especially when these infants are distressed . as a result of this history of maternal lack of consistency , these infants maximize attachment behaviors when distressed ; that is , they become clingier , and they cry and fuss more to get the caregiver 's attention . some evidence also supports the idea that caregivers of insecure - ambivalent infants inadvertently socialize them to maximize their expression of fearful negative emotions . for example , ambivalent infants show more fear and cry more in laboratory procedures during infancy and toddlerhood , and as preschoolers , these children are more fearful when exploring a new environment and when interacting with peers . since insecure attachment is related to children 's poor emotion regulation , it may also be related to children 's poor regulation of energy intake and thus be a risk factor for childhood obesity . in one of the only studies that has examined the relation between attachment and obesity , anderson and whitaker argued that since insecure attachment is related to children 's poor emotion regulation , it may be a risk factor for childhood obesity . they reasoned that emotion regulation should be related to obesity since poorly regulated children experience greater stress , and the stress response has been linked to obesity [ 83 , 84 ] . in addition , problems with regulating negative emotions such as fear , sadness , and anger have been found to predict eating in the absence of hunger and disinhibited eating [ 85 , 86 ] . using the large national sample from the early childhood longitudinal study , anderson and whitaker found that insecure attachment , assessed at 24 months of age , predicted an increased risk of childhood obesity at age 4.5 years . this was found even after controlling for potentially confounding variables , including the quality of mother - child interaction , parenting practices related to obesity ( e.g. , having regular family dinners , duration of breastfeeding , television / video viewing time , etc . ) , maternal health and bmi , and ses . however , this study did not investigate mechanisms that might explain the link between insecure attachment and an increased risk of obesity . the researchers suggested that poor emotion regulation mediates this relationship , but children 's emotion regulation was not assessed . moreover , different types of insecure attachment ( avoidant versus ambivalent ) might affect children 's emotion regulation in different ways . since both over- and underregulation of emotional expression are nonoptimal emotion regulation strategies , both types of insecure attachment may increase the risk of obesity by increasing children 's physiological stress . however , if disinhibited eating , rather than stress , is the key mediator of the relation between insecure attachment and obesity , and if disinhibited eating is related to emotional underregulation , it may be that anxious - ambivalent children are at particularly high risk for obesity . in a cross - sectional study done with adults , disinhibited eating was found to mediate the relation between anxious attachment style ( analogous to insecure - ambivalent attachment style in infancy ) and higher adult bmi . however , avoidant attachment was unrelated to both disinhibited eating and having a higher bmi . longitudinal studies are needed that directly examine children 's stress reactions and disinhibited eating as possible mediators of the relation between both types of insecure attachment and obesity . in addition , future research should investigate whether parent - child attachment security relates to differences in the ways that parents feed their children and socialize their eating habits . since research has found strong links between infants ' attachment security and parental sensitivity [ 8890 ] , it may be that parents of insecure infants are less sensitive in interactions surrounding food . for example , they may be more controlling or restrictive during such interactions , which may in turn lead to an increased risk for obesity . obesity is increasing in children at epidemic rates with one - third of children in the united states currently overweight or obese . given that increases in childhood obesity are not fully explained by genetics alone , researchers need to concentrate on the obesogenic environment of children including factors in the family environment . as evidenced thus far , parents are key figures in socializing their children 's behaviors and self - regulation of energy intake . however , little is known about the developmental process by which parents influence children 's self - regulation of energy intake . researchers interested in this process can learn from the large body of research on how parents influence children 's regulation of emotions . this research provides important insights regarding how parents might influence children 's development of self - regulation of energy intake and how variations in parenting , child temperament , and the parent - child relationship might affect this . to date , research examining the relation of parenting to children 's overweight status has found links between children 's overweight status and the ways that parents respond to their children 's eating , particularly in terms of restricting or controlling children 's food intake and the use of rewards and punishments to do this . however , whether or not children 's energy intake mediates the relationship between food parenting practices and children 's overweight status has rarely been examined directly . in addition , the role of parental modeling and parental coaching in the development of children 's self - regulation of energy intake has rarely been studied . future studies should include more observational research of parent - child interactions surrounding food since parents may underestimate their use of particular food parenting strategies , such as the use of sweets as a reward . parents also may be unaware of their emotional responses to children 's eating behaviors or of the ways they might coach children 's self - regulation of energy intake . parents ' socialization of children 's eating behaviors ( including modeling , responding to children 's eating behaviors , coaching , and using rewards and punishments ) is likely to change over time in response to developmental changes in their children 's self - regulation of eating and their children 's subsequent eating behaviors . thus , longitudinal studies are needed to examine bidirectional relationships between food parenting practices , children 's self - regulation , children 's eating behaviors , and children 's overweight status over time . longitudinal studies are also important for examining the role of how moderating factors such as parenting styles , child temperament , and parent - child attachment affect the relation between food parenting practices and children 's self - regulation of energy intake . to date , most studies have not gone beyond simply finding relations between these factors and children 's overweight status ; for example , insecure attachment has been found to be related to increased risk of child obesity . some of the research on parenting styles has gone further , finding relations between parenting styles , particular kinds of parental feeding practices , and children 's overweight status over time . in discussing the results of these studies , obesity researchers have often speculated that children 's self - regulation of energy intake may mediate these associations , but research specifically investigating children 's self - regulation of energy intake as a mediator that may explain the relation of food parenting practices and children 's overweight status is lacking . whereas researchers in the area of self - regulation of energy intake have many opportunities to learn from the literature on self - regulation of emotions , it is also important to note that the two domains do not function independently ; instead , there are a variety of overlapping contexts in which food and emotions exist . for this reason , researchers interested in the role of self - regulation of energy intake in childhood obesity should conduct assessments of children 's general self - regulation as well as self - regulation of energy intake to see if the two are related . future research should also examine the role of the broader economic and cultural context on how parents influence children 's development of self - regulation of energy intake . according to bronfenbrenner 's ecological model of development , parent - child interactions are nested within larger socioeconomic and cultural contexts and can not be fully understood apart from these larger contexts . parenting in low - ses samples differs from parenting in middle- and upper - income samples in a multitude of ways , including types of maternal employment , single - mother families , and lower maternal education . one key factor likely to be relevant to parents ' influence on children 's self - regulation of both emotions and energy intake is the high degree of stress present in low - ses environments . stress has been found to have a substantial impact of parenting , as it has been related to more insecure attachment and a greater use of authoritarian parenting practices . also , as noted previously , the relation of different parenting styles to child outcomes varies according to ses and cultural differences . low - income parents are also more likely to experience food insecurity , which may affect food parenting . for example , food insecurity has been associated with maternal reports of using compensatory feeding practices , that is , giving children extra food or more energy - dense foods such as soda . low - income families are also more likely to serve calorie - dense fast foods and processed foods to their children since they are less expensive than fresh fruits and vegetables , as well as more filling . additionally , low - income parents might be more likely to give their children calorie - rich comfort foods to help them deal with stress since low - income families face more economic stress and stressful life events . finally , different ethnic groups are likely to have different culturally based customs regarding serving and eating food . for example , hispanic mothers have been found to use a more indulgent feeding style in several studies , granting most of their children 's requests for food [ 60 , 100 ] . although it is common for researchers interested in self - regulation of energy intake to assess children 's ability to regulate energy intake with children alone , the process by which parents influence children 's development of self - regulation is important ; therefore , researchers interested in parental contributions to the development of children 's self - regulation of energy intake should study it within the context of parent - child relationships . multidisciplinary teams of researchers with knowledge of nutrition and physiology should collaborate with developmental psychologists in order to fully understand the complex process by which parents influence children 's self - regulation of energy - intake .

the following article examines the role of parents in the development of children 's self - regulation of energy intake . various paths of parental influence are offered based on the literature on parental influences on children 's emotion self - regulation . the parental paths include modeling , responses to children 's behavior , assistance in helping children self - regulate , and motivating children through rewards and punishments . additionally , sources of variation in parental influences on regulation are examined , including parenting style , child temperament , and child - parent attachment security . parallels in the nature of parents ' role in socializing children 's regulation of emotions and energy intake are examined . implications for future research are discussed .

1. Introduction 2. The Influence of Parents on Children's Self-Regulation of Emotions and Energy Intake 3. Sources of Variation in Parental Influences on Children's Emotion Regulation and Energy Intake 4. Summary

children 's development of emotional self - regulation is important for many aspects of their health and wellbeing , including their ability to tolerate frustration , curb aggressive impulses , delay gratification , and express emotions in socially acceptable ways . children 's deficits in the ability to self - regulate their own behavior have been linked to rapid weight gain and obesity in middle childhood . there is a strong body of evidence to support that parents play an important role in children 's development of self - regulation of emotions in the early years [ 7 , 10 , 11 ] . children 's self - regulation of energy intake refers to children 's ability ( inborn and socialized ) to eat and not eat in response to cues of hunger and satiety . the development of self - regulation of energy intake in childhood is important for many aspects of health and wellbeing . notably , overweight children have been found to have deficits in self - regulation of energy intake compared to normal weight peers [ 14 , 15 ] . researchers have demonstrated that preschool children are capable of self - regulation of energy intake [ 16 , 17 ] . at the same time , large individual differences in children 's self - regulation of energy intake have been found . to the extent that individuals are motivated by external cues , they may be less sensitive to internal signals of hunger and less able to self - regulate their energy intake in the presence of external food cues . given that socialized ( non - inborn ) aspects of self - regulation of energy intake develop in the context of parent - child interactions during feeding with parents , parents are likely to play an important role in the development of such individual differences . a small but growing body of research is indicating that this is the case ; individual differences in parents ' feeding practices have been linked to individual differences in children 's self - regulation of energy intake [ 2024 ] . at the same time , there are gaps in our knowledge of how parents influence children 's development of self - regulation of energy intake . there is a large body of research on parental influences around emotion regulation , and this represents an exemplary model system for understanding parenting influences on children 's development of self - regulation of energy intake . thus , the literature on how parents influence children 's emotion regulation can provide useful information for child obesity researchers concerning how parents may contribute to an obesogenic environment by influencing the development of children 's self - regulation of energy intake . the purpose of this paper is to examine the literature on children 's early development of emotion regulation , and in particular , parents ' role in this development , to provide insight into the developmental processes by which parents influence children 's self - regulation of energy intake . individual differences in children 's abilities to regulate their emotions are apparent by this time , and whether they intend to or not , parents substantially influence these individual differences . they include ( 1 ) modeling emotion regulation or specific emotion regulation strategies ; ( 2 ) responding to their children 's emotional expression by acknowledging emotions , helping children process their emotions , helping children calm down , or questioning / punishing emotional expression ; ( 3 ) assisting children in the moment by teaching them various emotion regulation strategies ( e.g. , attend to relevant stimuli , seek out appropriate information , analyze the situation , generate , and evaluate alternatives ) and ( 4 ) motivating children through various social and material rewards and punishments . this line of research on parental influences on children 's self - regulation of emotions provides a useful framework that researchers interested in parental influences on children 's self - regulation around food can utilize . parents ' reactions to children in these situations influence how children react to future experiences ; therefore , how parents interact with their children influences children 's self - regulation of both emotions and eating . the following discussion of each of these parenting processes shows how the literature on parental influences on children 's emotional regulation can inform our understanding of the impact of parents ' behaviors on children 's self - regulation of energy intake . the literature on self - regulation of emotions indicates that parents provide very important models by which children learn to express emotions and later learn to control emotional expressivity . similarly , parents can provide both positive and negative models of self - regulation of energy intake through their own eating practices . however , parents ' modeling behaviors may either be conducive or not conducive to children developing good self - regulation of energy intake . to this point , johnson and birch found parental self - report of disinhibited eating was correlated with children 's lessened ability to self - regulate energy intake . it may be that parents who report disinhibited eating are providing negative models of self - regulation of energy intake , thereby setting forth the pathway to poor self - regulation of energy intake in their children . although , to our knowledge , most of the literature on modeling and energy - intake deals with food acceptance , and little - to - no work has been conducted on parent modeling and self - regulation of energy intake with children . as discussed by the authors , one possible reason for the lessened amount consumed in the presence of peers for overweight children is that normal weight children were modeling lower energy intake amounts and overweight children modified their intake amounts accordingly when in the presence of normal weight peers . researchers interested in understanding parental contributions to children 's self - regulation of energy intake should further examine how parents model behaviors around their own regulation of energy intake and the influence of these parental behaviors on children . research in the area of emotional expressivity indicates that the specific ways in which parents respond to children 's emotions , beginning in infancy and continuing throughout early childhood , are influential in shaping children 's self - regulation of emotions . parents ' acceptance of positive and negative emotions is important for optimal development of emotional expression and emotional regulation because children whose parents are accepting of their negative and positive emotions feel free to express both types of emotions when distressed and are better able to eventually express these emotions in socially accepted ways [ 7 , 33 , 34 ] . parental responses to their children 's negative emotions should also be contingent on children 's behavior , as well as appropriate given the child 's behavior , age , and needs ( see black and aboud for review of responsive parenting ) [ 3538 ] . in contrast , the parental practice of over - control around emotions occurs when parents respond in punitive , distressed , or minimizing ways to children 's expressions of negative emotions [ 13 , 14 ] . parental over - restriction of emotional expressivity has been linked to children 's lessened capacity to regulate their own emotions and thus to undesirable outcomes for children . similarly , parental responses to children 's expressions of hunger and satiety cues are likely to be important influences in shaping their children 's self - regulation of energy intake . parents can respond to children 's cues in ways that encourage optimal self - regulation of energy intake ( e.g. , letting children leave the table when they say they are full , encouraging children to reflect on how full they are before they are served some more , encouraging children to slow down the rate of eating ) or in ways that interfere with self - regulation of energy intake ( e.g. just as parental restriction of children 's emotional expressivity has been thought to interfere with children 's ability to self - regulate their own emotions , parental restriction around food has been linked to deficits in self - regulation of energy intake in children . thus , researchers interested in the development of children 's self - regulation of energy intake should examine over - restriction of foods parents make available to children as well as overcontrol of the amount of food children can eat as a possible mechanism for children developing less than optimal self - regulation of energy intake . children who experience too much parental control around eating may not learn to identify their own satiety cues and thus may have poor abilities to self - regulate their energy intake . to this point , researchers have found associations between controlling parent feeding practices ( as assessed by survey measures ) and children 's lessened ability to self - regulate energy intake . thus , as parental overcontrol of young children 's emotional expressivity results in children not learning adaptive ways of expressing emotions ( either over- or underregulating their emotions ) , parental over - restrictiveness around food may diminish children 's ability to read their own hunger and satiety cues and respond with appropriate self - regulation of energy intake . in addition to affecting the development of their children 's abilities to self - regulate energy intake through controlling their children 's food intake or access to certain foods , parents may also affect their children 's energy intake through the ways in which they respond to children 's emotions surrounding food . this is an important process for researchers interested in self - regulation of energy intake to better understand because parental responses to children 's negative emotions around food have the potential to influence the child 's view of that particular food or the process of eating in general . , parents who engage in emotion coaching are highly aware of their children 's emotions , they are accepting of their children 's emotions ( positive and negative ) and assist them in the process of understanding their emotions and expressing their emotions in socially appropriate ways . these techniques used by parents have been associated with better self - regulation of emotions in children . these findings have great potential to inform researchers interested in understanding parental influences on children 's ability to self - regulate energy intake . parents can assist children in the process of self - regulation of energy intake by helping them attend to relevant stimuli ( e.g. additionally , given that portion sizes have increased in recent years , parents can control the amount of food presented to children when out at restaurants by modifying portions on their children 's plates before they begin eating . researchers should investigate whether these parental practices of intake - coaching , a concept similar to scaffolding energy intake , foster better self - regulation of energy intake in children . as noted above , punitive parental responses to children 's expression of negative emotion have negative consequences for children 's emotion regulation . similarly , some parents may try to motivate self - regulation of energy intake through external rewards and punishments . experiments have indicated better ability to self - regulate energy intake in children who have been encouraged by caregivers to pay attention to internal cues of hunger and satiety rather than external cues such as rewards . it has been theorized that contingency - based parent behaviors such as this impair children 's ability to self - regulate their energy intake . studies examining parental influences on children 's emotion regulation have examined the role of numerous sources of variation that relate to parent - child interaction patterns relevant to children 's development of healthy patterns of emotional self - regulation . , the child 's inborn temperament ) , and variations in the parent - child relationship ( e.g. , child - parent attachment security ) . we next explore implications of these sources of variation for the development of children 's energy intake . parenting styles are relevant to self - regulation of both emotions and eating because , as rhee suggested , they can be looked at as the although parenting styles were first conceptualized by baumrind , scholars further elaborated these styles using dimensions of demandingness and responsiveness . the authoritative style presents an optimal context for fostering children 's self - regulation in many situations since authoritative parents are more likely than other parents to model positive and socially appropriate emotional responses to frustrating situations and to provide adaptive emotional coaching . these parenting styles have also been hypothesized to be relevant for explaining differences in children 's self - regulation of energy intake . this would be conducive to children developing good self - regulation around eating because it would encourage them to pay attention to their own internal cues of hunger and fullness . additionally , parents responding to children 's cues of hunger and fullness should empower children to self - regulate energy intake instead of energy intake being regulated solely by parents . in contrast , it is unlikely that authoritarian parents would be responsive to children 's negative emotions around food or children 's requests to eat or not eat due to feelings of hunger or fullness . given what is known in the literature related to emotion regulation , parental lack of responsiveness to children 's cues of hunger and satiety paired with parents being demanding might result in a flattening of these cues and , as a result , be detrimental to self - regulation around eating in children of authoritarian parents . it is important for researchers to look further into the relation between parenting style and children 's overweight status to examine whether children 's self - regulation of energy intake does in fact mediate this association . understanding findings across low income samples of children is especially important given that , in the united states , people of low ses have a higher rate of obesity than people who are not low ses . highly permissive parenting ( including both indulgent and uninvolved ) has also been linked with young children 's poor emotion regulation and aggressive behavior , indicating that parents ' sensitive responsiveness alone is not sufficient for children 's development of optimal emotion regulation . similarly , they may be less likely to provide children with the motivation and guidance needed to help them develop effective self - regulation of energy intake . parents who engage in indulgent feeding place few demands on their children 's eating behavior , although the few demands they do make are nondirective and supportive . it is possible that these children lack the scaffolding provided by authoritative and authoritarian parents to help teach self - regulation of energy intake . again , research on whether or not self - regulation of energy intake and low parental scaffolding mediate the relationship between indulgent parenting / feeding and higher child bmi is needed . hughes and colleagues did not find differences in children 's energy intake as a function of parents having an uninvolved style of parenting . future research on the feeding patterns of uninvolved parents is needed . self - regulation of emotions and energy intake are both likely to be influenced by children 's inborn traits . thus , some researchers have studied temperament from a more developmental , cyclical perspective to better understand how infants ' inborn temperament influences their development of emotion self - regulation . for example , thompson approached the subject of parental influence on children 's self - regulation of emotion from a bidirectional point of view ; he argued that both intrinsic components ( e.g. , parental socialization ) are involved in the development of emotional regulation . borrowing from the literature on the development of self - regulation of emotions , it is evident that temperament is likely to be an important factor in the cyclical process of how interactions with parents influence children 's self - regulation of energy intake . agras and colleagues found child temperament to mediate the relationship between parent overweight and child overweight . specifically , children of overweight parents who were rated as highly emotional in the children 's behavior questionnaire were more than twice as likely to be overweight at 9.5 years than children of overweight parents who were not highly emotional . therefore , researchers interested in the developmental processes by which parents influence children 's self - regulation of energy intake should take child temperament into account . research about the influence of attachment on children 's interactions with their parents and children 's subsequent self - regulation of emotions is also likely to have important implications for understanding the relation between children 's attachment and self - regulation of food intake . cassidy has proposed that individual differences in children 's emotion regulation can be predicted from their attachment histories . since insecure attachment is related to children 's poor emotion regulation , it may also be related to children 's poor regulation of energy intake and thus be a risk factor for childhood obesity . in one of the only studies that has examined the relation between attachment and obesity , anderson and whitaker argued that since insecure attachment is related to children 's poor emotion regulation , it may be a risk factor for childhood obesity . in addition , problems with regulating negative emotions such as fear , sadness , and anger have been found to predict eating in the absence of hunger and disinhibited eating [ 85 , 86 ] . moreover , different types of insecure attachment ( avoidant versus ambivalent ) might affect children 's emotion regulation in different ways . in addition , future research should investigate whether parent - child attachment security relates to differences in the ways that parents feed their children and socialize their eating habits . given that increases in childhood obesity are not fully explained by genetics alone , researchers need to concentrate on the obesogenic environment of children including factors in the family environment . as evidenced thus far , parents are key figures in socializing their children 's behaviors and self - regulation of energy intake . however , little is known about the developmental process by which parents influence children 's self - regulation of energy intake . researchers interested in this process can learn from the large body of research on how parents influence children 's regulation of emotions . this research provides important insights regarding how parents might influence children 's development of self - regulation of energy intake and how variations in parenting , child temperament , and the parent - child relationship might affect this . to date , research examining the relation of parenting to children 's overweight status has found links between children 's overweight status and the ways that parents respond to their children 's eating , particularly in terms of restricting or controlling children 's food intake and the use of rewards and punishments to do this . in addition , the role of parental modeling and parental coaching in the development of children 's self - regulation of energy intake has rarely been studied . parents also may be unaware of their emotional responses to children 's eating behaviors or of the ways they might coach children 's self - regulation of energy intake . parents ' socialization of children 's eating behaviors ( including modeling , responding to children 's eating behaviors , coaching , and using rewards and punishments ) is likely to change over time in response to developmental changes in their children 's self - regulation of eating and their children 's subsequent eating behaviors . thus , longitudinal studies are needed to examine bidirectional relationships between food parenting practices , children 's self - regulation , children 's eating behaviors , and children 's overweight status over time . longitudinal studies are also important for examining the role of how moderating factors such as parenting styles , child temperament , and parent - child attachment affect the relation between food parenting practices and children 's self - regulation of energy intake . some of the research on parenting styles has gone further , finding relations between parenting styles , particular kinds of parental feeding practices , and children 's overweight status over time . in discussing the results of these studies , obesity researchers have often speculated that children 's self - regulation of energy intake may mediate these associations , but research specifically investigating children 's self - regulation of energy intake as a mediator that may explain the relation of food parenting practices and children 's overweight status is lacking . whereas researchers in the area of self - regulation of energy intake have many opportunities to learn from the literature on self - regulation of emotions , it is also important to note that the two domains do not function independently ; instead , there are a variety of overlapping contexts in which food and emotions exist . for this reason , researchers interested in the role of self - regulation of energy intake in childhood obesity should conduct assessments of children 's general self - regulation as well as self - regulation of energy intake to see if the two are related . future research should also examine the role of the broader economic and cultural context on how parents influence children 's development of self - regulation of energy intake . parenting in low - ses samples differs from parenting in middle- and upper - income samples in a multitude of ways , including types of maternal employment , single - mother families , and lower maternal education . one key factor likely to be relevant to parents ' influence on children 's self - regulation of both emotions and energy intake is the high degree of stress present in low - ses environments . although it is common for researchers interested in self - regulation of energy intake to assess children 's ability to regulate energy intake with children alone , the process by which parents influence children 's development of self - regulation is important ; therefore , researchers interested in parental contributions to the development of children 's self - regulation of energy intake should study it within the context of parent - child relationships . multidisciplinary teams of researchers with knowledge of nutrition and physiology should collaborate with developmental psychologists in order to fully understand the complex process by which parents influence children 's self - regulation of energy - intake .

the european prospective investigation of cancer ( epic)-norfolk is a population - based prospective study that monitors 25,639 men and women aged 4074 years residing in the norfolk region , u.k . details of the study have been described elsewhere ( 17 ) . in brief , between 1993 and 1997 , 77,630 individuals were recruited from general practice to participate in the study , and 25,639 ( 33% ) consented and attended a baseline health assessment . participants completed questionnaires about their personal and family history of disease , medication , and lifestyle factors , including smoking habits . they were asked whether a physician had ever told them that they had any of the conditions in a list that included diabetes , heart attack , and stroke . baseline diabetes status was ascertained by 1 ) self - report of diabetes medication , 2 ) diabetes medication brought to the baseline examination , 3 ) participants indicating modification of their diet in the past year because of diabetes , or 4 ) participants indicating adherence to a diabetic diet . anthropometric and blood pressure measurements , as well as nonfasting blood samples were taken at the health assessment . because funding for measurement of hba1c only became available in 1995 , about 50% of all participants had information on this measure at baseline . hba1c was measured on fresh edta blood samples using high - performance liquid chromatography ( diamat automated glycated hemoglobin analyzer ; bio - rad laboratories ltd . , hemel hemstead , u.k . ) , which was standardized to the diabetes control and complications trial ( dcct ) assay . participants were invited to attend a second health assessment after 3 years ( 19982001 ) , at which identical measurements were taken , and 15,028 participants ( 59% ) attended . general practitioners of participants whose hba1c test results exceeded 7.0% were notified so that they could assume responsibility for confirming diagnosis and arranging treatment . individuals who live the norfolk area are slightly healthier than those in the general u.k . population , with a standardized mortality ratio of 93 ( office for national statistics death registration data , 2008 ) . however , epic - norfolk participants are similar to a nationally representative sample regarding anthropometric indices , blood pressure , and serum lipid levels ( 17 ) . we report results for follow - up at the second health assessment , a median of 3 years . we limited our analyses to the 6,372 individuals with hba1c measurements at baseline and at the second health assessment . we used this study sample to estimate the prevalence of known ( clinically diagnosed diabetes , self - reported physician - diagnosed diabetes , and diabetes medication ) and previously undiagnosed diabetes at baseline ( baseline hba1c 6.5% ) . after excluding those with diabetes at baseline ( clinically diagnosed diabetes and diabetes defined using hba1c diagnostic criteria ) , we further excluded 335 individuals with missing data for other metabolic risk factors , including age , sex , a family history of diabetes , smoking , the use of corticosteroids and antihypertensive drugs , bmi , waist circumference , systolic blood pressure , cholesterol , and triglyceride , leaving 5,735 individuals for analyses of the incidence and risk of diabetes ( fig . 1 ) . schematic diagram demonstrates the numbers and percentages of individuals with prevalent and incidence diabetes in a cohort of 6,372 men and women over 3 years . individuals with clinically diagnosed diabetes and hba1c 6.5% were considered to have clinically diagnosed diabetes in this diagram . * self - reported diabetes , evidence of diabetes medications , and dietary modification due to diabetes . self - reported diabetes , evidence of diabetes medication , diabetes registers , hospitalizations with diabetes , and diabetes codes on death certificates . participants were identified as having incident diabetes if 1 ) they reported physician - diagnosed diabetes or diabetes medication , or brought diabetes medication to the second health assessment ( clinical diagnosis ) , 2 ) they were identified on medical records , diabetes registers , or death certificates ( clinical diagnosis ) or 3 ) they had an hba1c of 6.5% at the second health assessment ( hba1c - defined diabetes ) . participants were identified through their general practice diabetes register or the norfolk and norwich hospital diabetes register . participants admitted to a hospital with a diabetes - related condition were identified by their national health service number . hospitals were linked to the east norfolk health authority database , which identifies all hospital contacts throughout england and wales for norfolk residents . vital status for all epic - norfolk participants was obtained through death certification at the office for national statistics , and death certification with coding for diabetes was identified . previous validation studies in this cohort using capture recapture analysis indicated that the use of multiple sources of ascertainment information for diabetes detected 99% of incident cases when comparing with diagnostic information from a comprehensive review of medical records ( 18 ) . in 5,735 participants free of diabetes at baseline with data on hba1c for the baseline and second health assessments , we calculated the incidence of diabetes defined clinically and by using hba1c diagnostic criteria in the whole cohort and separately for different categories of baseline hba1c . baseline characteristics were summarized for groups defined by different categories of baseline hba1c ( < 5.0 , 5.05.4 , 5.55.9 , and 6.06.4% ) . we tested for differences between groups using the test for categoric variables and anova or kruskal - wallis tests for normally or nonnormally distributed continuous variables , respectively . we used logistic regression to estimate the risk of developing diabetes as measured by the odds ratios ( ors ) for every 0.5% increase in hba1c as well as for different categories of hba1c compared with the lowest hba1c category of < 5.0% . we examined ors adjusted for age only , age and sex only , and multiple risk factors , including age , sex , self - reported family history of diabetes , smoking , the use of antihypertensive drugs or corticosteroids , bmi , waist circumference , systolic blood pressure , and total cholesterol , hdl cholesterol , and triglyceride values . to inform alternative screening strategies , we investigated risk factors associated with incident diabetes in those with a baseline hba1c of < 6.0% . we also performed a sensitivity analysis using a more restricted definition of incident diabetes in which participants were not classified as having incident diabetes unless a self - reported diagnosis was supported by information on diabetes - specific medication or confirmed by information from clinical records , death certificates , or hba1c . the european prospective investigation of cancer ( epic)-norfolk is a population - based prospective study that monitors 25,639 men and women aged 4074 years residing in the norfolk region , u.k . details of the study have been described elsewhere ( 17 ) . in brief , between 1993 and 1997 , 77,630 individuals were recruited from general practice to participate in the study , and 25,639 ( 33% ) consented and attended a baseline health assessment . participants completed questionnaires about their personal and family history of disease , medication , and lifestyle factors , including smoking habits . they were asked whether a physician had ever told them that they had any of the conditions in a list that included diabetes , heart attack , and stroke . baseline diabetes status was ascertained by 1 ) self - report of diabetes medication , 2 ) diabetes medication brought to the baseline examination , 3 ) participants indicating modification of their diet in the past year because of diabetes , or 4 ) participants indicating adherence to a diabetic diet . anthropometric and blood pressure measurements , as well as nonfasting blood samples were taken at the health assessment . because funding for measurement of hba1c only became available in 1995 , about 50% of all participants had information on this measure at baseline . hba1c was measured on fresh edta blood samples using high - performance liquid chromatography ( diamat automated glycated hemoglobin analyzer ; bio - rad laboratories ltd . , hemel hemstead , u.k . ) , which was standardized to the diabetes control and complications trial ( dcct ) assay . participants were invited to attend a second health assessment after 3 years ( 19982001 ) , at which identical measurements were taken , and 15,028 participants ( 59% ) attended . general practitioners of participants whose hba1c test results exceeded 7.0% were notified so that they could assume responsibility for confirming diagnosis and arranging treatment . individuals who live the norfolk area are slightly healthier than those in the general u.k . population , with a standardized mortality ratio of 93 ( office for national statistics death registration data , 2008 ) . however , epic - norfolk participants are similar to a nationally representative sample regarding anthropometric indices , blood pressure , and serum lipid levels ( 17 ) . we report results for follow - up at the second health assessment , a median of 3 years . we limited our analyses to the 6,372 individuals with hba1c measurements at baseline and at the second health assessment . we used this study sample to estimate the prevalence of known ( clinically diagnosed diabetes , self - reported physician - diagnosed diabetes , and diabetes medication ) and previously undiagnosed diabetes at baseline ( baseline hba1c 6.5% ) . after excluding those with diabetes at baseline ( clinically diagnosed diabetes and diabetes defined using hba1c diagnostic criteria ) , we further excluded 335 individuals with missing data for other metabolic risk factors , including age , sex , a family history of diabetes , smoking , the use of corticosteroids and antihypertensive drugs , bmi , waist circumference , systolic blood pressure , cholesterol , and triglyceride , leaving 5,735 individuals for analyses of the incidence and risk of diabetes ( fig . 1 ) . schematic diagram demonstrates the numbers and percentages of individuals with prevalent and incidence diabetes in a cohort of 6,372 men and women over 3 years . individuals with clinically diagnosed diabetes and hba1c 6.5% were considered to have clinically diagnosed diabetes in this diagram . * self - reported diabetes , evidence of diabetes medications , and dietary modification due to diabetes . self - reported diabetes , evidence of diabetes medication , diabetes registers , hospitalizations with diabetes , and diabetes codes on death certificates . participants were identified as having incident diabetes if 1 ) they reported physician - diagnosed diabetes or diabetes medication , or brought diabetes medication to the second health assessment ( clinical diagnosis ) , 2 ) they were identified on medical records , diabetes registers , or death certificates ( clinical diagnosis ) or 3 ) they had an hba1c of 6.5% at the second health assessment ( hba1c - defined diabetes ) . participants were identified through their general practice diabetes register or the norfolk and norwich hospital diabetes register . participants admitted to a hospital with a diabetes - related condition were identified by their national health service number . hospitals were linked to the east norfolk health authority database , which identifies all hospital contacts throughout england and wales for norfolk residents . vital status for all epic - norfolk participants was obtained through death certification at the office for national statistics , and death certification with coding for diabetes was identified . previous validation studies in this cohort using capture recapture analysis indicated that the use of multiple sources of ascertainment information for diabetes detected 99% of incident cases when comparing with diagnostic information from a comprehensive review of medical records ( 18 ) . in 5,735 participants free of diabetes at baseline with data on hba1c for the baseline and second health assessments , we calculated the incidence of diabetes defined clinically and by using hba1c diagnostic criteria in the whole cohort and separately for different categories of baseline hba1c . baseline characteristics were summarized for groups defined by different categories of baseline hba1c ( < 5.0 , 5.05.4 , 5.55.9 , and 6.06.4% ) . we tested for differences between groups using the test for categoric variables and anova or kruskal - wallis tests for normally or nonnormally distributed continuous variables , respectively . we used logistic regression to estimate the risk of developing diabetes as measured by the odds ratios ( ors ) for every 0.5% increase in hba1c as well as for different categories of hba1c compared with the lowest hba1c category of < 5.0% . we examined ors adjusted for age only , age and sex only , and multiple risk factors , including age , sex , self - reported family history of diabetes , smoking , the use of antihypertensive drugs or corticosteroids , bmi , waist circumference , systolic blood pressure , and total cholesterol , hdl cholesterol , and triglyceride values . to inform alternative screening strategies , we investigated risk factors associated with incident diabetes in those with a baseline hba1c of < 6.0% . we also performed a sensitivity analysis using a more restricted definition of incident diabetes in which participants were not classified as having incident diabetes unless a self - reported diagnosis was supported by information on diabetes - specific medication or confirmed by information from clinical records , death certificates , or hba1c . table 1 summarizes baseline characteristics of participants in the epic - norfolk cohort by different hba1c categories . participants ( 45% men ) had a mean age of 57.4 ( sd 9.4 ) years . participants with a higher hba1c value were older , more likely to be male , obese , current smokers , and to come from a lower socioeconomic class than those with a lower hba1c value . they were also more likely to have higher blood pressure , higher total cholesterol and triglyceride values , and lower hdl cholesterol values . there was no difference in family history of diabetes and the use of corticosteroids among the groups . comparison of baseline characteristics across categories of baseline hba1c in 5,735 participants in the epic - norfolk cohort data are presented as the mean ( sd ) , unless specified otherwise . iqr , interquartile range ; tg , triglyceride . * differences between groups using tests for categoric variables , and anova or kruskal - wallis tests for normally or nonnormally distributed continuous variables . registrar general 's social class : class i = professional , etc . occupations ; ii = managerial and technical occupations ; iiia = skilled occupations ( nonmanual ) ; iiib = skilled occupations ( manual ) ; iv = partly - skilled occupations ; v = unskilled occupations . among 6,372 individuals with hba1c measurements at both health assessments , 302 ( 4.7% ) had prevalent diabetes at baseline ( fig . 178 ( 2.8% ) had known diabetes ( those identified clinically ) , and 124 ( 1.9% ) had previously undiagnosed diabetes ( those identified using hba1c criteria ) . among 5,735 participants free of diabetes at baseline , 72 developed diabetes over 3 years ( fig . 1 ) . the cumulative incidence was 1.3% ( 95% ci 1.01.5 ) over 3 years , an annual incidence of 0.4% . among these new cases of diabetes , 37 individuals ( 51% ) were identified clinically ( e.g. , by their response to the questionnaire at the second health check or through linkage to clinical records or diabetes registers ) , with an incidence of 0.6% ( 0.40.9 ) . the remaining 35 individuals ( 49% ) with incident diabetes were identified by their hba1c results at the second health assessment . table 2 reports the incidence of diabetes by baseline hba1c levels for clinical diagnosis only and for clinical or hba1c - defined diagnosis , or both . the incidence of diabetes increased progressively with increasing baseline hba1c levels . in those with a baseline hba1c of 6.06.4% , the incidence of clinically diagnosed or hba1c - defined diabetes was three times higher than that of clinically diagnosed diabetes , at 7.0 ( 95% ci 4.810.1 ) and 2.4 ( 1.34.6 ) , respectively . thirty - six percent of incident cases of diabetes arose from individuals with a baseline hba1c of 6.06.4% ( 6% of the total population ) , and just over 35% of incident cases arose among individuals with a baseline hba1c of < 5.5% ( 69% of the total population ) . incidence and risk ( or ) of diabetes over 3 years by baseline hba1c categories in the epic - norfolk cohort ( n = 5,735 ) * adjusted for age , sex , social class , self - reported family history of diabetes , smoking , use of corticosteroids and antihypertensive drugs , bmi , waist circumference , systolic blood pressure , cholesterol , hdl cholesterol , and triglyceride . significant positive associations were found between hba1c and the risk of developing diabetes ( table 2 ) . a 0.5% increase in baseline hba1c was associated with more than a twofold increase in the risk of clinically diagnosed or hba1c - defined diabetes , or both ( age - adjusted or 2.7 [ 95% ci 2.13.5 ] ) . participants with a baseline hba1c of 6.06.4% had about a sevenfold higher risk of clinically diagnosed diabetes than those with an hba1c of < 5.0% . the highest risk of clinically diagnosed or hba1c - defined diabetes was observed in the highest baseline hba1c category compared with those with a baseline hba1c of < 5.0% ( or 15.5 [ 7.233.3 ] ) . these ors remained unchanged after adjustment for other risk factors . among individuals with a baseline hba1c < 6.0% , a family history of diabetes and waist circumference were the strongest nonlaboratory predictors of incident diabetes over 3 years . in the sensitivity analysis using a more restricted definition of incident diabetes , the incidence was 1.0 ( 95% ci 0.81.3 ) over 3 years , an annual incidence of 0.3% . approximately 40% of incident cases of diabetes developed in individuals with a high baseline hba1c of 6.06.4% ( 6% of total population ) . a 27-fold higher risk of diabetes was observed in those with a baseline hba1c of 6.06.4% compared with those with a baseline hba1c of < 5.0% . among 6,372 individuals with hba1c measurements at both health assessments , 302 ( 4.7% ) had prevalent diabetes at baseline ( fig . 178 ( 2.8% ) had known diabetes ( those identified clinically ) , and 124 ( 1.9% ) had previously undiagnosed diabetes ( those identified using hba1c criteria ) . among 5,735 participants free of diabetes at baseline , 72 developed diabetes over 3 years ( fig . 1 ) . the cumulative incidence was 1.3% ( 95% ci 1.01.5 ) over 3 years , an annual incidence of 0.4% . among these new cases of diabetes , 37 individuals ( 51% ) were identified clinically ( e.g. , by their response to the questionnaire at the second health check or through linkage to clinical records or diabetes registers ) , with an incidence of 0.6% ( 0.40.9 ) . the remaining 35 individuals ( 49% ) with incident diabetes were identified by their hba1c results at the second health assessment . table 2 reports the incidence of diabetes by baseline hba1c levels for clinical diagnosis only and for clinical or hba1c - defined diagnosis , or both . the incidence of diabetes increased progressively with increasing baseline hba1c levels . in those with a baseline hba1c of 6.06.4% , the incidence of clinically diagnosed or hba1c - defined diabetes was three times higher than that of clinically diagnosed diabetes , at 7.0 ( 95% ci 4.810.1 ) and 2.4 ( 1.34.6 ) , respectively . thirty - six percent of incident cases of diabetes arose from individuals with a baseline hba1c of 6.06.4% ( 6% of the total population ) , and just over 35% of incident cases arose among individuals with a baseline hba1c of < 5.5% ( 69% of the total population ) . incidence and risk ( or ) of diabetes over 3 years by baseline hba1c categories in the epic - norfolk cohort ( n = 5,735 ) * adjusted for age , sex , social class , self - reported family history of diabetes , smoking , use of corticosteroids and antihypertensive drugs , bmi , waist circumference , systolic blood pressure , cholesterol , hdl cholesterol , and triglyceride . significant positive associations were found between hba1c and the risk of developing diabetes ( table 2 ) . a 0.5% increase in baseline hba1c was associated with more than a twofold increase in the risk of clinically diagnosed or hba1c - defined diabetes , or both ( age - adjusted or 2.7 [ 95% ci 2.13.5 ] ) . participants with a baseline hba1c of 6.06.4% had about a sevenfold higher risk of clinically diagnosed diabetes than those with an hba1c of < 5.0% . the highest risk of clinically diagnosed or hba1c - defined diabetes was observed in the highest baseline hba1c category compared with those with a baseline hba1c of < 5.0% ( or 15.5 [ 7.233.3 ] ) . these ors remained unchanged after adjustment for other risk factors . among individuals with a baseline hba1c < 6.0% , a family history of diabetes and waist circumference were the strongest nonlaboratory predictors of incident diabetes over 3 years . in the sensitivity analysis using a more restricted definition of incident diabetes , 59 individuals developed diabetes ( supplementary table 1 ) . the incidence was 1.0 ( 95% ci 0.81.3 ) over 3 years , an annual incidence of 0.3% . approximately 40% of incident cases of diabetes developed in individuals with a high baseline hba1c of 6.06.4% ( 6% of total population ) . a 27-fold higher risk of diabetes was observed in those with a baseline hba1c of 6.06.4% compared with those with a baseline hba1c of < 5.0% . we used data from a large population - based british prospective cohort to estimate the prevalence and incidence of diabetes defined clinically or by hba1c , or both , over 3 years . the incidence of diabetes increased progressively across baseline hba1c levels , with 36% of incident cases developing in individuals with a baseline hba1c of 6.06.4% . to the best of our knowledge , this study is the first to report the incidence of diabetes based on hba1c diagnostic criteria using repeated assessment of hba1c . our prevalence estimate is comparable with the prevalence of diabetes in england ( 4.4% ) estimated from an epidemiologic model in which individuals with known and previously undiagnosed diabetes were included ( 19 ) . we also found that approximately 40% of prevalent cases were identified using hba1c diagnostic criteria and , hence , were previously undiagnosed . this is consistent with previous studies using an ogtt as a screening test ( 20 ) . a number of studies have estimated diabetes incidence by using longitudinal repeat ogtt or fasting plasma glucose measurements ( 15,16 ) . the incidence of diabetes in these studies varied from 6 to 10% over 9 to 10 years . data from a british population in ely , cambridgeshire , showed that the cumulative incidence of diabetes using repeat ogtt measurements was 5.9% over 10 years , corresponding to an annual incidence of 0.6% ( 16 ) . the higher incidence in the earlier study may be explained by enhanced case detection from repeated testing by ogtt over a longer period ( ogtt testing at baseline , 4.5 , and 10 years in the earlier ely study vs. hba1c at baseline and after 3 years of follow - up in the epic - norfolk study ) and the different contributions of the healthy volunteer effect in each study ( response rates of 74% in the ely study and 33% in the epic - norfolk study ) . few studies have examined the incidence and relative risk of diabetes in individuals or groups defined by different baseline hba1c levels . ( 21 ) examined the incidence of self - reported diabetes in american men and women with different baseline hba1c values . the 15-year cumulative incidence of diabetes was 6 , 12 , 21 , and 44% in individuals with an hba1c of < 5.0 , 5.05.4 , 5.55.9 , and 6.06.4% , respectively . the estimated annual incidence was higher than those observed across all hba1c categories in our study . this might be explained by the differences in levels of other risk factors ( higher bmi , smoking , and family history of diabetes in the selvin study ) , follow - up time ( 15 vs. 3 years , if incidence rates are not consistent across different durations of follow - up ) , and in particular , the different definitions of diabetes used in each study . hba1c has been shown to be a useful tool for the early detection of diabetes ( 21 ) . a few studies have demonstrated that hba1c predicts future risk of diabetes in high - risk individuals with glucose intolerance ( 22 ) . we have shown that hba1c predicts risk of diabetes in healthy middle - aged men and women . an hba1c of 6.06.4% identified 24% of clinically incident diabetes , and 36% of clinically incident or hba1c - defined diabetes . these figures were even higher when a more restricted definition of incident diabetes was used . recent evidence shows that this predictive ability also holds true in low - risk nondiabetic men and women ( 21 ) and in elderly individuals ( 23 ) . however , given that the associations between hba1c and the risk of diabetes were hardly changed after adjustment for multiple risk factors , there may not be much to gain from including data on multiple risk factors alongside hba1c for prediction of diabetes risk . ada suggested that there was no specific threshold that defines individuals who might be offered preventive interventions and that any such threshold would vary between countries with different heath care priorities ( 12 ) . however , ada suggested that individuals with an hba1c between 6.0 and 6.4% might represent a group in whom the risk of development of diabetes was very high and who could therefore be targeted for individual prevention interventions ( 12 ) . ada also suggested that this range should not be considered an absolute threshold and that interventions may be appropriate in other individuals on the basis of other risk information . our findings support this statement by demonstrating that most new cases of diabetes developed in those with a baseline hba1c of < 6.0% . the selection of a population for a high - risk prevention strategy is by the level of risk identified , the proportion of the population to be targeted , and the proportion of future cases that might therefore be prevented . our study showed that 36% of new cases of diabetes arose from the 6% of the study population who had the highest glycemic levels ( hba1c 6.06.4% ) . indeed , if previously proven intensive prevention interventions ( 2,3 ) were targeted at this middle - aged population , approximately 20% of new cases of diabetes could be prevented over 3 years . strategies for identifying which individuals should have an hba1c measurement , including simple risk scores using easily measured or routinely available risk factors , are needed . although the category of people with an hba1c of 6.06.4% identifies a high - risk group , most new cases of diabetes developed in individuals whose baseline hba1c values were < 6% . complementary strategies to identify high - risk individuals among those without raised hba1c may therefore be necessary . our subgroup analysis in individuals with an hba1c of < 6.0% suggested that those with central obesity and a family history of diabetes might represent another relatively easily identifiable subgroup to whom preventive interventions could be targeted . this also suggests that in addition to high - risk approaches , we need to develop a complementary population - based strategy aimed at shifting the whole distribution of hba1c in the population to reduce the risk of both diabetes and its complications ( 24 ) . however , although there is some evidence for the cost - effectiveness of prevention interventions among high risk individuals ( 25 ) , evidence on the cost - effectiveness of population - based strategies is very limited , making judgments about the balance of investment in high - risk and population - based approaches difficult . we have reported the incidence of diabetes in a large prospective british cohort using clinical ascertainment and newly proposed hba1c diagnostic criteria . participants included in this analysis were healthier than those excluded ; hence , our findings are likely to underestimate the incidence of diabetes in the whole cohort . given the 33% recruitment rate in this study , it is possible that participants might be more health - conscious and more likely to engage in healthy behaviors and to take up existing preventive services compared with nonparticipants . we might therefore have underestimated the overall incidence of diabetes in the population , which might consequently influence estimates of the relative risk for hba1c . because the epic - norfolk study collected nonfasting blood samples , it is not possible to compare the predictive ability for incident diabetes of different measures of glycemia . similar to other diagnostic tests , the use of a single measure of hba1c for diagnosing diabetes might lead to some degree of misclassification . however , given that hba1c is more reliable compared with an ogtt and the fasting plasma glucose test , the misclassification is likely to be only modest . a relatively short follow - up period and relatively small number of events mean that our finding should be interpreted with caution . furthermore , the follow - up of 3 years is still a plausible and important timeframe for identifying those at high risk of diabetes , because in addition to long - term risk information , one might also be interested in and more persuaded for behavior modification by information on the short - term risk of diabetes . our findings were specific to a population aged 4074 years , and may not represent the burden and risk of diabetes in relation to hba1c levels in younger people . lastly , most of the epic - norfolk participants are of european descent , which limits the generalizability of our findings to other ethnic groups and populations . in conclusion , the cumulative incidence of diabetes defined using a newly proposed hba1c threshold in this middle - aged british cohort was 1.3% over 3 years ( 0.4% per year ) . hba1c independently predicted the risk of incident diabetes , with each 0.5% difference in hba1c being associated with more than doubling of the risk of diabetes . because 36% of incident cases of diabetes came from the 6% of the population with a baseline hba1c of between 6 and 6.5% , this may be an easily identifiable subgroup to whom preventive interventions could be targeted . alternative strategies to identify high - risk individuals may be necessary , however , and complementary population - based approaches need to be developed to shift the underlying distribution of glycemia .

objectiveto evaluate the incidence and relative risk of type 2 diabetes defined by the newly proposed hba1c diagnostic criteria in groups categorized by different baseline hba1c levels.research design and methodsusing data from the european prospective investigation of cancer ( epic)-norfolk cohort with repeat hba1c measurements , we estimated the prevalence of known and previously undiagnosed diabetes at baseline ( baseline hba1c 6.5% ) and the incidence of diabetes over 3 years . we also examined the incidence and corresponding odds ratios ( ors ) by different levels of baseline hba1c . incident diabetes was defined clinically ( self - report at follow - up , prescribed diabetes medication , or inclusion on a diabetes register ) or biochemically ( hba1c 6.5% at the second health assessment ) , or both.resultsthe overall prevalence of diabetes was 4.7% ; 41% of prevalent cases were previously undiagnosed . among 5,735 participants without diabetes at baseline ( identified clinically or using hba1c criteria , or both ) , 72 developed diabetes over 3 years ( 1.3% [ 95% ci 1.01.5 ] ) , of which 49% were identified using the hba1c criteria . in 6% of the total population , the baseline hba1c was 6.06.4% ; 36% of incident cases arose in this group . the incidence of diabetes in this group was 15 times higher than in those with a baseline hba1c of < 5.0% ( or 15.5 [ 95% ci 7.233.3]).conclusionsthe cumulative incidence of diabetes defined using a newly proposed hba1c threshold in this middle - aged british cohort was 1.3% over 3 years . targeting interventions to individuals with an hba1c of 6.06.4% might represent a feasible preventive strategy , although complementary population - based preventive strategies are also needed to reduce the growing burden of diabetes .

RESEARCH DESIGN AND METHODS Study design and population Ascertainment of incident diabetes Statistical analyses RESULTS Prevalence of diabetes at baseline Incidence of diabetes over 3 years Risk of developing diabetes in groups defined by different HbA CONCLUSIONS Supplementary Material

the european prospective investigation of cancer ( epic)-norfolk is a population - based prospective study that monitors 25,639 men and women aged 4074 years residing in the norfolk region , u.k . baseline diabetes status was ascertained by 1 ) self - report of diabetes medication , 2 ) diabetes medication brought to the baseline examination , 3 ) participants indicating modification of their diet in the past year because of diabetes , or 4 ) participants indicating adherence to a diabetic diet . participants were invited to attend a second health assessment after 3 years ( 19982001 ) , at which identical measurements were taken , and 15,028 participants ( 59% ) attended . we report results for follow - up at the second health assessment , a median of 3 years . we limited our analyses to the 6,372 individuals with hba1c measurements at baseline and at the second health assessment . we used this study sample to estimate the prevalence of known ( clinically diagnosed diabetes , self - reported physician - diagnosed diabetes , and diabetes medication ) and previously undiagnosed diabetes at baseline ( baseline hba1c 6.5% ) . after excluding those with diabetes at baseline ( clinically diagnosed diabetes and diabetes defined using hba1c diagnostic criteria ) , we further excluded 335 individuals with missing data for other metabolic risk factors , including age , sex , a family history of diabetes , smoking , the use of corticosteroids and antihypertensive drugs , bmi , waist circumference , systolic blood pressure , cholesterol , and triglyceride , leaving 5,735 individuals for analyses of the incidence and risk of diabetes ( fig . individuals with clinically diagnosed diabetes and hba1c 6.5% were considered to have clinically diagnosed diabetes in this diagram . participants were identified as having incident diabetes if 1 ) they reported physician - diagnosed diabetes or diabetes medication , or brought diabetes medication to the second health assessment ( clinical diagnosis ) , 2 ) they were identified on medical records , diabetes registers , or death certificates ( clinical diagnosis ) or 3 ) they had an hba1c of 6.5% at the second health assessment ( hba1c - defined diabetes ) . in 5,735 participants free of diabetes at baseline with data on hba1c for the baseline and second health assessments , we calculated the incidence of diabetes defined clinically and by using hba1c diagnostic criteria in the whole cohort and separately for different categories of baseline hba1c . baseline characteristics were summarized for groups defined by different categories of baseline hba1c ( < 5.0 , 5.05.4 , 5.55.9 , and 6.06.4% ) . we used logistic regression to estimate the risk of developing diabetes as measured by the odds ratios ( ors ) for every 0.5% increase in hba1c as well as for different categories of hba1c compared with the lowest hba1c category of < 5.0% . to inform alternative screening strategies , we investigated risk factors associated with incident diabetes in those with a baseline hba1c of < 6.0% . we also performed a sensitivity analysis using a more restricted definition of incident diabetes in which participants were not classified as having incident diabetes unless a self - reported diagnosis was supported by information on diabetes - specific medication or confirmed by information from clinical records , death certificates , or hba1c . the european prospective investigation of cancer ( epic)-norfolk is a population - based prospective study that monitors 25,639 men and women aged 4074 years residing in the norfolk region , u.k . baseline diabetes status was ascertained by 1 ) self - report of diabetes medication , 2 ) diabetes medication brought to the baseline examination , 3 ) participants indicating modification of their diet in the past year because of diabetes , or 4 ) participants indicating adherence to a diabetic diet . participants were invited to attend a second health assessment after 3 years ( 19982001 ) , at which identical measurements were taken , and 15,028 participants ( 59% ) attended . we report results for follow - up at the second health assessment , a median of 3 years . we limited our analyses to the 6,372 individuals with hba1c measurements at baseline and at the second health assessment . we used this study sample to estimate the prevalence of known ( clinically diagnosed diabetes , self - reported physician - diagnosed diabetes , and diabetes medication ) and previously undiagnosed diabetes at baseline ( baseline hba1c 6.5% ) . after excluding those with diabetes at baseline ( clinically diagnosed diabetes and diabetes defined using hba1c diagnostic criteria ) , we further excluded 335 individuals with missing data for other metabolic risk factors , including age , sex , a family history of diabetes , smoking , the use of corticosteroids and antihypertensive drugs , bmi , waist circumference , systolic blood pressure , cholesterol , and triglyceride , leaving 5,735 individuals for analyses of the incidence and risk of diabetes ( fig . individuals with clinically diagnosed diabetes and hba1c 6.5% were considered to have clinically diagnosed diabetes in this diagram . self - reported diabetes , evidence of diabetes medication , diabetes registers , hospitalizations with diabetes , and diabetes codes on death certificates . participants were identified as having incident diabetes if 1 ) they reported physician - diagnosed diabetes or diabetes medication , or brought diabetes medication to the second health assessment ( clinical diagnosis ) , 2 ) they were identified on medical records , diabetes registers , or death certificates ( clinical diagnosis ) or 3 ) they had an hba1c of 6.5% at the second health assessment ( hba1c - defined diabetes ) . in 5,735 participants free of diabetes at baseline with data on hba1c for the baseline and second health assessments , we calculated the incidence of diabetes defined clinically and by using hba1c diagnostic criteria in the whole cohort and separately for different categories of baseline hba1c . baseline characteristics were summarized for groups defined by different categories of baseline hba1c ( < 5.0 , 5.05.4 , 5.55.9 , and 6.06.4% ) . we used logistic regression to estimate the risk of developing diabetes as measured by the odds ratios ( ors ) for every 0.5% increase in hba1c as well as for different categories of hba1c compared with the lowest hba1c category of < 5.0% . to inform alternative screening strategies , we investigated risk factors associated with incident diabetes in those with a baseline hba1c of < 6.0% . we also performed a sensitivity analysis using a more restricted definition of incident diabetes in which participants were not classified as having incident diabetes unless a self - reported diagnosis was supported by information on diabetes - specific medication or confirmed by information from clinical records , death certificates , or hba1c . among 6,372 individuals with hba1c measurements at both health assessments , 302 ( 4.7% ) had prevalent diabetes at baseline ( fig . 178 ( 2.8% ) had known diabetes ( those identified clinically ) , and 124 ( 1.9% ) had previously undiagnosed diabetes ( those identified using hba1c criteria ) . among 5,735 participants free of diabetes at baseline , 72 developed diabetes over 3 years ( fig . the cumulative incidence was 1.3% ( 95% ci 1.01.5 ) over 3 years , an annual incidence of 0.4% . among these new cases of diabetes , 37 individuals ( 51% ) were identified clinically ( e.g. , by their response to the questionnaire at the second health check or through linkage to clinical records or diabetes registers ) , with an incidence of 0.6% ( 0.40.9 ) . the remaining 35 individuals ( 49% ) with incident diabetes were identified by their hba1c results at the second health assessment . table 2 reports the incidence of diabetes by baseline hba1c levels for clinical diagnosis only and for clinical or hba1c - defined diagnosis , or both . the incidence of diabetes increased progressively with increasing baseline hba1c levels . in those with a baseline hba1c of 6.06.4% , the incidence of clinically diagnosed or hba1c - defined diabetes was three times higher than that of clinically diagnosed diabetes , at 7.0 ( 95% ci 4.810.1 ) and 2.4 ( 1.34.6 ) , respectively . thirty - six percent of incident cases of diabetes arose from individuals with a baseline hba1c of 6.06.4% ( 6% of the total population ) , and just over 35% of incident cases arose among individuals with a baseline hba1c of < 5.5% ( 69% of the total population ) . incidence and risk ( or ) of diabetes over 3 years by baseline hba1c categories in the epic - norfolk cohort ( n = 5,735 ) * adjusted for age , sex , social class , self - reported family history of diabetes , smoking , use of corticosteroids and antihypertensive drugs , bmi , waist circumference , systolic blood pressure , cholesterol , hdl cholesterol , and triglyceride . a 0.5% increase in baseline hba1c was associated with more than a twofold increase in the risk of clinically diagnosed or hba1c - defined diabetes , or both ( age - adjusted or 2.7 [ 95% ci 2.13.5 ] ) . participants with a baseline hba1c of 6.06.4% had about a sevenfold higher risk of clinically diagnosed diabetes than those with an hba1c of < 5.0% . the highest risk of clinically diagnosed or hba1c - defined diabetes was observed in the highest baseline hba1c category compared with those with a baseline hba1c of < 5.0% ( or 15.5 [ 7.233.3 ] ) . among individuals with a baseline hba1c < 6.0% , a family history of diabetes and waist circumference were the strongest nonlaboratory predictors of incident diabetes over 3 years . in the sensitivity analysis using a more restricted definition of incident diabetes , the incidence was 1.0 ( 95% ci 0.81.3 ) over 3 years , an annual incidence of 0.3% . approximately 40% of incident cases of diabetes developed in individuals with a high baseline hba1c of 6.06.4% ( 6% of total population ) . a 27-fold higher risk of diabetes was observed in those with a baseline hba1c of 6.06.4% compared with those with a baseline hba1c of < 5.0% . among 6,372 individuals with hba1c measurements at both health assessments , 302 ( 4.7% ) had prevalent diabetes at baseline ( fig . 178 ( 2.8% ) had known diabetes ( those identified clinically ) , and 124 ( 1.9% ) had previously undiagnosed diabetes ( those identified using hba1c criteria ) . among 5,735 participants free of diabetes at baseline , 72 developed diabetes over 3 years ( fig . the cumulative incidence was 1.3% ( 95% ci 1.01.5 ) over 3 years , an annual incidence of 0.4% . , by their response to the questionnaire at the second health check or through linkage to clinical records or diabetes registers ) , with an incidence of 0.6% ( 0.40.9 ) . the remaining 35 individuals ( 49% ) with incident diabetes were identified by their hba1c results at the second health assessment . table 2 reports the incidence of diabetes by baseline hba1c levels for clinical diagnosis only and for clinical or hba1c - defined diagnosis , or both . the incidence of diabetes increased progressively with increasing baseline hba1c levels . in those with a baseline hba1c of 6.06.4% , the incidence of clinically diagnosed or hba1c - defined diabetes was three times higher than that of clinically diagnosed diabetes , at 7.0 ( 95% ci 4.810.1 ) and 2.4 ( 1.34.6 ) , respectively . thirty - six percent of incident cases of diabetes arose from individuals with a baseline hba1c of 6.06.4% ( 6% of the total population ) , and just over 35% of incident cases arose among individuals with a baseline hba1c of < 5.5% ( 69% of the total population ) . incidence and risk ( or ) of diabetes over 3 years by baseline hba1c categories in the epic - norfolk cohort ( n = 5,735 ) * adjusted for age , sex , social class , self - reported family history of diabetes , smoking , use of corticosteroids and antihypertensive drugs , bmi , waist circumference , systolic blood pressure , cholesterol , hdl cholesterol , and triglyceride . a 0.5% increase in baseline hba1c was associated with more than a twofold increase in the risk of clinically diagnosed or hba1c - defined diabetes , or both ( age - adjusted or 2.7 [ 95% ci 2.13.5 ] ) . participants with a baseline hba1c of 6.06.4% had about a sevenfold higher risk of clinically diagnosed diabetes than those with an hba1c of < 5.0% . the highest risk of clinically diagnosed or hba1c - defined diabetes was observed in the highest baseline hba1c category compared with those with a baseline hba1c of < 5.0% ( or 15.5 [ 7.233.3 ] ) . among individuals with a baseline hba1c < 6.0% , a family history of diabetes and waist circumference were the strongest nonlaboratory predictors of incident diabetes over 3 years . the incidence was 1.0 ( 95% ci 0.81.3 ) over 3 years , an annual incidence of 0.3% . approximately 40% of incident cases of diabetes developed in individuals with a high baseline hba1c of 6.06.4% ( 6% of total population ) . a 27-fold higher risk of diabetes was observed in those with a baseline hba1c of 6.06.4% compared with those with a baseline hba1c of < 5.0% . we used data from a large population - based british prospective cohort to estimate the prevalence and incidence of diabetes defined clinically or by hba1c , or both , over 3 years . the incidence of diabetes increased progressively across baseline hba1c levels , with 36% of incident cases developing in individuals with a baseline hba1c of 6.06.4% . to the best of our knowledge , this study is the first to report the incidence of diabetes based on hba1c diagnostic criteria using repeated assessment of hba1c . our prevalence estimate is comparable with the prevalence of diabetes in england ( 4.4% ) estimated from an epidemiologic model in which individuals with known and previously undiagnosed diabetes were included ( 19 ) . we also found that approximately 40% of prevalent cases were identified using hba1c diagnostic criteria and , hence , were previously undiagnosed . data from a british population in ely , cambridgeshire , showed that the cumulative incidence of diabetes using repeat ogtt measurements was 5.9% over 10 years , corresponding to an annual incidence of 0.6% ( 16 ) . the higher incidence in the earlier study may be explained by enhanced case detection from repeated testing by ogtt over a longer period ( ogtt testing at baseline , 4.5 , and 10 years in the earlier ely study vs. hba1c at baseline and after 3 years of follow - up in the epic - norfolk study ) and the different contributions of the healthy volunteer effect in each study ( response rates of 74% in the ely study and 33% in the epic - norfolk study ) . few studies have examined the incidence and relative risk of diabetes in individuals or groups defined by different baseline hba1c levels . ( 21 ) examined the incidence of self - reported diabetes in american men and women with different baseline hba1c values . the 15-year cumulative incidence of diabetes was 6 , 12 , 21 , and 44% in individuals with an hba1c of < 5.0 , 5.05.4 , 5.55.9 , and 6.06.4% , respectively . this might be explained by the differences in levels of other risk factors ( higher bmi , smoking , and family history of diabetes in the selvin study ) , follow - up time ( 15 vs. 3 years , if incidence rates are not consistent across different durations of follow - up ) , and in particular , the different definitions of diabetes used in each study . we have shown that hba1c predicts risk of diabetes in healthy middle - aged men and women . an hba1c of 6.06.4% identified 24% of clinically incident diabetes , and 36% of clinically incident or hba1c - defined diabetes . however , ada suggested that individuals with an hba1c between 6.0 and 6.4% might represent a group in whom the risk of development of diabetes was very high and who could therefore be targeted for individual prevention interventions ( 12 ) . our findings support this statement by demonstrating that most new cases of diabetes developed in those with a baseline hba1c of < 6.0% . our study showed that 36% of new cases of diabetes arose from the 6% of the study population who had the highest glycemic levels ( hba1c 6.06.4% ) . indeed , if previously proven intensive prevention interventions ( 2,3 ) were targeted at this middle - aged population , approximately 20% of new cases of diabetes could be prevented over 3 years . although the category of people with an hba1c of 6.06.4% identifies a high - risk group , most new cases of diabetes developed in individuals whose baseline hba1c values were < 6% . our subgroup analysis in individuals with an hba1c of < 6.0% suggested that those with central obesity and a family history of diabetes might represent another relatively easily identifiable subgroup to whom preventive interventions could be targeted . this also suggests that in addition to high - risk approaches , we need to develop a complementary population - based strategy aimed at shifting the whole distribution of hba1c in the population to reduce the risk of both diabetes and its complications ( 24 ) . we have reported the incidence of diabetes in a large prospective british cohort using clinical ascertainment and newly proposed hba1c diagnostic criteria . participants included in this analysis were healthier than those excluded ; hence , our findings are likely to underestimate the incidence of diabetes in the whole cohort . we might therefore have underestimated the overall incidence of diabetes in the population , which might consequently influence estimates of the relative risk for hba1c . furthermore , the follow - up of 3 years is still a plausible and important timeframe for identifying those at high risk of diabetes , because in addition to long - term risk information , one might also be interested in and more persuaded for behavior modification by information on the short - term risk of diabetes . in conclusion , the cumulative incidence of diabetes defined using a newly proposed hba1c threshold in this middle - aged british cohort was 1.3% over 3 years ( 0.4% per year ) . hba1c independently predicted the risk of incident diabetes , with each 0.5% difference in hba1c being associated with more than doubling of the risk of diabetes . because 36% of incident cases of diabetes came from the 6% of the population with a baseline hba1c of between 6 and 6.5% , this may be an easily identifiable subgroup to whom preventive interventions could be targeted .

tiotropium bromide is an anticholinergic drug , categorized as a long - acting muscarinic antagonist ( lama ) or long - acting anticholinergic bronchodilator.1 these drugs are used primarily in the management of chronic obstructive pulmonary disease ( copd ) , but interest in lama for the treatment of asthma has increased in recent years , particularly for patients whose asthma is poorly controlled with inhaled corticosteroids ( ics ) and long - acting 2 agonists ( laba).14 despite clear guidelines and a range of available treatments , at least 50% of asthma patients continue to experience poor control of their current symptoms or future exacerbations , which significantly compromises the patient s quality of life and places considerable strain on health care systems.1,4 thus , there is a need for additional therapeutic options . the mechanisms by which lama have the potential to improve asthma control have recently been reviewed.1 briefly , lama may : induce bronchodilation , or inhibit cholinergically - mediated bronchoconstriction , and with regular use inhibit the airway smooth muscle hypertrophy and hyper - responsiveness characteristic of chronic asthma ; reduce cholinergically - mediated mucus secretion and inhibit goblet cell hyperplasia and mucus gland hypertrophy ; and moderate leukocyte responses in the lower airways as well as proinflammatory gene expression by airway smooth muscle and bronchial epithelium . in addition , lama such as tiotropium may act as steroid - sparing agents in patients with severe or poorly controlled asthma,5,6 which may alleviate some of the burden of corticosteroid use on health - related quality of life in these patients.7 a formulation of tiotropium delivered by soft mist inhaler has recently been approved in the uk8 and in several other countries for add - on maintenance therapy in adults with asthma , specifically for patients who are currently treated with ics and laba yet experienced at least one severe exacerbation in the past year . regulatory approval was based on the results of randomized controlled trials ( rcts ) in patients with asthma which showed that addition of tiotropium to the existing controller regimen ( ics laba ) improved lung function,2,3,913 and may improve symptom control3,11,13 and lower the patient s exacerbation risk,3 although it did not alter the use of rescue medications such as short - acting 2 agonists ( sabas).2,3,10,13 however , these rcts involved highly selected patient populations ( for example , nonsmokers with no serious comorbidities ) managed in tightly controlled settings , in most cases with relatively small patient numbers and short study periods.2,913 their broader applicability requires the support of clinical studies that better represent real - life populations and situations.14 clinical studies on the effectiveness of lama as add - on therapy in real - life asthma care are still needed . to that end , we examined the clinical effects of add - on therapy with tiotropium in a diverse group of over 2,000 asthma patients treated in uk primary care practice . study patients were at least 18 years of age and had a physician - recorded diagnosis of asthma ; patients with a recorded diagnosis of copd were excluded , but no other clinically relevant exclusions were applied . by comparing exacerbation rates and other measures of asthma control in the year before and after the addition of tiotropium to the patient s asthma regimen , our goal was to determine whether the addition of lama improves asthma control in routine clinical practice . this historical , observational study of uk asthma patients used data obtained from the optimum patient care research database ( opcrd ; http://www.optimumpatientcare.org ) , which contains more than 5 million anonymized , longitudinal patient records from approximately 400 participating medical practices in the uk and is focused on patients with evidence of respiratory disease . the opcrd has been approved by the trent multicentre research ethics committee for use in clinical research , and the protocol for this study was approved by the anonymised data ethics protocols and transparency committee , which is the independent scientific advisory committee for the opcrd . the medical record of each study patient was examined for the 12 months before ( baseline ) and 12 months after ( outcome ) the date of the patient s first prescription for tiotropium ( index prescription date ) ; hence , the index prescription dates spanned 20022012 . other lama drugs became available in the uk towards the end of the study period , but in the interest of investigating lama use in as many asthma patients as possible while using well - defined criteria , we limited the index prescription to tiotropium . two formulations of tiotropium bromide for inhalation were available in the uk during the study period : a dry - powder inhaler with a recommended dosage of 10 g ( the dose delivered from one 18 g capsule ) once a day15 and a soft mist inhaler with a recommended dosage of 5 g ( two puffs of 2.5 g ) once a day.8 although equivalence data are not yet available for patients with asthma , these formulations and dosages have comparable bronchodilator efficacy in patients with copd,16,17 so we chose to study the patients as a group , regardless of formulation / dosage . the study patients had at least one prescription for tiotropium during the study window , had a recorded diagnosis of asthma , had no recorded diagnosis of copd , were at least 18 years of age at the index prescription date , and had two continuous years of data ( 12 months each of baseline and outcome data ) . the baseline year was used for patient characterization and the outcome year for evaluation of asthma - related effectiveness measures . a complete list of the baseline variables examined is provided in the supplementary materials ( table s1 ) , along with their definitions and methods of calculation . the effectiveness measures compared before and after addition of tiotropium are described in table 1 . we used the american thoracic society and european respiratory society definition of an exacerbation ( asthma - related urgent hospital attendance / admission or acute course of systemic corticosteroids),18 although we limited systemic corticosteroid use to an acute course of oral corticosteroids ( ocs ) accompanied by a lower respiratory consultation ( physician visit coded as asthma , copd , lower respiratory tract infection , or lung function / asthma monitoring , with further respiratory examination , thoracic radiographs , or referral ) . an acute respiratory event was defined as either an exacerbation or an antibiotic prescription accompanied by a lower respiratory consultation ( which was taken to indicate that the physician treated the episode as a lower respiratory tract infection ) . multiple qualifying events within a 2-week window were counted as a single exacerbation or acute respiratory event . secondary measures included the risk domain of asthma control ( a proxy measure of asthma control as it pertains to exacerbation risk ) , specific components of the primary effectiveness measures ( asthma - related acute ocs and antibiotic use ) , peak expiratory flow ( pef ) , forced expiratory volume in one second ( fev1 ) , the ratio of fev1 to forced vital capacity ( fvc ) , and saba usage . asthma control in the risk domain was achieved if there were no episodes of asthma - related hospital , ocs , or antibiotic usage in the year of interest . as the measure of saba usage , the prescribed saba dosage was averaged over the year of interest , with all saba dosages converted to salbutamol ( albuterol ) equivalents ( table s1 ) . the statistical analysis was conducted using statistical package for the social sciences version 21 software ( ibm corporation , armonk , ny , usa ) and microsoft excel 2007 ( microsoft corporation , redmond , wa , usa ) . mean values are presented with their standard deviations and median values with their interquartile ranges ( 25th and 75th percentiles ) . where data were not normally distributed , median ( interquartile range ) values are reported . the number of exacerbations in the year before and after initiating tiotropium was then compared using the wilcoxon signed - rank test for exacerbation counts measured on the interval scale and the marginal homogeneity test for categorized exacerbation data . these tests were also used to compare baseline and outcome years for acute respiratory events , acute ocs courses , antibiotic prescriptions , and saba usage . the paired t - test was used to compare baseline and outcome years for pef and fev1 , and the marginal homogeneity test was used for comparison of categorized fev1/fvc ratios . the proportion of patients achieving asthma control in the risk domain was compared between baseline and outcome years using the mcnemar test . although the selection process deliberately excluded asthma patients who also had a recorded diagnosis of copd , some patients with undiagnosed or unrecorded copd may have been included , given the average age of the study patients and the prevalence of current or former smokers . for this reason , the primary analyses ( exacerbations and acute respiratory events ) were repeated for a subset of patients who were unlikely to have copd based on the following criteria : 40 years of age , > 40 years of age but never smoked , and > 40 years of age with no evidence of airway obstruction ( baseline fev1/fvc ratio 0.7 ) . the baseline year was used for patient characterization and the outcome year for evaluation of asthma - related effectiveness measures . a complete list of the baseline variables examined is provided in the supplementary materials ( table s1 ) , along with their definitions and methods of calculation . the effectiveness measures compared before and after addition of tiotropium are described in table 1 . we used the american thoracic society and european respiratory society definition of an exacerbation ( asthma - related urgent hospital attendance / admission or acute course of systemic corticosteroids),18 although we limited systemic corticosteroid use to an acute course of oral corticosteroids ( ocs ) accompanied by a lower respiratory consultation ( physician visit coded as asthma , copd , lower respiratory tract infection , or lung function / asthma monitoring , with further respiratory examination , thoracic radiographs , or referral ) . an acute respiratory event was defined as either an exacerbation or an antibiotic prescription accompanied by a lower respiratory consultation ( which was taken to indicate that the physician treated the episode as a lower respiratory tract infection ) . multiple qualifying events within a 2-week window were counted as a single exacerbation or acute respiratory event . secondary measures included the risk domain of asthma control ( a proxy measure of asthma control as it pertains to exacerbation risk ) , specific components of the primary effectiveness measures ( asthma - related acute ocs and antibiotic use ) , peak expiratory flow ( pef ) , forced expiratory volume in one second ( fev1 ) , the ratio of fev1 to forced vital capacity ( fvc ) , and saba usage . asthma control in the risk domain was achieved if there were no episodes of asthma - related hospital , ocs , or antibiotic usage in the year of interest . as the measure of saba usage , the prescribed saba dosage was averaged over the year of interest , with all saba dosages converted to salbutamol ( albuterol ) equivalents ( table s1 ) . the statistical analysis was conducted using statistical package for the social sciences version 21 software ( ibm corporation , armonk , ny , usa ) and microsoft excel 2007 ( microsoft corporation , redmond , wa , usa ) . mean values are presented with their standard deviations and median values with their interquartile ranges ( 25th and 75th percentiles ) . where data were not normally distributed , median ( interquartile range ) values are reported . the number of exacerbations in the year before and after initiating tiotropium was then compared using the wilcoxon signed - rank test for exacerbation counts measured on the interval scale and the marginal homogeneity test for categorized exacerbation data . these tests were also used to compare baseline and outcome years for acute respiratory events , acute ocs courses , antibiotic prescriptions , and saba usage . the paired t - test was used to compare baseline and outcome years for pef and fev1 , and the marginal homogeneity test was used for comparison of categorized fev1/fvc ratios . the proportion of patients achieving asthma control in the risk domain was compared between baseline and outcome years using the mcnemar test . although the selection process deliberately excluded asthma patients who also had a recorded diagnosis of copd , some patients with undiagnosed or unrecorded copd may have been included , given the average age of the study patients and the prevalence of current or former smokers . for this reason , the primary analyses ( exacerbations and acute respiratory events ) were repeated for a subset of patients who were unlikely to have copd based on the following criteria : 40 years of age , > 40 years of age but never smoked , and > 40 years of age with no evidence of airway obstruction ( baseline fev1/fvc ratio 0.7 ) . the dry powder formulation of tiotropium was prescribed in 1,898 patients ( 93% ) and the soft mist inhaler in 144 patients ( 7% ) . key baseline patient characteristics are summarized in tables 2 and 3 ; complete baseline data are provided in the supplementary materials ( tables s2s6 ) . the mean age was 63 years ; 59% of patients were female ; the mean body mass index was 29 kg / m ; and of those whose smoking history was known , 54% were either current ( 17% ) or former ( 37% ) smokers . in the patients with baseline lung function data , the mean pef was 69% of predicted normal , the mean fev1 was 59% of predicted normal , and the mean fev1/fvc ratio was 0.61 ; only 40% of the patients with fev1 and fvc data had a ratio 0.7 . at baseline , most patients were being treated according to british thoracic society guidelines19 step 3 ( 21.5% ) or step 4 ( 49.5% ; table s5 ) . an ics was prescribed in 83% of patients and a laba in 68% of patients ; 67% were prescribed both ( table s5 ) . median adherence to ics therapy ( table s1 ) was 100% , with 51% of patients having 100% adherence ( table s5 ) . the median controller - reliever ratio ( table s1 ) was 0.5 , indicating approximately equal use of controller ( ics or leukotriene receptor antagonist ) and reliever ( saba ) medications , although 59% of patients had a ratio 0.5 ( table s5 ) , indicating relatively more controller than reliever use . during their baseline year , 37% of patients had at least one exacerbation , 58% had at least one acute respiratory event , and 41% achieved asthma control in the risk domain ( table 3 ) . comparisons between baseline and outcome years for the various effectiveness measures are summarized in table 3 and illustrated in figures 24 , with further details provided in table s6 . addition of tiotropium was associated with significantly fewer exacerbations ( figure 2 ) and acute respiratory events ( figure 3 ) ; 27% of patients had at least one exacerbation , 47% had at least one acute respiratory event , and 52% achieved asthma control in the risk domain during the outcome year ( table 3 ) . in addition , tiotropium use was associated with significantly less asthma - related acute ocs and antibiotic use ( tables 3 and s6 ) . comparing baseline and outcome years , saba usage significantly increased ( figure 4 ) , but there were no significant changes in pef , fev1 , or fev1/fvc ratio in the patients with paired baseline and outcome data for those variables ( table 3 ) . the sensitivity analysis was conducted on 928 patients ( 45% of the full study group ) with the least likelihood of also having copd . baseline characteristics are summarized in table 4 , distributed by age group ( 40 and > 40 years ) . compared with the full study group , proportionately more patients in this subset were female ( 65% ) and nonsmokers ( 76% ) , and fewer were current ( 9% ) or former ( 15% ) smokers . the rates of exacerbations and acute respiratory events during the baseline year were comparable with those of the full study group , and the addition of tiotropium was associated with a similar decrease in exacerbations and acute respiratory events during the outcome year ( tables 5 and s7 ) . comparisons between baseline and outcome years for the various effectiveness measures are summarized in table 3 and illustrated in figures 24 , with further details provided in table s6 . addition of tiotropium was associated with significantly fewer exacerbations ( figure 2 ) and acute respiratory events ( figure 3 ) ; 27% of patients had at least one exacerbation , 47% had at least one acute respiratory event , and 52% achieved asthma control in the risk domain during the outcome year ( table 3 ) . in addition , tiotropium use was associated with significantly less asthma - related acute ocs and antibiotic use ( tables 3 and s6 ) . comparing baseline and outcome years , saba usage significantly increased ( figure 4 ) , but there were no significant changes in pef , fev1 , or fev1/fvc ratio in the patients with paired baseline and outcome data for those variables ( table 3 ) . the sensitivity analysis was conducted on 928 patients ( 45% of the full study group ) with the least likelihood of also having copd . baseline characteristics are summarized in table 4 , distributed by age group ( 40 and > 40 years ) . compared with the full study group , proportionately more patients in this subset were female ( 65% ) and nonsmokers ( 76% ) , and fewer were current ( 9% ) or former ( 15% ) smokers . the rates of exacerbations and acute respiratory events during the baseline year were comparable with those of the full study group , and the addition of tiotropium was associated with a similar decrease in exacerbations and acute respiratory events during the outcome year ( tables 5 and s7 ) . our study showed that primary care physicians in the uk have been prescribing lama for the treatment of asthma since 2002 , even though there were no uk license or guideline recommendations for lama use in patients with asthma until september 2014.15 it was not possible with our study design to determine why a lama was prescribed in these patients . however , 93% of the patients were over 40 years of age and 62% were over 60 years of age , more than 50% of whom were current or former smokers ( table s3 ) . most had significant comorbidities ( table s2 ) , and many had impairments of lung function and asthma control in the risk domain despite treatment with moderate to high doses of ics laba , 100% adherence to their ics prescriptions , and controller - reliever ratios indicative of equal or greater use of controller medications throughout the year . thus , it appears that physicians were prescribing lama predominantly as add - on therapy in older patients with poorly controlled asthma despite good treatment compliance , particularly in those who were current or former smokers . in this population , addition of tiotropium was associated with a significant decrease in the incidence of exacerbations and other acute respiratory events ( specifically , antibiotic prescriptions for lower respiratory tract infection ) , and a significant increase in the rate of asthma control in the risk domain over the following year . whereas most of the aforementioned clinical trials emphasized current symptom control , our study was focused primarily on the effects of lama on the patient s exacerbation risk , as exacerbations have such a negative impact on the patient s quality of life and on health care resources.20 the rct by kerstjens et al3 showed a significant reduction in exacerbation risk with the addition of tiotropium ; however , their patient population was younger than our study group by about 10 years ( mean age , 53 years ) , 76% of their patients had never smoked , the remainder had a smoking history of < 10 pack - years , and none of their patients had serious coexisting illnesses . thus , it may be particularly noteworthy that in our study lama therapy was associated with a decrease in exacerbations even under less - than - ideal conditions . also of note , lama use in our study was associated with a significant reduction in acute ocs use , which may be expected to reduce the multifaceted treatment burden documented for corticosteroid use in patients with severe or poorly controlled asthma.7 given our study design , it may be argued that regression toward the mean could have contributed to the differences found between baseline and outcome years . this issue may have been obviated by using a matched control group not prescribed tiotropium , although such an approach would almost certainly have led to the loss of unmatched but otherwise qualified patients prescribed tiotropium and thus to a smaller number and perhaps less diverse group of patients for study . as it was , our study design involved data collected over 2 consecutive years within a continuum of ongoing therapy in long - term asthma patients that , for the group , spanned over a decade ( 20012013 ) ; and with the exception of the lung function indices , the baseline and outcome values each represented a full year of data , not repeated measures of single , isolated points in time . thus , if regression toward the mean was a factor in our study results , its influence was likely small . furthermore , our findings are supported by those of kerstjens et al,3 who showed in two replicate , randomized , double - blind , placebo - controlled , parallel - group clinical trials involving 48-week study periods and 912 patients in 15 countries that addition of tiotropium significantly increased the time to first exacerbation and decreased the exacerbation risk in patients whose asthma was poorly controlled despite maintenance treatment with ics and laba . a legitimate concern , given our study design and patient demographics , is that the group may have included some asthma patients who also had undiagnosed or unrecorded copd . the sensitivity analysis , limited to the patients least likely to have copd , was conducted to address this issue . in this subset , addition of tiotropium was associated with decreases in the incidence of exacerbations and other acute respiratory events ( specifically , antibiotic prescriptions for lower respiratory tract infection ) similar to that found in the full study group . thus , it is reasonable to conclude that lama therapy may be associated with a decrease in asthma exacerbations whether or not the patient may also have copd . a further criticism is that no separate control or comparator group was included , and treatment was not randomized . our study purpose was to retrospectively examine the outcomes of clinical decisions made by primary care physicians with regard to the use of lama therapy in real - life patients with asthma . there was therefore no opportunity for us to randomize treatment , and as discussed above , our decision not to include a matched control group was made deliberately and with the goal of generating as large and diverse a treatment group as possible . thus , our study patients served as their own controls , the comparisons in asthma - related effectiveness measures being made between the year before and the year after initiating lama therapy . despite this and other limitations imposed by our study design , our findings are generally consistent with those of the rcts that included placebo2,3,912 or comparator drug groups.10,13 yet another potential shortcoming of our study design is that not all study patients had lung function data , and in those who did , no constraints could be placed on the timing of the lung function tests with regard to time of day ( morning or evening ) or bronchodilator administration ( before or after bronchodilation ) , when the tests were performed in the outcome year relative to initiation of tiotropium therapy , nor on quality control of the measurements . in comparison , licensing studies require centralized spirometry over - reading to ensure high quality spirometry . in order to maximize patient numbers , we elected not to limit the study group to only those patients with paired lung function data . physicians evidently were prescribing lama for patients with asthma even in the absence of lung function tests , so our full study group reflects recent clinical practices and outcomes . the inability to dictate the timing and performance of the lung function tests in our study likely contributed to the large variations found in lung function values . along with patient age and inclusion of smokers , that may explain the lack of significant changes in lung function with the addition of tiotropium , when improvements in lung function were a consistent rct finding.2,3,913 it is worth noting , however , that the rct improvements in lung function with the addition of tiotropium , while statistically significant , often were small , and they were not consistently accompanied by improvements in asthma control or quality of life.2,3,9,12,13 between baseline and outcome years , the change in saba usage in our study group , while statistically significant , was small and may be considered of little clinical relevance . this finding is consistent with rct results for tiotropium use in patients with asthma,2,3,10,13 but it is somewhat at odds with the classification of tiotropium as a long - acting bronchodilator that is administered once daily,8,15 and with the proposed mechanisms by which lama may improve asthma control.1 a longer investigation period may be required to determine whether or not lama perform in practice as these mechanisms propose . in the meantime , it is useful to know that addition of tiotropium to the current treatment regimen may reduce the asthma patient s exacerbation risk within the first year of treatment . in this real - life population of adults with asthma treated in routine clinical practice , addition of a lama to the current treatment regimen was associated with a decrease in exacerbations and other acute respiratory events ( specifically , antibiotic prescriptions for lower respiratory tract infections ) in the following year . baseline variables examined date of the patient s first tiotropium prescription aylin p , bottle a , jen mh , et al . london , uk : doctor foster research ; 2010 . available from : http://www.nhs.uk/scorecard/documents/hsmr%20methodology%2009%20november.pdf . accessed on march 15 , 2013 any lower respiratory consultation ( asthma , copd , or lrti code , or lung function / asthma monitoring ) with additional respiratory examinations , chest x - rays , and referrals british guideline on the management of asthma . london , uk : british thoracic society ; 2014 . available from : https://www.brit - thoracic.org.uk / document - library / clinical - information / asthma / btssign - asthma - guideline-2014/. abbreviations : a&e , accident and emergency department ; bdp , beclomethasone dipropionate ; bts , british thoracic society ; copd , chronic obstructive pulmonary disease ; fev1 , forced expiratory volume in one second ; fp , fluticasone propionate ; fvc , forced vital capacity ; ics , inhaled corticosteroid ; laba , long - acting 2 agonist ; ltra , leukotriene receptor antagonist ; lrti , lower respiratory tract infection ; nsaids , nonsteroidal anti - inflammatory drugs ; ocs , oral corticosteroid ; pef , peak expiratory flow ; saba , short - acting 2 agonist . baseline patient characteristics : demographic and clinical variables notes : n=2,042 , unless otherwise noted ; diagnosis at any time or nasal spray prescribed during baseline or outcome year diagnosis or cardiac drugs prescribed at any time . abbreviations : bmi , body mass index ; cci , charlson comorbidity index ; fev1 , forced expiratory volume in one second , expressed as percentage of predicted normal ; fvc , forced vital capacity ; gerd , gastroesophageal reflux disease ; nsaid , nonsteroidal anti - inflammatory drug ; pef , peak expiratory flow , expressed as percentage of predicted normal ; sd , standard deviation . baseline smoking status by age group distribution of baseline fev1/fvc ratio in patients over 60 years of age , by smoking status abbreviations : fev1 , forced expiratory volume in one second , expressed as percentage of predicted normal ; fvc , forced vital capacity . baseline patient characteristics : asthma treatment and control notes : n=2,042 , unless otherwise noted ; nonspecialist primary care consultations where asthma was recorded prescribed ics dosage , averaged over the baseline year in fluticasone - propionate equivalents dosage prescribed at most recent consultation before ipd at least one asthma - related hospital visit during baseline year ( categorized ) . abbreviations : a&e , accident and emergency department ; bts , british thoracic society ; ics , inhaled corticosteroid ; ipd , index prescription date ( date of first tiotropium script ) ; iqr , interquartile range ; laba , long - acting 2 agonist ; ltra , leukotriene receptor antagonist ; saac , short - acting anticholinergic ; saba , short - acting 2 agonist . comparison of effectiveness measures before ( baseline ) and after ( outcome ) addition of tiotropium notes : n=2,042 , unless otherwise noted ; wilcoxon signed - rank test marginal homogeneity test in salbutamol ( albuterol ) equivalents . abbreviations : fev1 , forced expiratory volume in one second , expressed as percentage of predicted normal ; fvc , forced vital capacity ; iqr , interquartile range ; ocs , oral corticosteroid ; pef , peak expiratory flow , expressed as percentage of predicted normal ; saba , short - acting 2 agonist ; sd , standard deviation . sensitivity analysis : comparison of primary measures before ( baseline ) and after ( outcome ) addition of tiotropium wilcoxon signed - rank test marginal homogeneity test .

backgroundrandomized controlled trials indicate that addition of a long - acting muscarinic antagonist ( lama ) such as tiotropium may improve asthma control and reduce exacerbation risk in patients with poorly controlled asthma , but broader clinical studies are needed to investigate the effectiveness of lama in real - life asthma care.methodsmedical records of adults with asthma ( aged 18 years ) prescribed tiotropium were obtained from the uk optimum patient care research database for the period 20012013 . patients diagnosed with chronic obstructive pulmonary disease were excluded , but no other clinical exclusions were applied . two primary outcomes were compared in the year before ( baseline ) and the year after ( outcome ) addition of tiotropium : exacerbations ( asthma - related hospital emergency department attendance or inpatient admission , or acute oral corticosteroid course ) and acute respiratory events ( exacerbation or antibiotic prescription with lower respiratory consultation ) . secondary outcomes included lung function test results and short - acting 2 agonist usage . the wilcoxon signed - rank test was used for variables measured on the interval scale , the marginal homogeneity test for categorized variables , and the paired t - test for lung function indices.resultsof the 2,042 study patients , 83% were prescribed an inhaled corticosteroid and 68% a long - acting 2 agonist during the baseline year ; 67% were prescribed both . comparing baseline and outcome years , the percentage of patients having at least one exacerbation decreased from 37% to 27% ( p<0.001 ) and the percentage having at least one acute respiratory event decreased from 58% to 47% ( p<0.001 ) . there were no significant changes in lung function , and usage of short - acting 2 agonists ( in salbutamol / albuterol equivalents ) increased from a median ( interquartile range ) of 274 ( 110 , 548 ) to 329 ( 110 , 603 ) g / day ( p=0.01).conclusionin this real - life asthma population , addition of lama therapy was associated with significant decreases in the incidence of exacerbations and antibiotic prescriptions for lower respiratory tract infections in the following year .

Introduction Materials and methods Effectiveness measures Statistical analysis Sensitivity analysis Results Outcomes Sensitivity analysis Discussion Conclusion Supplementary materials

tiotropium bromide is an anticholinergic drug , categorized as a long - acting muscarinic antagonist ( lama ) or long - acting anticholinergic bronchodilator.1 these drugs are used primarily in the management of chronic obstructive pulmonary disease ( copd ) , but interest in lama for the treatment of asthma has increased in recent years , particularly for patients whose asthma is poorly controlled with inhaled corticosteroids ( ics ) and long - acting 2 agonists ( laba).14 despite clear guidelines and a range of available treatments , at least 50% of asthma patients continue to experience poor control of their current symptoms or future exacerbations , which significantly compromises the patient s quality of life and places considerable strain on health care systems.1,4 thus , there is a need for additional therapeutic options . in addition , lama such as tiotropium may act as steroid - sparing agents in patients with severe or poorly controlled asthma,5,6 which may alleviate some of the burden of corticosteroid use on health - related quality of life in these patients.7 a formulation of tiotropium delivered by soft mist inhaler has recently been approved in the uk8 and in several other countries for add - on maintenance therapy in adults with asthma , specifically for patients who are currently treated with ics and laba yet experienced at least one severe exacerbation in the past year . regulatory approval was based on the results of randomized controlled trials ( rcts ) in patients with asthma which showed that addition of tiotropium to the existing controller regimen ( ics laba ) improved lung function,2,3,913 and may improve symptom control3,11,13 and lower the patient s exacerbation risk,3 although it did not alter the use of rescue medications such as short - acting 2 agonists ( sabas).2,3,10,13 however , these rcts involved highly selected patient populations ( for example , nonsmokers with no serious comorbidities ) managed in tightly controlled settings , in most cases with relatively small patient numbers and short study periods.2,913 their broader applicability requires the support of clinical studies that better represent real - life populations and situations.14 clinical studies on the effectiveness of lama as add - on therapy in real - life asthma care are still needed . study patients were at least 18 years of age and had a physician - recorded diagnosis of asthma ; patients with a recorded diagnosis of copd were excluded , but no other clinically relevant exclusions were applied . by comparing exacerbation rates and other measures of asthma control in the year before and after the addition of tiotropium to the patient s asthma regimen , our goal was to determine whether the addition of lama improves asthma control in routine clinical practice . this historical , observational study of uk asthma patients used data obtained from the optimum patient care research database ( opcrd ; http://www.optimumpatientcare.org ) , which contains more than 5 million anonymized , longitudinal patient records from approximately 400 participating medical practices in the uk and is focused on patients with evidence of respiratory disease . the medical record of each study patient was examined for the 12 months before ( baseline ) and 12 months after ( outcome ) the date of the patient s first prescription for tiotropium ( index prescription date ) ; hence , the index prescription dates spanned 20022012 . two formulations of tiotropium bromide for inhalation were available in the uk during the study period : a dry - powder inhaler with a recommended dosage of 10 g ( the dose delivered from one 18 g capsule ) once a day15 and a soft mist inhaler with a recommended dosage of 5 g ( two puffs of 2.5 g ) once a day.8 although equivalence data are not yet available for patients with asthma , these formulations and dosages have comparable bronchodilator efficacy in patients with copd,16,17 so we chose to study the patients as a group , regardless of formulation / dosage . the study patients had at least one prescription for tiotropium during the study window , had a recorded diagnosis of asthma , had no recorded diagnosis of copd , were at least 18 years of age at the index prescription date , and had two continuous years of data ( 12 months each of baseline and outcome data ) . the baseline year was used for patient characterization and the outcome year for evaluation of asthma - related effectiveness measures . we used the american thoracic society and european respiratory society definition of an exacerbation ( asthma - related urgent hospital attendance / admission or acute course of systemic corticosteroids),18 although we limited systemic corticosteroid use to an acute course of oral corticosteroids ( ocs ) accompanied by a lower respiratory consultation ( physician visit coded as asthma , copd , lower respiratory tract infection , or lung function / asthma monitoring , with further respiratory examination , thoracic radiographs , or referral ) . an acute respiratory event was defined as either an exacerbation or an antibiotic prescription accompanied by a lower respiratory consultation ( which was taken to indicate that the physician treated the episode as a lower respiratory tract infection ) . secondary measures included the risk domain of asthma control ( a proxy measure of asthma control as it pertains to exacerbation risk ) , specific components of the primary effectiveness measures ( asthma - related acute ocs and antibiotic use ) , peak expiratory flow ( pef ) , forced expiratory volume in one second ( fev1 ) , the ratio of fev1 to forced vital capacity ( fvc ) , and saba usage . asthma control in the risk domain was achieved if there were no episodes of asthma - related hospital , ocs , or antibiotic usage in the year of interest . the number of exacerbations in the year before and after initiating tiotropium was then compared using the wilcoxon signed - rank test for exacerbation counts measured on the interval scale and the marginal homogeneity test for categorized exacerbation data . these tests were also used to compare baseline and outcome years for acute respiratory events , acute ocs courses , antibiotic prescriptions , and saba usage . the paired t - test was used to compare baseline and outcome years for pef and fev1 , and the marginal homogeneity test was used for comparison of categorized fev1/fvc ratios . the proportion of patients achieving asthma control in the risk domain was compared between baseline and outcome years using the mcnemar test . for this reason , the primary analyses ( exacerbations and acute respiratory events ) were repeated for a subset of patients who were unlikely to have copd based on the following criteria : 40 years of age , > 40 years of age but never smoked , and > 40 years of age with no evidence of airway obstruction ( baseline fev1/fvc ratio 0.7 ) . the baseline year was used for patient characterization and the outcome year for evaluation of asthma - related effectiveness measures . we used the american thoracic society and european respiratory society definition of an exacerbation ( asthma - related urgent hospital attendance / admission or acute course of systemic corticosteroids),18 although we limited systemic corticosteroid use to an acute course of oral corticosteroids ( ocs ) accompanied by a lower respiratory consultation ( physician visit coded as asthma , copd , lower respiratory tract infection , or lung function / asthma monitoring , with further respiratory examination , thoracic radiographs , or referral ) . an acute respiratory event was defined as either an exacerbation or an antibiotic prescription accompanied by a lower respiratory consultation ( which was taken to indicate that the physician treated the episode as a lower respiratory tract infection ) . secondary measures included the risk domain of asthma control ( a proxy measure of asthma control as it pertains to exacerbation risk ) , specific components of the primary effectiveness measures ( asthma - related acute ocs and antibiotic use ) , peak expiratory flow ( pef ) , forced expiratory volume in one second ( fev1 ) , the ratio of fev1 to forced vital capacity ( fvc ) , and saba usage . asthma control in the risk domain was achieved if there were no episodes of asthma - related hospital , ocs , or antibiotic usage in the year of interest . the number of exacerbations in the year before and after initiating tiotropium was then compared using the wilcoxon signed - rank test for exacerbation counts measured on the interval scale and the marginal homogeneity test for categorized exacerbation data . these tests were also used to compare baseline and outcome years for acute respiratory events , acute ocs courses , antibiotic prescriptions , and saba usage . the paired t - test was used to compare baseline and outcome years for pef and fev1 , and the marginal homogeneity test was used for comparison of categorized fev1/fvc ratios . the proportion of patients achieving asthma control in the risk domain was compared between baseline and outcome years using the mcnemar test . for this reason , the primary analyses ( exacerbations and acute respiratory events ) were repeated for a subset of patients who were unlikely to have copd based on the following criteria : 40 years of age , > 40 years of age but never smoked , and > 40 years of age with no evidence of airway obstruction ( baseline fev1/fvc ratio 0.7 ) . in the patients with baseline lung function data , the mean pef was 69% of predicted normal , the mean fev1 was 59% of predicted normal , and the mean fev1/fvc ratio was 0.61 ; only 40% of the patients with fev1 and fvc data had a ratio 0.7 . during their baseline year , 37% of patients had at least one exacerbation , 58% had at least one acute respiratory event , and 41% achieved asthma control in the risk domain ( table 3 ) . addition of tiotropium was associated with significantly fewer exacerbations ( figure 2 ) and acute respiratory events ( figure 3 ) ; 27% of patients had at least one exacerbation , 47% had at least one acute respiratory event , and 52% achieved asthma control in the risk domain during the outcome year ( table 3 ) . comparing baseline and outcome years , saba usage significantly increased ( figure 4 ) , but there were no significant changes in pef , fev1 , or fev1/fvc ratio in the patients with paired baseline and outcome data for those variables ( table 3 ) . the rates of exacerbations and acute respiratory events during the baseline year were comparable with those of the full study group , and the addition of tiotropium was associated with a similar decrease in exacerbations and acute respiratory events during the outcome year ( tables 5 and s7 ) . addition of tiotropium was associated with significantly fewer exacerbations ( figure 2 ) and acute respiratory events ( figure 3 ) ; 27% of patients had at least one exacerbation , 47% had at least one acute respiratory event , and 52% achieved asthma control in the risk domain during the outcome year ( table 3 ) . in addition , tiotropium use was associated with significantly less asthma - related acute ocs and antibiotic use ( tables 3 and s6 ) . comparing baseline and outcome years , saba usage significantly increased ( figure 4 ) , but there were no significant changes in pef , fev1 , or fev1/fvc ratio in the patients with paired baseline and outcome data for those variables ( table 3 ) . the rates of exacerbations and acute respiratory events during the baseline year were comparable with those of the full study group , and the addition of tiotropium was associated with a similar decrease in exacerbations and acute respiratory events during the outcome year ( tables 5 and s7 ) . our study showed that primary care physicians in the uk have been prescribing lama for the treatment of asthma since 2002 , even though there were no uk license or guideline recommendations for lama use in patients with asthma until september 2014.15 it was not possible with our study design to determine why a lama was prescribed in these patients . most had significant comorbidities ( table s2 ) , and many had impairments of lung function and asthma control in the risk domain despite treatment with moderate to high doses of ics laba , 100% adherence to their ics prescriptions , and controller - reliever ratios indicative of equal or greater use of controller medications throughout the year . in this population , addition of tiotropium was associated with a significant decrease in the incidence of exacerbations and other acute respiratory events ( specifically , antibiotic prescriptions for lower respiratory tract infection ) , and a significant increase in the rate of asthma control in the risk domain over the following year . whereas most of the aforementioned clinical trials emphasized current symptom control , our study was focused primarily on the effects of lama on the patient s exacerbation risk , as exacerbations have such a negative impact on the patient s quality of life and on health care resources.20 the rct by kerstjens et al3 showed a significant reduction in exacerbation risk with the addition of tiotropium ; however , their patient population was younger than our study group by about 10 years ( mean age , 53 years ) , 76% of their patients had never smoked , the remainder had a smoking history of < 10 pack - years , and none of their patients had serious coexisting illnesses . also of note , lama use in our study was associated with a significant reduction in acute ocs use , which may be expected to reduce the multifaceted treatment burden documented for corticosteroid use in patients with severe or poorly controlled asthma.7 given our study design , it may be argued that regression toward the mean could have contributed to the differences found between baseline and outcome years . as it was , our study design involved data collected over 2 consecutive years within a continuum of ongoing therapy in long - term asthma patients that , for the group , spanned over a decade ( 20012013 ) ; and with the exception of the lung function indices , the baseline and outcome values each represented a full year of data , not repeated measures of single , isolated points in time . furthermore , our findings are supported by those of kerstjens et al,3 who showed in two replicate , randomized , double - blind , placebo - controlled , parallel - group clinical trials involving 48-week study periods and 912 patients in 15 countries that addition of tiotropium significantly increased the time to first exacerbation and decreased the exacerbation risk in patients whose asthma was poorly controlled despite maintenance treatment with ics and laba . in this subset , addition of tiotropium was associated with decreases in the incidence of exacerbations and other acute respiratory events ( specifically , antibiotic prescriptions for lower respiratory tract infection ) similar to that found in the full study group . our study purpose was to retrospectively examine the outcomes of clinical decisions made by primary care physicians with regard to the use of lama therapy in real - life patients with asthma . thus , our study patients served as their own controls , the comparisons in asthma - related effectiveness measures being made between the year before and the year after initiating lama therapy . despite this and other limitations imposed by our study design , our findings are generally consistent with those of the rcts that included placebo2,3,912 or comparator drug groups.10,13 yet another potential shortcoming of our study design is that not all study patients had lung function data , and in those who did , no constraints could be placed on the timing of the lung function tests with regard to time of day ( morning or evening ) or bronchodilator administration ( before or after bronchodilation ) , when the tests were performed in the outcome year relative to initiation of tiotropium therapy , nor on quality control of the measurements . along with patient age and inclusion of smokers , that may explain the lack of significant changes in lung function with the addition of tiotropium , when improvements in lung function were a consistent rct finding.2,3,913 it is worth noting , however , that the rct improvements in lung function with the addition of tiotropium , while statistically significant , often were small , and they were not consistently accompanied by improvements in asthma control or quality of life.2,3,9,12,13 between baseline and outcome years , the change in saba usage in our study group , while statistically significant , was small and may be considered of little clinical relevance . this finding is consistent with rct results for tiotropium use in patients with asthma,2,3,10,13 but it is somewhat at odds with the classification of tiotropium as a long - acting bronchodilator that is administered once daily,8,15 and with the proposed mechanisms by which lama may improve asthma control.1 a longer investigation period may be required to determine whether or not lama perform in practice as these mechanisms propose . in the meantime , it is useful to know that addition of tiotropium to the current treatment regimen may reduce the asthma patient s exacerbation risk within the first year of treatment . in this real - life population of adults with asthma treated in routine clinical practice , addition of a lama to the current treatment regimen was associated with a decrease in exacerbations and other acute respiratory events ( specifically , antibiotic prescriptions for lower respiratory tract infections ) in the following year . accessed on march 15 , 2013 any lower respiratory consultation ( asthma , copd , or lrti code , or lung function / asthma monitoring ) with additional respiratory examinations , chest x - rays , and referrals british guideline on the management of asthma . abbreviations : a&e , accident and emergency department ; bdp , beclomethasone dipropionate ; bts , british thoracic society ; copd , chronic obstructive pulmonary disease ; fev1 , forced expiratory volume in one second ; fp , fluticasone propionate ; fvc , forced vital capacity ; ics , inhaled corticosteroid ; laba , long - acting 2 agonist ; ltra , leukotriene receptor antagonist ; lrti , lower respiratory tract infection ; nsaids , nonsteroidal anti - inflammatory drugs ; ocs , oral corticosteroid ; pef , peak expiratory flow ; saba , short - acting 2 agonist . baseline patient characteristics : asthma treatment and control notes : n=2,042 , unless otherwise noted ; nonspecialist primary care consultations where asthma was recorded prescribed ics dosage , averaged over the baseline year in fluticasone - propionate equivalents dosage prescribed at most recent consultation before ipd at least one asthma - related hospital visit during baseline year ( categorized ) . abbreviations : a&e , accident and emergency department ; bts , british thoracic society ; ics , inhaled corticosteroid ; ipd , index prescription date ( date of first tiotropium script ) ; iqr , interquartile range ; laba , long - acting 2 agonist ; ltra , leukotriene receptor antagonist ; saac , short - acting anticholinergic ; saba , short - acting 2 agonist . comparison of effectiveness measures before ( baseline ) and after ( outcome ) addition of tiotropium notes : n=2,042 , unless otherwise noted ; wilcoxon signed - rank test marginal homogeneity test in salbutamol ( albuterol ) equivalents . abbreviations : fev1 , forced expiratory volume in one second , expressed as percentage of predicted normal ; fvc , forced vital capacity ; iqr , interquartile range ; ocs , oral corticosteroid ; pef , peak expiratory flow , expressed as percentage of predicted normal ; saba , short - acting 2 agonist ; sd , standard deviation . sensitivity analysis : comparison of primary measures before ( baseline ) and after ( outcome ) addition of tiotropium wilcoxon signed - rank test marginal homogeneity test .

omic tools enable simultaneous and large - scale study of molecules of exposed organisms to extract underlying alterations caused by environmental stressors . state - of - the - art omic techniques include microarray - based and sequencing techniques , nuclear magnetic resonance spectroscopy , and mass spectrometry . however , another technique valid for omics , providing rapid and nondestructive analyses , is ir spectroscopy . over the last few decades , ir has become a powerful methodology to study agriculture - related products and plant materials . recently , ir has provided excellent results both in clinical research [ e.g. , discriminating benign from malignant tumors in tissue samples such as the breast , colon , lung , or prostate , and examining biofluids , including urine , saliva , serum , or whole blood ] and in the environmental field . because of its capacity to interrogate biochemical signals of stressed organisms , attenuated total reflection fourier - transform ir ( atr - ftir ) spectroscopy shows great potential . the use of atr - ftir spectroscopy to address biological questions is viable since biomolecules with chemical bonds having an electric dipole moment absorb in the mid - ir region through their vibrations , giving rise to a detailed biomolecular fingerprint in the form of an ir spectrum . the acquisition of such fingerprints allows subsequent spectral classification with computational methods and possibly permits biomarker detection . various chemometric methods are suitable for ir data sets , both for exploratory or modeling purposes , including principal component analysis ( pca ) and linear discriminant analysis ( lda ) . these methods allow data reduction to facilitate the identification of wavenumber - related spectral alterations associated with glycogen content , lipid content , conformational changes and phosphorylation characteristics in proteins or structural alterations in dna / rna . another chemometric technique suited for the analysis of ir data sets , especially for those obtained in multifactorial designs , such as that hereby presented , is anova - simultaneous component analysis ( asca ) . omic experiments focused on molecules with high environmental persistence [ e.g. , perfluoroalkylated substances ( pfass ) ] permit the investigation of unknown effects of xenobiotics in target organisms . preferred doses of exposure are usually in the nanomolar scale , so as to reproduce real - world low - doses . pfass represent a large group of compounds highly used in a variety of consumer products , very resistant to degradation and with a high accumulation potential . concerns about pfass have risen due to their widespread distribution and persistence in humans and the environment but also due to their toxicity and ability to act as endocrine - disrupting chemicals ( edcs ) and obesogens . other biosystems might be affected by the presence of pfass , such as the a6 cell line . the renal epithelial a6 cell line was produced in 1969 from the renal uriniferous tubule of the adult african clawed frog xenopus laevis . it expresses the properties of tight epithelium , renal distal tubules , and collecting ducts . at confluence , a6 cells can form an epithelial monolayer ( figure 1a , c , e ) and spontaneously differentiate into a dome structure ( figure 1b , d , f ) . renal epithelial cells are specialized for absorption or secretion , where the membrane facing the culture media is the apical membrane , the membrane attached to the plastic culture flask is the basement membrane , and the membrane lying along the basement surface is the basolateral membrane ( figure 1a , b ) . the apical membrane of a6 cells contains the epithelial na channel ( enac ) , while na / k - atpase is in the basolateral membrane . the incorporation of na ions by enac and their expulsion into the extracellular space by na / k - atpases causes an accumulation of na ions in the space between a6 cells and the plastic culture flask . subsequent osmotic water inflow produces an elevation of the cell layer and results in a dome formation ( figure 1b ) , a structure easily seen by the microscope ( figure 1d , f ) . a6 cells forming domes have distinct physiological and structural properties ( e.g. , changes in the cytoskeleton ) than a6 cells forming monolayers . because it is easy to culture , the a6 cell line is commonly used in space studies , and several studies investigate the effects of gravitational forces on dome formation . however , little research has been conducted into the effects of xenobiotics on a6 cells as a toxicological model to simulate the effects on amphibians . schematic representation of xenopus laevis a6 kidney epithelial cells forming a monolayer ( a ) and a dome ( b ) . direct inversion images of giemsa - stained a6 cells disposed on a monolayer ( c ) and forming a dome ( d ) . phase - contrast images of a6 cells disposed on a monolayer ( e ) and forming a dome ( f ) . ( g ) experimental design for the study of pfas - induced effects ( refer to section 2.3 ) . the aim of this study was to examine the alterations induced in a6 cells , forming monolayers or differentiated into domes , exposed to four pfas substances [ i.e. , perfluorobutanesulfonate ( pfbs ) , perfluorooctanesulfonate ( pfos ) , perfluorooctanoic acid ( pfoa ) , and perfluorononanoic acid ( pfna ) ] , using atr - ftir spectroscopy and chemometric analysis [ i.e. , pca - lda and asca ; see supporting information ( esi ) for a short description of these methods ] . in addition , a growth - curve experiment was developed to determine whether the four distinct pfas - exposures differentially altered dose- and time - related cell number increases in culture . pfbs and pfos were obtained from fluka ( austria ) , whereas pfoa and pfna were purchased from sigma - aldrich ( steinheim , germany ) . cell culture consumables were obtained from invitrogen life technologies ( paisley , uk ) , unless otherwise stated . xenopus laevis a6 kidney epithelial cells were obtained from american type culture collection ( atcc ccl-102 ) . they were cultured in modified l15 culture medium consisting of 70% leibovitz media , 19% milli - q water sterile filtered through a 0.2 m syringe filter , 10% fetal bovine serum ( fbs ) , and 1% penicillin / streptomycin , at 5% co2 and 26 c . cells were trypsinized before the incorporation of cell aliquots for routine culture in t75 polystyrene flasks . toward experiments , a6 cells were disaggregated , resuspended in complete medium , and then seeded in t25 flasks at a rate of 500,000 cells per flask whereupon they were grown for the time required depending on the experiment ( see section 2.3 ) . for pfas - treatment , 25 l of stock solutions were added to 5 ml of the culture medium so that final exposure - doses ranged from 0 to 10 m [ considering 0 m as zero - dose control , in which cells were only exposed to the carrier solvent ( dmso ) ] . following treatment , cells were disaggregated into cell suspensions and immediately fixed with 70% etoh . the effects of the four pfas substances were studied on a6 cells forming monolayers or domes in three distinct experiments [ figure 1g1,2,3 ] . in experiment 1 [ figure 1g1 ] , pfas - induced effects were evaluated in cells forming confluent monolayers . toward this , cells were seeded for 1-day prior to pfas - exposure for a further 1-day ( 2-day experiment ) and final fixation . in this experiment , cells were exposed to chemicals at six concentrations ( 0 , 10 , 10 , 10 , 10 , or 10 m ) . in experiments 2 and 3 [ figure 1g2,3 , respectively ] , pfas - effects were evaluated in cells forming domes ( 9-day experiment ) . however , the introduction of pfas - treatment differed between them . in experiment 2 [ figure 1g2 ] , following 1-day seeding and 1-day pfas - exposure , the medium was aspirated and cells were grown for further 7 days in fresh medium , allowing dome formation , before final fixation . in contrast , in experiment 3 [ figure 1g3 ] , cells were grown for 8 days to allow dome formation , followed by 1-day treatment exposure prior to fixation . in experiment 2 , cells were pfas - exposed at four concentrations ( 0 , 10 , 10 , or 10 m ) , while in experiment 3 , three concentrations ( 0 , 10 , or 10 m ) were tested . five independent experiments were performed for each treatment at conditions 1 and 2 ( i.e. , 5 samples per category ) . thus , the total number of samples was 120 ( i.e. , 5 experiments 4 pfass 6 doses ) and 80 ( i.e. , 5 experiments 4 pfass 4 doses ) at conditions 1 and 2 , respectively . in the third conditions , hence , the total number of samples in the latter case was 24 ( i.e. , 2 experiments 4 pfass 3 doses ) . low number of replicates was performed in experiment 3 , as the results obtained are used to extract tentative conclusions about long - term pfas - induced effects considering the physiological conditions of cells forming domes . cellular material in 70% etoh was applied to 1 cm 1 cm low - e - reflective glass microscope slides ( kevley technologies , chesterland , oh , usa ) ( figure 1 g ) and allowed to air - dry prior to storage in marked 30 mm petri dishes kept in a desiccated environment until analysis . ir spectra were obtained using a bruker vector 22 ftir spectrometer with a helios atr attachment containing an 250 m 250 m aperture diamond crystal ( bruker optics ltd . , coventry , uk ) . the atr crystal was cleaned with sodium dodecyl sulfate ( sds ; sigma chemical co. ) ; a new background was taken prior the analysis of each new sample . from each treatment flask ( generating one slide ) , 10 ir spectra were acquired from different locations across each sample . spectra were coadded for 32 scans ; these were converted into absorbance units by bruker opus software . raw ir spectra obtained from exposed and control samples were preprocessed prior to chemometric analysis ( see figure s1 ) . initially , using opus software , ir spectra were individually cut to include only wavelengths between 1,800 and 900 cm ( 235 wavenumbers at 4 cm data spacing ) , the area associated with the biological spectral fingerprints . then , the baseline of the resulting data set was corrected by applying rubberband correction methods , and spectra were subsequently normalized to amide i ( i.e. , 1,650 cm ) . afterward , spectra were mean - centered , and finally , the class of the sample was defined . pca - lda was applied to the spectral data sets using matlab 8.3.0 r2014a ( the math works , natick , ma , usa ) and the irootlab toolbox ( http://irootlab.googlecode.com ) . as stated in the theory section , pca allows for the reduction of the number of variables in the spectral data set , whose small number of principal components ( pcs ) can capture 95% of the variance present in the original data set . in this study , a total of 12 pca - lda analyses were performed , considering 4 pfass and 3 experiments . for each model , the results of the analysis were visualized through one - dimensional ( 1-d ) scores plots ( figure 2 ) and cluster vectors plots ( see figure s2 ) . response effects of pfass , by examining the proximity in multivariate distance between exposed and control samples . response effects of pfbs , pfoa , pfos , and pfna in the three experiments . asca ( see esi ) was applied to three well - balanced spectral data sets ( see figure s3 ) by using pls toolbox 7.8 ( eigenvector research inc . , wenatche , wa , usa ) working in a matlab 8.3.0 r2014a environment ( the math works , natick , ma , usa ) . in these asca models , the effects of two categorical factors [ i.e. , type of chemical , with four levels ( pfbs , pfos , pfoa , or pfna ) , and dose of exposure , with a number of levels differing among experiments ( see section 2.3 ) ] and interaction were studied . statistical significances of the two factors and interaction were evaluated by a permutation test , using 10,000 permutations . a6 cells coming from 90% confluent t75 flasks were disaggregated , resuspended in complete medium , and seeded in plastic culture tissue coverslips ( sarstedt , usa ) in 30 mm petri dishes at a rate of 200,000 cells per petri dish and allowed to attach overnight at 26 c . then , cells were grown for 2 days , when they formed a confluent monolayer , or for 9 days , when they formed domes . finally , cells were fixed using 70% etoh ( 40 min ) , washed twice with 70% etoh , and kept at 4 c until microscopy visualization . cells fixed in the coverslips were air - dried for 24 h , transferred to new 30 mm petri dishes containing 3 ml of a solution of 5% giemsa ( sigma - aldrich , uk ) , and left for 20 min . then , the coverslips were washed twice with distilled water and allowed to air - dry . a nikon coolpix 950 camera , mounted via a nikon coolpix mdc lens 0.820.29 adapter to a nikon eclipse te300 inverted microscope , fitted with a nikon plan apo 60/1.20 water immersion objective was used to obtain images of giemsa - stained cells ( figure 1c , d ) . also , a confocal microscope , leica dmire2 inverted microscope connected to a leica tcs sp2 scan head and phase contrast settings , was used to obtain images of figure 1e , f . a6 cells were seeded at a ratio of 500,000 in t25 flasks in 5 ml of complete medium containing individual chemicals ( pfbs , pfos , pfoa , or pfna ) at concentrations of 10 , 10 , or 0 m ( control ) . this point was taken as time zero ( t0 ) , and duplicate cell counts in triplicate flasks were acquired . these t0 cell counts ( n = 6 per category ) were averaged and normalized to 100% . cells were washed , trypsinized , resuspended , and the cell number determined at indicated time points employing a hemocytometer . the acquired values for each experimental condition were averaged , and these contributed to the mean sd of the three separate experiments . results were expressed as relative cell number [ % ; i.e. , ratio of the cell number at the indicated time point relative to that determined at t0 ( normalized to 100% ) 100 ] . in the present study , ir data sets were first evaluated with 12 pca - lda models and further examined with 3 asca models . response effects of each pfass in the three experiments , whereas asca allowed data analysis considering the underlying experimental design . results of both analyses are presented below . results of the 12 pca - lda ( figure 2 ) evidenced a distinct dose response pattern in experiment 1 compared to experiments 2 and 3 . in the first experiment , the highest distinction between treated and control cell populations was observed at the lowest concentration tested ( 10 m ) in all pfas - treatments . conversely , higher pfas concentrations were responsible for marked effects in experiments 2 and 3 , especially in the latter conditions . a similar tendency was observed in the two last experiments : pfos and pfna caused in both conditions the highest alterations at 10 m , and pfoa produced the highest impact at low doses of 10 and 10 m in experiments 2 and 3 , respectively . only pfbs behaved inversely in these two experiments , producing major effects at the lowest dose tested ( 10 m ) in experiment 2 and at the highest ( 10 m ) in experiment 3 . primary wavenumbers important for discrimination of pfas - treatments at the concentration producing more effects in the three experiments together with the molecular entities associated with them are shown in table s1 and can be visualized in the cluster vectors plots of figure s2 . a first impression of the amount of variation related to the design factors can be obtained by separating this variation into contributions from the different factors . in this study , the statistical significances of the two categorical factors ( i.e. , chemical and dose ) and of their interaction were evaluated separately in the three experiments ( table 1 ) . results of this evaluation were attributed to the dominant part of variation to natural variability ( residuals 85% ) and the minor part to factors and interaction ( 12% ) , observing higher effects in experiment 3 . results of the permutation test showed larger significances ( p - values 0.05 ) of factors in experiments 1 and 2 . despite the observed small pfas - induced effects on a6 cells , the good reproducibility of the atr - ftir technique allows the extraction of reliable conclusions about their impact on a6 cells in the present study . percentage of variation expressed as sums of squared deviations from the overall mean and not variances . [ tc values of equation s2 ] shown in figure 3 indicate that chemicals producing more effects were pfos , pfoa , and pfbs in experiments 1 , 2 , and 3 , respectively . for data sets 1 , 2 , and 3 , the first component explains 56% , 82% , and 90% of variation , respectively . chemical was significant in experiments 1 and 2 ( p - values of 1 10 and 4 10 , respectively ) but not significant in experiment 3 ( p - value = 3 10 ) , according to the permutation test ( table 1 ) . asca score plots of the first component for the factors chemical and dose of the three experiments . scores of the first component of factor dose are shown in figure 3 for data sets acquired in experiments 1 , 2 , and 3 , respectively [ td values of equation s2 ] . for data sets 1 , 2 , and 3 , the first component explains 78% , 93% , and 94% of variation , respectively . scores of these figures indicate that concentrations of exposure producing more effects were 10 , 10 , and 10 m in experiments 1 , 2 , and 3 , respectively . the results of the permutation test evidenced that the pfas - dose was significant in experiments 1 and 2 with p - values of 1 10 and 1 10 , respectively , but not significant in experiment 3 , with a p - value of 8 10 ( table 1 ) . scores of the first component of the interaction showed no pattern related to the interaction of factors : there was no increasing or decreasing trend of scores of the different doses of exposure respect to the chemical treatment . for this reason , the plot of these scores is not provided in this study . the effects of the four pfass on the increases in a6 cell number are shown in figure 4 . the behavior observed was similar between pairs of chemicals pfbs and pfna vs pfos and pfoa . for the first two chemicals , following 24-h or 48-h treatment , no marked differences in cell population were observed with respect to the control . however , 24-h and 48-h treatments with pfos and pfoa caused a significant decrease in cell number , which became higher with time and gave rise to maximum cell depletion after 48 h of exposure . effects of pfbs , pfna , pfos , and pfoa on dose- and time - related cell number increases in culture . our study evidenced a primary effect of low - dose ( 10 m ) pfas - treatment on a6 cells forming monolayers ( experiment 1 ) ( figure 2 ) . predominant effects of environmental stressors at low - doses of exposure have often been reported in the literature , especially for edcs , such as pfass . a review by vandenberg et al . big effects at low doses , a theory primarily defined by the national toxicology program ( http://ntp.niehs.nih.gov/ ) , and second , the nonmonotonicity ( i.e. , nonlinear relationship between dose and effect ) thus declaring the dogma it is important to stress that our findings in experiment 1 ( cells forming a monolayer ) followed this low - dose theory , while results of experiments 2 and 3 ( cells differentiated into dome structures ) showed an opposing tendency ( major alterations at high doses ) . such differences can be explained by the specific physiological status of cells forming monolayers or domes , the latter having enhanced cell excretion function and requiring higher pfas - doses to produce an effect . also , the differential dose response effects of pfas substances depending on cell differentiation status were to some extent related to the cytotoxicity of most influential chemicals ( according to asca results ) in each experiment . considering the median effective doses ( ec50 ) of pfass reported in a previous study on jeg-3 cells [ i.e. , pfos ( 107125 m ) < pfna ( 213220 m ) < pfoa ( 594647 m ) pfbs ( n.d . ) ] , it was observed that major effects in experiment 1 ( primarily influenced by high - cytotoxic pfos ) occurred at low doses , whereas larger effects in experiments 2 and 3 ( primarily influenced by less - cytotoxic pfoa and pfbs , respectively ) were observed at higher doses . considering overall effects , a greater impact of pfas substances was evident in experiment 3 , presenting the treated samples to maximum cluster segregation compared to that of the control in 1-d pca - lda score plots ( figure 2 ) . the higher effects of pfas substances in experiment 3 vs experiment 2 might be attributed to a cellular recuperation after the chemical stress possible in the second conditions since cells were allowed to grow for 7 days in fresh medium after pfas - exposure , a time that cells might use to eliminate those pfas substances previously incorporated . interestingly , distinct spectral alterations were induced by pfas substances in the three experiments , suggesting three mechanisms of action of the chemicals depending on cell differentiation ( i.e. , monolayer or dome ) , moment of exposure ( i.e. , pre- or postdome formation ) , and cell population . in experiment 1 , all pfas substances produced alterations associated with dna / rna ( e.g. , spo2 ) ( see figure s2 and table s1 ) . in fact , pfoa and pfna are suspected genotoxic carcinogens through induction of reactive oxygen species that are responsible for oxidative dna damage . recently , yahia et al . demonstrated that pfoa and pfna induced dna damage in tk6 cells , observing that pfna produced high levels of 8-hydroxy-2-deoxyguanosine ( 8-ohdg ) , a biomarker of oxidative dna damage . in contrast , in experiment 2 , all pfas substances caused alterations associated with secondary structures of proteins ( amide i , amide ii , and amide iii ) ( see figure s2 and table s1 ) . the alteration of proteins observed under conditions 2 ( 9-day experiment ) might be attributed to a direct consequence of the effects produced on dna in the first conditions ( 2-day experiment ) since effects on dna are ultimately expressed in the proteins that it encodes . in experiment 3 , all pfas substances produced alterations associated with fatty acids ( see figure s2 and table s1 ) . in this experiment , one could expect similar effects as in experiment 1 since in both cases cells are analyzed right after pfas exposure . however , the observed differences might be attributed to the different numbers of cells between both experiments and their dissimilar physiological properties ( i.e. , monolayer vs domes ) . continuing with the study of distinguishing features induced by pfas treatments , special attention was focused on lipids , due to the reported capacity of pfas substances to alter lipid species of cellular membranes . a previous study performed on human placental chroriocarcinoma jeg-3 cells exposed to a mixture of eight pfas substances by lc - ms revealed that increased levels of the major components of cell membranes [ i.e. , phosphatidylcholine ( pc ) , lyso plasmalogen pc , and plasmalogen pc ] and a relatively low increase in triacylglicerols ( tag ) were induced by pfas substances . similarly , our findings showed some effects of pfas on lipids of a6 cells . as observed in table s1 , in experiment 1 pfos and pfoa produced effects at 1,736 cm ( c = o stretching lipids ) and at 1,444 cm ( lipids ) , respectively , and in experiment 2 pfbs induced alterations at 1,750 cm , associated with a c = c stretching in lipids , as occurred with cells exposed to pfna in experiment 3 . the findings of this study demonstrate that pfas substances pose a significant effect on the metabolome and lipidome of xenopus laevis a6 cells . however , more information is needed in order to detect and identify potential biomarkers of lipid and metabolite disruption and to find most altered biochemical pathways , and future studies should focus on this point . liquid chromatography coupled to high resolution mass spectrometry ( lc - hrms ) techniques , which enable the analysis of compounds at low doses with high accuracy mass determination , are highly suitable for such purposes . moreover , the fusion of ir data with lc - ms data is also worthy since it can provide a more comprehensive knowledge of the effects of pfas substances in this amphibian cell model . finally , results of the growth - curve experiment showed different responses of a6 cells exposed to pfbs / pfna compared to pfos / pfoa ( figure 4 ) . pfbs and pfna did not induce distinguishable a6 cell proliferation or depletion compared to those of the control . in contrast , pfos and pfoa induced a decrease in a6 cell number compared to that of the control , in a time- and dose - specific manner : after 48 h of treatment , a time when the two pfas substances presented maximum effects , high - dose pfos produced the most profound cell decreases , whereas low - dose pfoa caused the maximum decline in cell population . the capacity of pfos and pfoa to inhibit cell proliferation has been reported in other studies . recently , cui et al . found that 80% inhibitory concentration ( ic80 ) of pfoa ( 150.97 g / ml ) and 50% inhibitory concentration ( ic50 ) of pfos ( 27.92 g / ml ) blocked cell cycle and proliferation of zebrafish ( danio rerio ) liver cells ( zfl ) . also , other studies showed pfoa capacity to induce apoptosis in hepatoma hepg2 cells . overall , this work contributes to the better knowledge of pfas substance effects on xenopus laevis a6 kidney epithelial cells indicating an overall interference with dna / rna , secondary structures of proteins , lipids , and fatty acids at concentrations well below those associated with other adverse effects , such as cytotoxicity or endocrine disruption . this work also highlights ( a ) the differential effects of pfas substances depending on cell - differentiation , presenting a nonmonotonic - behavior on a6 cells forming monolayers and ( b ) the ability of pfos and pfoa to induce cell death .

the effects of four perfluoroalkylated substances ( pfass ) , namely , perfluorobutanesulfonate ( pfbs ) , perfluorooctanoic acid ( pfoa ) , perfluorooctanesulfonate ( pfos ) , and perfluorononanoic acid ( pfna ) were assessed in xenopus laevis a6 kidney epithelial cells by attenuated total reflection fourier - transform infrared ( atr - ftir ) spectroscopy and chemometric analysis . principal component analysis linear discriminant analysis ( pca - lda ) was used to visualize wavenumber - related alterations and anova - simultaneous component analysis ( asca ) allowed data processing considering the underlying experimental design . both analyses evidenced a higher impact of low - dose pfas - treatments ( 109 m ) on a6 cells forming monolayers , while there was a larger influence of high - dose pfas - treatments ( 105 m ) on a6 cells differentiated into dome structures . the observed dose response pfas - induced effects were to some extent related to their cytotoxicity : the ec50-values of most influential pfas - treatments increased ( pfos < pfna < pfoa pfbs ) , and higher - doses of these chemicals induced a larger impact . major spectral alterations were mainly attributed to dna / rna , secondary protein structure , lipids , and fatty acids . finally , pfos and pfoa caused a decrease in a6 cell numbers compared to controls , whereas pfbs and pfna did not significantly change cell population levels . overall , this work highlights the ability of pfass to alter a6 cells , whether forming monolayers or differentiated into dome structures , and the potential of pfos and pfoa to induce cell death .

Introduction Materials and Methods Results Discussion

because of its capacity to interrogate biochemical signals of stressed organisms , attenuated total reflection fourier - transform ir ( atr - ftir ) spectroscopy shows great potential . the use of atr - ftir spectroscopy to address biological questions is viable since biomolecules with chemical bonds having an electric dipole moment absorb in the mid - ir region through their vibrations , giving rise to a detailed biomolecular fingerprint in the form of an ir spectrum . various chemometric methods are suitable for ir data sets , both for exploratory or modeling purposes , including principal component analysis ( pca ) and linear discriminant analysis ( lda ) . these methods allow data reduction to facilitate the identification of wavenumber - related spectral alterations associated with glycogen content , lipid content , conformational changes and phosphorylation characteristics in proteins or structural alterations in dna / rna . another chemometric technique suited for the analysis of ir data sets , especially for those obtained in multifactorial designs , such as that hereby presented , is anova - simultaneous component analysis ( asca ) . , perfluoroalkylated substances ( pfass ) ] permit the investigation of unknown effects of xenobiotics in target organisms . preferred doses of exposure are usually in the nanomolar scale , so as to reproduce real - world low - doses . concerns about pfass have risen due to their widespread distribution and persistence in humans and the environment but also due to their toxicity and ability to act as endocrine - disrupting chemicals ( edcs ) and obesogens . other biosystems might be affected by the presence of pfass , such as the a6 cell line . the renal epithelial a6 cell line was produced in 1969 from the renal uriniferous tubule of the adult african clawed frog xenopus laevis . renal epithelial cells are specialized for absorption or secretion , where the membrane facing the culture media is the apical membrane , the membrane attached to the plastic culture flask is the basement membrane , and the membrane lying along the basement surface is the basolateral membrane ( figure 1a , b ) . the apical membrane of a6 cells contains the epithelial na channel ( enac ) , while na / k - atpase is in the basolateral membrane . the incorporation of na ions by enac and their expulsion into the extracellular space by na / k - atpases causes an accumulation of na ions in the space between a6 cells and the plastic culture flask . a6 cells forming domes have distinct physiological and structural properties ( e.g. , changes in the cytoskeleton ) than a6 cells forming monolayers . because it is easy to culture , the a6 cell line is commonly used in space studies , and several studies investigate the effects of gravitational forces on dome formation . however , little research has been conducted into the effects of xenobiotics on a6 cells as a toxicological model to simulate the effects on amphibians . schematic representation of xenopus laevis a6 kidney epithelial cells forming a monolayer ( a ) and a dome ( b ) . ( g ) experimental design for the study of pfas - induced effects ( refer to section 2.3 ) . the aim of this study was to examine the alterations induced in a6 cells , forming monolayers or differentiated into domes , exposed to four pfas substances [ i.e. , perfluorobutanesulfonate ( pfbs ) , perfluorooctanesulfonate ( pfos ) , perfluorooctanoic acid ( pfoa ) , and perfluorononanoic acid ( pfna ) ] , using atr - ftir spectroscopy and chemometric analysis [ i.e. , pca - lda and asca ; see supporting information ( esi ) for a short description of these methods ] . in addition , a growth - curve experiment was developed to determine whether the four distinct pfas - exposures differentially altered dose- and time - related cell number increases in culture . pfbs and pfos were obtained from fluka ( austria ) , whereas pfoa and pfna were purchased from sigma - aldrich ( steinheim , germany ) . xenopus laevis a6 kidney epithelial cells were obtained from american type culture collection ( atcc ccl-102 ) . they were cultured in modified l15 culture medium consisting of 70% leibovitz media , 19% milli - q water sterile filtered through a 0.2 m syringe filter , 10% fetal bovine serum ( fbs ) , and 1% penicillin / streptomycin , at 5% co2 and 26 c . toward experiments , a6 cells were disaggregated , resuspended in complete medium , and then seeded in t25 flasks at a rate of 500,000 cells per flask whereupon they were grown for the time required depending on the experiment ( see section 2.3 ) . for pfas - treatment , 25 l of stock solutions were added to 5 ml of the culture medium so that final exposure - doses ranged from 0 to 10 m [ considering 0 m as zero - dose control , in which cells were only exposed to the carrier solvent ( dmso ) ] . the effects of the four pfas substances were studied on a6 cells forming monolayers or domes in three distinct experiments [ figure 1g1,2,3 ] . in experiment 1 [ figure 1g1 ] , pfas - induced effects were evaluated in cells forming confluent monolayers . in experiments 2 and 3 [ figure 1g2,3 , respectively ] , pfas - effects were evaluated in cells forming domes ( 9-day experiment ) . in experiment 2 , cells were pfas - exposed at four concentrations ( 0 , 10 , 10 , or 10 m ) , while in experiment 3 , three concentrations ( 0 , 10 , or 10 m ) were tested . low number of replicates was performed in experiment 3 , as the results obtained are used to extract tentative conclusions about long - term pfas - induced effects considering the physiological conditions of cells forming domes . raw ir spectra obtained from exposed and control samples were preprocessed prior to chemometric analysis ( see figure s1 ) . pca - lda was applied to the spectral data sets using matlab 8.3.0 r2014a ( the math works , natick , ma , usa ) and the irootlab toolbox ( http://irootlab.googlecode.com ) . response effects of pfass , by examining the proximity in multivariate distance between exposed and control samples . response effects of pfbs , pfoa , pfos , and pfna in the three experiments . in these asca models , the effects of two categorical factors [ i.e. , type of chemical , with four levels ( pfbs , pfos , pfoa , or pfna ) , and dose of exposure , with a number of levels differing among experiments ( see section 2.3 ) ] and interaction were studied . a6 cells coming from 90% confluent t75 flasks were disaggregated , resuspended in complete medium , and seeded in plastic culture tissue coverslips ( sarstedt , usa ) in 30 mm petri dishes at a rate of 200,000 cells per petri dish and allowed to attach overnight at 26 c . finally , cells were fixed using 70% etoh ( 40 min ) , washed twice with 70% etoh , and kept at 4 c until microscopy visualization . a nikon coolpix 950 camera , mounted via a nikon coolpix mdc lens 0.820.29 adapter to a nikon eclipse te300 inverted microscope , fitted with a nikon plan apo 60/1.20 water immersion objective was used to obtain images of giemsa - stained cells ( figure 1c , d ) . also , a confocal microscope , leica dmire2 inverted microscope connected to a leica tcs sp2 scan head and phase contrast settings , was used to obtain images of figure 1e , f . a6 cells were seeded at a ratio of 500,000 in t25 flasks in 5 ml of complete medium containing individual chemicals ( pfbs , pfos , pfoa , or pfna ) at concentrations of 10 , 10 , or 0 m ( control ) . this point was taken as time zero ( t0 ) , and duplicate cell counts in triplicate flasks were acquired . cells were washed , trypsinized , resuspended , and the cell number determined at indicated time points employing a hemocytometer . in the present study , ir data sets were first evaluated with 12 pca - lda models and further examined with 3 asca models . response effects of each pfass in the three experiments , whereas asca allowed data analysis considering the underlying experimental design . results of the 12 pca - lda ( figure 2 ) evidenced a distinct dose response pattern in experiment 1 compared to experiments 2 and 3 . in the first experiment , the highest distinction between treated and control cell populations was observed at the lowest concentration tested ( 10 m ) in all pfas - treatments . a similar tendency was observed in the two last experiments : pfos and pfna caused in both conditions the highest alterations at 10 m , and pfoa produced the highest impact at low doses of 10 and 10 m in experiments 2 and 3 , respectively . primary wavenumbers important for discrimination of pfas - treatments at the concentration producing more effects in the three experiments together with the molecular entities associated with them are shown in table s1 and can be visualized in the cluster vectors plots of figure s2 . results of this evaluation were attributed to the dominant part of variation to natural variability ( residuals 85% ) and the minor part to factors and interaction ( 12% ) , observing higher effects in experiment 3 . despite the observed small pfas - induced effects on a6 cells , the good reproducibility of the atr - ftir technique allows the extraction of reliable conclusions about their impact on a6 cells in the present study . [ tc values of equation s2 ] shown in figure 3 indicate that chemicals producing more effects were pfos , pfoa , and pfbs in experiments 1 , 2 , and 3 , respectively . scores of these figures indicate that concentrations of exposure producing more effects were 10 , 10 , and 10 m in experiments 1 , 2 , and 3 , respectively . the results of the permutation test evidenced that the pfas - dose was significant in experiments 1 and 2 with p - values of 1 10 and 1 10 , respectively , but not significant in experiment 3 , with a p - value of 8 10 ( table 1 ) . scores of the first component of the interaction showed no pattern related to the interaction of factors : there was no increasing or decreasing trend of scores of the different doses of exposure respect to the chemical treatment . the effects of the four pfass on the increases in a6 cell number are shown in figure 4 . the behavior observed was similar between pairs of chemicals pfbs and pfna vs pfos and pfoa . however , 24-h and 48-h treatments with pfos and pfoa caused a significant decrease in cell number , which became higher with time and gave rise to maximum cell depletion after 48 h of exposure . effects of pfbs , pfna , pfos , and pfoa on dose- and time - related cell number increases in culture . our study evidenced a primary effect of low - dose ( 10 m ) pfas - treatment on a6 cells forming monolayers ( experiment 1 ) ( figure 2 ) . predominant effects of environmental stressors at low - doses of exposure have often been reported in the literature , especially for edcs , such as pfass . big effects at low doses , a theory primarily defined by the national toxicology program ( http://ntp.niehs.nih.gov/ ) , and second , the nonmonotonicity ( i.e. , nonlinear relationship between dose and effect ) thus declaring the dogma it is important to stress that our findings in experiment 1 ( cells forming a monolayer ) followed this low - dose theory , while results of experiments 2 and 3 ( cells differentiated into dome structures ) showed an opposing tendency ( major alterations at high doses ) . such differences can be explained by the specific physiological status of cells forming monolayers or domes , the latter having enhanced cell excretion function and requiring higher pfas - doses to produce an effect . also , the differential dose response effects of pfas substances depending on cell differentiation status were to some extent related to the cytotoxicity of most influential chemicals ( according to asca results ) in each experiment . considering the median effective doses ( ec50 ) of pfass reported in a previous study on jeg-3 cells [ i.e. , pfos ( 107125 m ) < pfna ( 213220 m ) < pfoa ( 594647 m ) pfbs ( n.d . ) ] , it was observed that major effects in experiment 1 ( primarily influenced by high - cytotoxic pfos ) occurred at low doses , whereas larger effects in experiments 2 and 3 ( primarily influenced by less - cytotoxic pfoa and pfbs , respectively ) were observed at higher doses . considering overall effects , a greater impact of pfas substances was evident in experiment 3 , presenting the treated samples to maximum cluster segregation compared to that of the control in 1-d pca - lda score plots ( figure 2 ) . the higher effects of pfas substances in experiment 3 vs experiment 2 might be attributed to a cellular recuperation after the chemical stress possible in the second conditions since cells were allowed to grow for 7 days in fresh medium after pfas - exposure , a time that cells might use to eliminate those pfas substances previously incorporated . interestingly , distinct spectral alterations were induced by pfas substances in the three experiments , suggesting three mechanisms of action of the chemicals depending on cell differentiation ( i.e. , pre- or postdome formation ) , and cell population . in experiment 1 , all pfas substances produced alterations associated with dna / rna ( e.g. demonstrated that pfoa and pfna induced dna damage in tk6 cells , observing that pfna produced high levels of 8-hydroxy-2-deoxyguanosine ( 8-ohdg ) , a biomarker of oxidative dna damage . however , the observed differences might be attributed to the different numbers of cells between both experiments and their dissimilar physiological properties ( i.e. continuing with the study of distinguishing features induced by pfas treatments , special attention was focused on lipids , due to the reported capacity of pfas substances to alter lipid species of cellular membranes . , phosphatidylcholine ( pc ) , lyso plasmalogen pc , and plasmalogen pc ] and a relatively low increase in triacylglicerols ( tag ) were induced by pfas substances . similarly , our findings showed some effects of pfas on lipids of a6 cells . as observed in table s1 , in experiment 1 pfos and pfoa produced effects at 1,736 cm ( c = o stretching lipids ) and at 1,444 cm ( lipids ) , respectively , and in experiment 2 pfbs induced alterations at 1,750 cm , associated with a c = c stretching in lipids , as occurred with cells exposed to pfna in experiment 3 . the findings of this study demonstrate that pfas substances pose a significant effect on the metabolome and lipidome of xenopus laevis a6 cells . moreover , the fusion of ir data with lc - ms data is also worthy since it can provide a more comprehensive knowledge of the effects of pfas substances in this amphibian cell model . finally , results of the growth - curve experiment showed different responses of a6 cells exposed to pfbs / pfna compared to pfos / pfoa ( figure 4 ) . pfbs and pfna did not induce distinguishable a6 cell proliferation or depletion compared to those of the control . in contrast , pfos and pfoa induced a decrease in a6 cell number compared to that of the control , in a time- and dose - specific manner : after 48 h of treatment , a time when the two pfas substances presented maximum effects , high - dose pfos produced the most profound cell decreases , whereas low - dose pfoa caused the maximum decline in cell population . the capacity of pfos and pfoa to inhibit cell proliferation has been reported in other studies . overall , this work contributes to the better knowledge of pfas substance effects on xenopus laevis a6 kidney epithelial cells indicating an overall interference with dna / rna , secondary structures of proteins , lipids , and fatty acids at concentrations well below those associated with other adverse effects , such as cytotoxicity or endocrine disruption . this work also highlights ( a ) the differential effects of pfas substances depending on cell - differentiation , presenting a nonmonotonic - behavior on a6 cells forming monolayers and ( b ) the ability of pfos and pfoa to induce cell death .

cyclin - dependent kinases ( cdks ) regulate cell cycle progression and rna transcription in different cell types . cdks form complexes that influence several upstream and downstream pathways regulating cell cycle , cell proliferation , and apoptosis . since alterations in cell cycle progression occur in several malignancies , inhibition of cdks is regarded as a promising target for cancer treatment . among the cdks responsible for cell cycle progression cdk2 is an inherently flexible protein with many conformations needed for interactions with various ligands . cdk2 regulates cell cycle progression by forming ( a ) cyclin e - cdk2 complexes at the boundary of g1 to s transition and ( b ) cyclin a - cdk2 complexes for orderly s phase progression and g2 to m phase transition . using structural - drug design several small molecules and peptides have been developed to target atp binding subsites or other important binding sites needed for active confirmation of cdk2 . creating highly selective cdk2 compounds is a challenge due to the identity of atp binding subsites within cdk1 , cdk2 , and cdk3 molecules ; cdk2 also possesses 92% and 80% sequence identity in cdk5 and cdk6 molecules , respectively ( rcsb protein data bank code : 1b38 ) . in order to affect cdk2 binding to a specific ligand various specific cdk2 inhibitors have been shown to be effective in inducing apoptosis and reducing proliferation of various cancer cells . in normal cells an induced cell cycle arrest has been shown to be reversible [ 3 , 4 ] . only a weak g1 arrest has been observed in cdk2/ mefs [ 5 , 6 ] or after sirna ablation in established tumor cell lines . an arrest of the cell cycle in the g1 phase has however been detected in cells that have been synchronized and released from a nocodazole - induced mitotic block . additionally the cdk2 inhibitor flavopiridol was more cytotoxic to transformed cells when treated within the s phase . cells in certain cell cycle phases are thus likely more sensitive to cdk2 inhibition . some cancer cells however possess resistance to cdk2 inhibition , as shown by a unique upregulation of cdk2 target proteins and preexisting cellular polyploidy in cancer cells . among cdk2 inhibitors those with purine - based structures ( nu6140 and its derivatives ) have shown higher specificity to inhibit cdk2 interaction with cyclin a compared to other interactions ( cdk1/cyclin b , cdk4/cyclin d , cdk5/p25 , and cdk7/cyclin h ) [ 11 , 12 ] . nu6140 induces apoptosis in hela cervical carcinoma cells , arrests cells in the g2/m phase , and reduces cell survival both by itself and in combination with paclitaxel . in epithelial cells exactly how nu6140 affects the cell cycle in carcinoma - derived cells and whether the effect is reversible have remained unclear . several specific features of human embryonic stem ( he s ) cells are of special interest in studying the effect of cdk2 inhibition . first , he s cells are characterized by both unlimited proliferative potential and pluripotency , providing them with the capacity to differentiate into all three cell lineages ectoderm , endoderm , and mesoderm [ 1517 ] . the capacity to differentiate provides an opportunity to investigate whether cdk2 inhibition could alter the differentiation potential of these cells . second , he s cells possess a unique cell cycle profile with an abbreviated g1 phase and long s phase . third , a recent study on phosphoproteome of he s cells during differentiation revealed that cdk2 and cdc2 activities were central in promoting pluripotency and self - renewal . these three features taken together suggest he s cells can be a useful model for investigating the outcome of cdk2 inhibition . the broad - spectrum inhibitor of cdks roscovitine has been shown in he s cells to inhibit cdk1 , -2 , and -5 , cause g1/s arrest , accumulate hypophosphorylated rb , result in smaller cell colonies , and downregulate the pluripotency marker oct4 . modulating cdk2 expression by using small interfering rna ( sirna ) can result in the arrest of cells within the g1 phase and induce differentiation of cells into extraembryonic lineages . expression of p21 , a natural inhibitor of cdk2 , is associated with g1 arrest in he s cells . previous studies indicate the important role of cdk2 activity in modulating the he s cell cycle . it is important therefore to clarify how the cdk2 inhibitor nu6140 changes cell cycle and pluripotency of he s cells . pluripotency of he s cells is generally maintained by a transcriptional network coordinated by the three transcription factors oct4 , nanog , and sox2 . transcription factors ( tfs ) have been shown to function as cellular rheostats ; that is , small changes in tf expression result in big changes in self - renewal and pluripotency of he s cells . he s cell pluripotency and self - renewal are maintained and regulated by cell cycle regulators . expression of transcription factors characteristic to he s cells has been found in several cancers and is often associated with a poor clinical outcome . embryonal carcinoma - derived cells ( hec , ntera-2 , bg01v , and 2102ep ) have several advantages as a tool to study stem - cell - like signatures in carcinoma cells as they generally show expressions of proteins and mrnas specific to he s cells and possess low rates of spontaneous differentiation . the effect of cdk2 inhibitors on the proliferation of various cancer cells has been extensively studied , but less attention has been paid to the ability of cdk2 inhibitors to modulate the expression of transcription factors responsible for pluripotency . in this study we aimed to characterise the effect of the cdk2 inhibitor nu6140 on the cell cycle in carcinoma - derived cells compared to he s cells . we applied multicolor flow cytometric methods to a single cell analysis of the expression of cdk2 and pluripotency markers in different cell cycle phases . as a consequence of our first experiment another important question we wanted to answer was whether the modulation of transcription factors responsible for pluripotency ( nanog , oct4 , and sox2 ) was reversible and long lasting . in addition we also estimated the effect of cdk2 inhibition on the differentiation potential of he s cells . this study was conducted using a commercially available human embryonic stem cell line ( wa09-h9 , national stem cell bank , madison , wi , usa ) ; no in vivo experiments on animals or humans were performed and therefore approval from an ethics committee was unnecessary . the human embryonal carcinoma - derived ( hec ) cell line 2102ep ( globalstem , usa ) was maintained in a dmem medium ( containing glucose 4.5 g / l ) ( gibco life technologies ) containing 10% fetal bovine serum ( paa laboratories , linz , austria ) and mem nonessential - amino - acids - solution ( 0.1 mm , invitrogen , usa ) . ( wa09 , national stem cell bank , madison , wi , usa ) was maintained on matrigel ( bd biosciences , san jose , ca , usa ) coated plates in a mtesr1 maintenance medium ( stemcell technologies inc . , after 3 - 4 days of growth cell colonies were detached mechanically with a micropipette tip and broken up with gentle pipetting and the resulting individual he s cell clumps were plated onto fresh matrigel coated plates . anti - nanog ( pe conjugate ) , anti - oct4 ( alexa fluor 647 conjugate and percp - cy5.5 conjugate ) , and anti - sox2 ( percp - cy5.5 conjugate ) antibodies , plus their isotype control antibodies , were purchased from bd biosciences . anti - cleaved - caspase 3 and anti - histone 3 antibodies detecting phosphorylated serine 10 were purchased from cell signaling ( usa ) . anti - cdk2 antibodies were obtained from cell signaling and from santa cruz biotechnology ( san diego , ca , usa ) . anti - sox1 ( northernlights conjugate , nl-493 ) , anti - otx-2 ( nl-557 ) , anti - brachyury ( nl-557 ) , anti - hand1 ( nl-637 ) , anti - gata4 ( nl-493 ) , and anti - sox17 ( nl-637 ) antibodies were purchased from r&d systems ( abingdon , oxon , uk ) . nocodazole and nu6140 ( sigma - aldrich chemicals , st . louis , mo , usa ) were dissolved and diluted in dmso ( sigma - aldrich chemicals ) . cells with confluence levels of approximately 6070% ( 3 - 4 days after passage for he s cells , 1 - 2 days for hec cells ) on 6-well plates were treated for further 24 h with 100 ng / ml nocodazole , 10 m ( or various concentrations ) nu6140 , or 0.1% dmso as a control ( corresponding to the concentration of dmso in 10 m nu6140 solution ) or grown without treatment at 37c in 5% co2 humidified atmosphere . a dmso concentration of 0.1% used in assays is reported to have less influence in he s cell differentiation than higher concentrations of dmso [ 26 , 27 ] . after harvesting with 0.05% trypsin - edta solution ( paa laboratories , linz , austria ) and washing with pbs , single he s cell suspensions were fixed using 1.6% paraformaldehyde ( pfa , sigma - aldrich ) for 10 min at room temperature ( rt ) as described for the detection of intracellular phosphoproteins [ 28 , 29 ] . cells were then washed with permeabilisation buffer ( foxp3 staining buffer set , e - biosciences ) . he s cells were blocked using 2% goat serum ( paa laboratories ) in a permeabilisation buffer ( 10 min at rt ) and stained with appropriate antibodies or their isotype controls for 30 min at rt . for cell cycle analysis cells were further stained with dapi ( cystain dna , partec gmbh , mnster , germany ) . flow cytometry data were acquired with facsaria using facsdiva software ( bd biosciences ) . for analysing 2102ep cells after fixation with 1.6% pfa cells were permeabilized with ice - cold methanol for 20 min at 4c , washed with pbs containing 5 mm edta , blocked with 6% goat serum in a permeabilisation buffer , and then stained with antibodies as previously described for he s cells . for resuspension of 2102ep cells the pbs buffer containing 5 mm edta was used . when cells were harvested only for cell cycle analysis they were stained with propidium iodide after ethanol fixation . cell permeabilisation , fixation , staining , and data acquisition for all samples were done on the same day . for more accurate analysis we used sequential selection of cell populations only single cells were selected for detecting cells in different cell cycle phases and only these cells were used to analyse for the expression of various parameters ( see figures 16 ) , allowing the minimisation of nonspecific signals . protein samples were electrophoresed on sds polyacrylamide gel ( 10% ) and transblotted ( minitransblot cell , bio - rad , hercules , ca ) onto a polyvinylidene difluoride membrane ( millipore , billerica , ma ) . membranes were probed with rabbit anti - nanog antibodies ( aviva systems biology , san diego , ca , usa ) , mouse anti - oct4 antibodies ( santa cruz biotechnology ) , and anti - sox2 antibodies ( abcam , cambridge , ma , usa ) followed by horseradish peroxidase - conjugated goat anti - rabbit ( cell signaling ) or goat anti - mouse secondary antibodies ( labas , tartu , estonia ) . a mouse anti - beta - actin antibody ( abcam ) was used for detecting loading control . binding of antibodies was detected with ecl reagent ( western lightning plus - ecl , perkinelmer inc . , waltham , ma , usa ) and exposure of blots by using bioscpectrum 510 imaging system with visionworks ls software ( both from ultra - violet products ltd . , upland , ca , usa ) . he s cells with confluence levels of approximately 6070% ( 3 - 4 days after passage ) on matrigel were treated with 10 m nu6140 for 24 h and then a further 72 h with fresh mtesr1 maintenance medium . embryoid bodies ( ebs ) were formed by growing he s cells in suspension in a low attachment culture plate with knockout dmem / f12 media ( gibco , life technologies ) supplemented by 20% knockout serum replacement , nonessential amino acids ( 0.1 mm ) , glutamax ( 2 mm ) , and beta - mercaptoethanol ( 0.1 mm ) . the ability to form ebs was assessed visually using a microscope with a 37c heated stage . a two - tailed paired t - test with a confidence interval of 95% the distribution of cells in each phase of the cell cycle was calculated using modfit software . first we assessed the ability of the cdk2 inhibitor to influence the expression of pluripotency markers in he s cells . using flow cytometric analysis the detection of the expression of transcription factors ( nanog , oct4 , and sox2 ) was combined with the analysis of cell cycle profiles . in untreated and dmso treated he s cells the expression of nanog and oct4 was high ( 95.6% and 98.1% nanog+oct4 + cells in dmso treated or untreated cells population ) . treatment of cells with nocodazole or nu6140 decreased nanog+oct4 + cell population ( 4.2% and 77% after nocodazole and nu6140 treatment , resp . , figure 1 ) and increased the number of cells without nanog and oct4 expression ( 91.1% and 11.3% after nocodazole and nu6140 treatment , resp . ) . the significant correlation between nanog and oct4 expression was similar to that detected in our previous study . in dmso treated cells all nanog+oct4 + cells expressed sox2 , indicating that sox2 is important for pluripotency . in nocodazole treated cells three subpopulations of cells were detected , including those expressing as follows : ( i ) all three transcription factors ; ( ii ) only sox2 without nanog and oct4 expression ( 3234% ) ; ( iii ) no detectable sox2 expression ( 5863% , figure 1(a ) ) . nu6140 treatment had a minimal effect on reducing sox2 expression ( 2 - 3% of cells were without sox2 expression ) and the number of surviving cells expressing all three markers ( sox2 , nanog , and oct4 ) was higher compared to cells treated with nocodazole ( 7786% and 47% with nu6140 and nocodazole , resp . ) . changes in tfs expression were also detected at the population level using the western blot technique where all adherent cells were collected for analysis ( supplementary material figure 1(c ) available online at http://dx.doi.org/10.1155/2014/280638 ) . the number of he s cells decreasing after treatment neared significance with nu6140 ( p = 0.06 ) and was significant for nocodazole ( p < 0.01 ) when they were compared to dmso treated cells , indicating that both agents were able to inhibit cell proliferation ( supplementary material figure 1(a ) ) . to compare the effects of nu6140 on he s and hec cells we carried out a similar experiment on 2012ep cells to the previous experiment on he s cells . for accurate detection of tfs in 2102ep cells we found that a fixation with 1.6% pfa and permeabilisation with ice - cold methanol resulted in optimal detection of tfs and minimisation of nonspecific signals . a decreased expression of nanog and oct4 in surviving cells ( 51% of nanog+oct4 + cells , figure 2(a ) ) was detected in nocodazole treated cells compared to those treated with dmso ( 94% of nanog+oct4 + cells ) . the expression of nanog and oct4 was high in surviving cells after nu6140 treatment ( 81% compared to 94% nanog+oct4 + cells using dmso , figure 2(a ) ) . after nocodazole treatment the number of sox2 expressing cells coexpressing nanog and oct4 was lower ( 5559% versus 8490% ) and a population of cells expressing only sox2 higher , when compared with nu6140 treatment ( 2630% versus 410% ) . the number of cells without sox2 expression was higher in nocodazole treated cells ( 13 - 14% ) than in nu6140 treated cells ( 3 - 4% ) . western blot analysis of adherent cells showed notable changes in the expression of tfs ( supplementary material figure 1(d ) ) . nu6140 and nocodazole were significantly ( p < 0.05 , supplementary materials figure 1(b ) ) effective in reducing the proliferation of hec cells . the effects of nocodazole and nu6140 were similar in he s and hec cells , but he s cells were more sensitive than hec cells . another important issue from a clinical point of view is the recovery of cells after the removal of an inhibitor and the further culture of these cells under normal conditions . we investigated whether the expression of transcription factors is restored after the washout of nocodazole or nu6140 and a further culturing of cells in a fresh medium for 24 h. after release from the nocodazole - block an increase in nanog and oct4 expression was detected ( from 4% to 77% nanog+oct4 + cells , figure 1(a ) ) in he s cells , accompanied by a decrease in the subpopulation without nanog and oct4 expression ( from 91% to 14% , figure 1(a ) ) . this rather quick recovery in nanog and oct4 expressions detected after 24 h was different from our previous report where no significant increase was noticed after removal of nocodazole . since he s cells in this study were cultured on matrigel feeder surface but in previous experiments we used he s cells first cultured on mef feeder and then plated on matrigel coated plates , these differences in culturing could affect the growing rate , colony formation , and recovery of cells . nu6140 application and its release increased the population of he s cells expressing nanog and oct4 from 77% to 98% ( figure 1(a ) ) . similar changes in sox2 expression were also detectable in he s cells both with treatment of nocodazole or nu6140 and after their release from these agents . in treatments using nocodazole or nu6140 we detected amongst the released cells a high number of he s cells expressing sox2 and a smaller subpopulation expressing only sox2 without nanog and oct4 expression . the number of sox2 expressing cells without nanog and oct4 expression changed from 5863% to 11 - 12% in nocodazole treated and released cells and from 2 - 3% to 0.3% in nu6140 treated and released cells . when 2102ep ( hec ) cells were used instead of he s cells , the removal of nocodazole resulted in similar effects ; however removal of nu6140 had a different effect on the expression of all three tfs in hec cells . the number of cells coexpressing nanog and oct4 remained almost the same after nu6140 treatment and removal ( 81% and 80% , resp . , figure 2(a ) ) , but an increase in the cell subpopulation without sox2 was noticed after release ( from 4% to 11% ) though the number of cells expressing all three tfs remained at a similar level throughout . he s cells were able to restore pluripotency marker expression after the removal of nocodazole and nu6140 , with the number of cells remaining unchanged and no significant increase in proliferation of cells detected ( supplementary material figure 1 ) . similar effects of nocodazole treatment and its removal were found in hec cells , but nu6140 caused long lasting inhibition in the pluripotency marker sox2 expression . after washout of nocodazole or nu6140 , he s cells recovered more efficiently than hec cells , indicating that different mechanisms regulate the expression of pluripotency markers in he s and hec cells . as a result of finding the differences in he s and hec recovery after treatment with nu6140 , we investigated the correlation between the levels of pluripotency markers and cdk2 expression in he s and hec cells . the focus was primarily on the pluripotency markers nanog and sox2 , because the expression of oct4 correlated well with the expression of nanog . first it was confirmed that up to 95% of cells ( untreated and control dmso treated cells ) expressed cdk2 and among these two populations of he s cells the following were detected : ( i ) a majority population expressing nanog and cdk2 ( 9699% ) and ( ii ) a smaller cell population expressing only cdk2 without nanog expression ( 1 - 2% ) . nocodazole treatment of he s cells resulted in ( i ) a decrease in cdk2 and nanog coexpressing cells ( from 99% to 11% ) ; ( ii ) an increase in cells expressing cdk2 without nanog expression ( from 0.4% to 81% , figure 1(b ) ) ; ( iii ) an increase in the number of cells without nanog and cdk2 expression ( from 0.2% to 7% , figure 1(b ) ) . after release from nocodazole arrest and further culturing in fresh medium , all he s cells restored the expression of cdk2 , 79% of cells expressed cdk2 and nanog , and 15% of cells expressed cdk2 without nanog expression ( figure 1(b ) ) . when nu6140 was used instead of nocodazole , the number of cdk2 and nanog coexpressing cells increased from 82% to 98% after removal of nu6140 and the cdk2+nanog subpopulation decreased from 16% to 0.9% . cells without cdk2 expression were negative for sox2 expression and this subpopulation of cells appeared only with nocodazole treatment and not after its removal ( figure 1(b ) ) . similar to he s cells almost all untreated 2102ep cells expressed cdk2 . for 2102ep cells both agents nocodazole and nu6140 downregulated cdk2 expression compared to dmso treated cells ( 2630% and 57% cdk2 cells after nocodazole or nu6140 treatment , resp . cdk2 expression was restored after the removal of inhibiting agents nocodazole ( 2 - 3% cdk2 cells ) or nu6140 ( 35% cdk2 cells ) . a correlation was found between gradual downregulation of cdk2 expression levels and expression level of nanog and sox2 ( figure 2(b ) ) . nocodazole and nu6140 both affected cdk2 expression in hec cells , which was different from nu6140 treatment of he s cells where no change in cdk2 expression was detected . as a consequence of our results showing the effect of nu6140 on cdk2 expression , we analysed the effect of nu6140 on the distribution of cells within the different cell cycle phases in he s and hec cells , which possess different cell cycle profiles . in he s cells a low concentration ( 1 m ) of nu6140 had a minimal effect on cell cycles . higher concentrations ( 5 and 10 m ) of nu6140 increased the number of he s cells in the g2/m phase and caused a decrease of cells in the g1 phase ( figure 3(a ) ) . higher concentrations of nu6140 also decreased the number of he s cells within the s phase . after treatment with nocodazole , which arrests cells in the g2/m phases by interfering with the polymerization of microtubules , most of the cells were in the g2/m phase ( figure 3(a ) ) . similar effects in cell cycle distribution in he s cells were also found for 2102ep cells with nu6140 and nocodazole treatment ( figure 3(a ) ) . both nu6140 and nocodazole increased the number of cells within the g2/m phase and decreased it within the g1 phase , with the effect on the s phase minimal . to distinguish the cells belonging to the g2 or m phases we used the phosphorylation of histone 3 ( h3 ) at serine 10 as a specific marker of cells in m phase . nocodazole increased the number of he s cells within the m phase ( 17% of cells possessed phosphorylated h3 at serine 10 after nocodazole treatment compared to 2% after dmso treatment , figure 3(b ) ) . there was no increase in phosphorylation of h3 at ser10 ( 1.92.5% ) after nu6140 treatment , showing these cells accumulate preferably in the g2 , not the m phase ; this effect was similar in hec cells ( data not shown ) . to find out whether nu6140 or nocodazole had any effect on the expression of cdk2 in cells within the m phase we estimated cdk2 expression level in cells with phosphorylated h3 . in he s cells treated with nocodazole the expression level of cdk2 was lower in m phase cells ( which expressed both cdk2 and phospho - ser10-histone ) and significantly higher in cells within other cell cycle phases ( figure 3(b ) ) . a similar low level of cdk2 in the m phase was detected by applying increasing concentrations of nu6140 . when the mean fluorescence values of the two cell populations ( cdk2 + cells with and without phosphorylated h3 ) were compared , a lower expression of cdk2 in cells within the m phase compared to cells in other cell cycle phases was detected after nu6140 application ( figure 3(c ) ) . since nu6140 treatment decreased the number of adherent he s cells and downregulated expression of pluripotency markers nanog , oct4 , and sox2 , we asked whether these effects could be due to apoptosis of he s cells . cleaved caspase 3 was used as a late apoptosis marker to detect apoptotic cells within adherent cell populations expressing the pluripotency marker nanog . nu6140 treatment induced cleavage of caspase 3 in 4.3% of he s cells without nanog expression and in 2.2% of cells that still expressed nanog ( figure 4(a ) ) . nocodazole induced apoptosis in he s cells ( 17.7% of cleaved caspase 3 positive cells , figure 4(a ) ) , but nanog expression was not detectable in most apoptotic cells . in hec cells nu6140 increased the number of apoptotic cells with 0.6% of cleaved caspase 3 positive cells not expressing nanog and 7.8% of cells still expressing nanog ( figure 4(b ) ) . in nocodazole treated hec cells 4.4% cleaved - caspase-3-positive cells were without nanog expression and 9.8% of cells expressed nanog . since cleaved caspase 3 has been reported to mediate differentiation of he s cells , we analyzed nu6140 treated he s cells for the expression of gata4 as a marker of early differentiation [ 34 , 35 ] . in our study a small number of gata4 expressing cells was detected in dmso treated and untreated he s cells . after nu6140 treatment the number of cells expressing gata4 slightly increased ( 4.3% compared to 2.3% gata4 + cells after nu6140 or dmso treatment , resp . most of the gata4 expressing cells also expressed nanog in nu6140 , dmso , and untreated cells , indicating that expression of both pluripotency markers and differentiation markers can be detected in the same cell during early stages of differentiation . cdk2 inhibitors have been reported to possess the ability to arrest cells in the g1 phase after synchronization with a nocodazole - induced mitotic block . we tested the effect of nu6140 on nocodazole - arrested cells after a shortened treatment period with nocodazole ( 10 h instead of 24 h ) in order to obtain a higher number of surviving cells . in both he s and hec cells the number of cells coexpressing nanog and oct4 decreased slightly as a result of nocodazole arrest ( figure 5 ) . after careful washing with fresh mtesr1 maintenance medium cells were treated for a further 14 h with a medium containing 10 m nu6140 . this further treatment of cells with nu6140 decreased more efficiently the expression of nanog and oct4 . combined treatment with nocodazole followed by nu6140 resulted in 25% he s and 26% hec cells without expression of any of three tfs ( nanogoct4sox2 cells ) among surviving cells . treatments with nocodazole or nu6140 alone were not as effective in decreasing expression of all three tfs . combined treatment with nocodazole followed by nu6140 was more effective in decreasing expression of all three tfs in he s cells than hec cells , though the tendencies were similar . we also tested the recovery of cells to express nanog , oct4 , and sox2 after washout of nocodazole or nu6140 after 10 h treatments ( figure 5 ) and results were similar to those obtained with 24 h treatment period ( figures 1(a ) , and 2(a ) ) . figure 5(e ) shows the changes in he s cell colony structures after treatment with nocodazole and nu6140 . nocodazole treatment caused significant changes in colony structure and the surviving cells possessed specific characteristics of cells arrested in m phase ( round - shape larger cells ) . after removal of nocodazole fewer round - shape cells were detected and the formation of new small colonies was recognized . nu6140 treatment following nocodazole treatment did not reverse the effect of nocodazole , but rounded cells were detectable and the remaining colonies were similar to those after treatment with nu6140 alone . nu6140 treatment alone removed the single - cell layer from the colony edges and cells located in the center of the multicell layer survived . after removal of nu6140 he s cell colonies consisted only of multicell layers . cells treated with nu6140 had a compact , tightly packed colony structure that appeared to protect he s cells and support cell survival ( figure 5(e ) ) . the next important question to answer was whether nu6140 treated and released he s cells could maintain pluripotency and form colonies . it is known that colony formation consists of 4 stages : attachment , migration , aggregation , and formation . after removal of nu6140 and a further 3 days of culture no further propagation in colony formation was noticed ( figure 6(a ) ) . when he s cells were analysed for expression of pluripotency factors , 42% of cells expressed nanog , oct4 , and sox2 3 days after treatment with nu6140 . among untreated he s cells cultured for the same time period 60% of he s cells expressed all three tfs ( figure 6(b ) ) . in order to distinguish undifferentiated cells from differentiated cells , differentiation markers gata4 ( endodermal lineage ) , otx-2 ( ectodermal lineage ) , and hand1 ( mesodermal lineage ) were used . otx-2 expressing cells ( 13.2% ) and some gata4 expressing cells ( 1.8% ) were detected after release from nu6140 and further culture for 3 days , indicating some differentiation process had already started . in comparison , among untreated cells cultured for the same time period 12.8% otx-2 expressing cells were detected , but no gata4 and hand1 expressing cells . in both untreated and nu6140 treated cells differentiation into ectodermal lineage ( otx-2 expression ) was detected , when he s cells were cultured in conditions which supported pluripotent state . next we applied common embryoid bodies ( ebs ) formation protocols in order to characterize the differentiation ability of nu6140 treated and released he s cells . nu6140 treatment of he s cells had no effect on formation of ebs ( figure 6(a ) ) . after 4 days , embryoid bodies were analysed for the expression of ectoderm ( sox1 ) , mesoderm ( brachyury ) , and endoderm ( sox17 ) markers and pluripotency marker oct4 simultaneously by using flow cytometric method . we could detect lower number of ectodermal marker sox1 expressing cells from ebs formed from nu6140 treated cells compared to ebs formed from untreated he s cells ( 39% versus 78% , figure 6(c ) ) . the number of mesodermal marker brachyury expressing cells was also lower in ebs formed from nu6140 treated cells ( 39% versus 71% of untreated ebs ) . endodermal marker sox17 expression was detected in 4.5% of ebs formed from nu6140 treated he s cells , but in untreated ebs sox17 expression was detected only in 2.7% of cells . the number of oct4 expressing cells was higher in ebs formed from nu6140 treated he s cells ( 1721% ) than from untreated he s cells ( 1.92.8% ) . oct4 expression was not detected in these cells , which expressed brachyury , sox1 , or sox17 ( figure 6(c ) ) . the distribution of cells within cell cycle phases was similar in both ebs formed from nu6140 treated he s cells or from untreated cells ( 62% , 23% , and 15% versus 61% , 18% , and 21% in g0g1 , s , and g2/m phases , resp . ) . flexibility of cdk2 molecules allows interactions with various ligands to regulate progression and proper dynamics of cell cycles . in this study we focused on the small molecule nu6140 , which competes with atp for binding to the complex of cdk2 and our experiments using nu6140 treatment focused on changes in the expression patterns of cell pluripotency markers for human embryonic h9 stem cells ( he s ) and embryonal carcinoma - derived 2102ep cells ( hec ) . we found that both he s and hec cells were sensitive in suppressing the expression of nanog and oct4 with nocodazole or nu6140 , whereas the effect on sox2 expression was more pronounced after nocodazole treatment . after the removal of nocodazole or nu6140 , the expression of nanog , oct4 , and sox2 was restored in he s cells ; a similar tendency was detected in hec cells after nocodazole treatment and release . analysing embryoid bodies ( ebs ) revealed that differentiation into ectodermal and mesodermal lineages was affected in ebs formed from nu6140 treated he s cells compared to ebs from untreated he s cells . several cdk inhibitors have been shown to be effective in inhibiting the proliferation of and inducing apoptosis in various cancer cells . our finding that application of nu6140 also increased apoptosis and inhibited growth of he s and hec cells was therefore expected . the effect of nu6140 on the expression of pluripotency markers sox2 , nanog , and oct4 had not however been reported before this study . the expression of stem - cell - like tfs has been found in many cancers with poor treatment outcomes . it is therefore important to understand the abilities of potential medical agents in modulating the expression profiles of stem - cell - like tfs using in vitro testing . the differences in sensitivity to nu6140 in he s and hec cells found in this study point to the different roles of cdk2-cyclin a complex formation and its importance in regulating the expression of the three transcription factors . cells of all three germ layers were detected in embryoid bodies ( ebs ) formed from nu6140 treated he s cells ; however commitment into the endodermal lineage was somewhat higher and ectoderm and mesoderm lineages lower when compared to ebs from untreated cells . by applying a flow cytometric method to the analysis of ebs we were able to simultaneously detect both differentiation and pluripotency markers in single cells . before initiation of ebs and three days after release from nu6140 , he s cells displayed a tendency to differentiate with decreasing expression of nanog and oct4 ( 43% ) and with increased expression of early ectodermal marker otx-2 ( 13% ) . the increase of otx-2 expression in ebs has been reported to coincide with the downregulation of the pluripotency marker oct4 . we found in this study that up to 20% of ebs formed from nu6140 treated cells expressed oct4 by day 4 , whilst only 2 - 3% ebs from untreated cells expressed oct4 . cells expressing sox1 ( ectodermal marker ) , sox17 ( endodermal marker ) , or brachyury ( mesodermal marker ) did not express oct4 , indicating that ebs contained differentiated as well as some oct4 expressing cells . another study found a long persistence in the expression of the pluripotency marker oct4 in differentiating ebs by day 12 after initiation of ebs . our finding that differentiation and pluripotency markers can be detected during the early stages of cell differentiation accords with our previous study that revealed a gradual decrease in the expression of pluripotency markers which was accompanied by a gradual increase in the expression of endodermal markers . the differences we found in commitment of cells into endodermal , ectodermal , and mesodermal lineages between untreated he s cells and those treated with nu6140 indicates that inhibition of cdk2 with nu6140 may have long term consequences . treatment of he s cells with nu6140 might also induce epigenic changes , but further studies are required to answer this question . it has been reported that cells arrested and released from the m phase , as well as cells within the s phase , are more sensitive to cdk2 inhibitors [ 8 , 9 ] . results of our study confirm that he s cells have a longer s phase and are more sensitive to treatment with nu6140 than hec cells . we also tested combined treatments where cells were first treated with nocodazole ( arrests cells in m phase ) and then with nu6140 . a combined treatment of nocodazole and then nu6140 was more efficient in decreasing expression of nanog , oct4 , and sox2 in both he s and hec cells than either treatment alone . he s cells however possessed higher sensitivity than hec cells to treatments with individual agents or agents combined . there are several differences between he s and hec cells that might explain their different sensitivity . higher expression levels of sox2 and oct4 have been found in hec cells , with expression of oct4 isoforms ( oct4a , oct4b , etc . ) in hec cells [ 41 , 42 ] , overexpression of sox2 in some hec cell lines , and a low differentiation ability indicating that the control of pluripotency factors is more complicated in hec cells than in he s cells . in in testicular germ cell tumors the presence of mirna-372 and mirna-373 has been reported , whereas a mirna302 cluster was undetected . these authors also reported that mirna-372 and mirna-373 can neutralize p53-mediated cdk inhibition and allow the growth of tumor cells . taken together all these differences at various levels of regulating pluripotency indicate that the expression of tfs in hec cells is not as tightly controlled as it is in he s cells . in both he s and hec cells a correlation between the expressions of cdk2 and sox2 was found in surviving cells after treatment with nu6140 and most of cells expressing sox2 also expressed cdk2 . our findings argue that cdk2 expression is needed for transcription factor expression and any alterations in activity / expression influence firstly nanog and oct4 and then sox2 expression in he s cells , pointing to possible additional roles of sox2 in cell self - renewal and proliferation . a gradual downregulation of cdk2 and sox2 was detected in nocodazole or nu6140 treated he s and hec cells . additionally only a few gata4 expressing cells were detected after nu6140 treatment of he s cells , which could indicate that cdk2 activity is needed for cell survival and proper differentiation . analysis of he s cell phosphorylation dynamics during differentiation into extraembryonic lineages ( endoderm , etc . ) has shown that high expression of transcription factors and normal cell cycle profiles are crucial for initiation of he s cell differentiation . in this study we have shown that the expression of cdk2 in m phase arrested cells ( nocodazole treatment ) was lower than in normally cycling cells . in untreated cells and in dmso treated cells we detected similar expression levels of cdk2 independently of the cell cycle phases . it is possible that mammalian cells can tolerate loss of cdk2 ( by additional support from other cdks as cdk1 , cdk4 , or cdk6 ) , but cdk2 activity is required for cell proliferation . cdk1/cyclin a complexes have been specifically implicated in attenuating the expression of histone genes at the end of the s phase . in solid tumors , however , including breast cancers , the overexpression of cyclin e and overexpression of cyclin a have been linked with adverse outcomes . it has been shown that paclitaxel ( which disrupts microtubule dynamic and induces mitotic arrest ) can increase specific activity in cdk1 cells , though some cells are resistant . both cdk1 and cdk2 could therefore be required for proper m phase entry . in our study m phase arrested cells ( application of nocodazole ) decreased expression of nanog , oct4 , and sox2 in both he s and hec cells . release from nocodazole - arrest however restored expression of all three tfs in surviving cells . there was no increase though in the number of surviving cells , indicating that these cells , which had no detectable cdk2 and sox2 expression , could not survive . why nu6140 treatment and its removal caused a long lasting effect on decreasing expression of tfs in further colony formation could be explained by the gradual downregulation of cdk2 expression . the effects of nu6140 to inhibit other interactions of various cdks might also contribute to inhibiting the expression of pluripotency factors and needs further investigation . the effects of cdk2 inhibitor nu6140 to increase apoptosis and accumulate cells in the g2/m phase has been reported earlier for hela cervical carcinoma cells . a novel finding of our study was that histone 3 phosphorylation decreased after treatment with nu6140 in both he s cells and hec cells , indicating that nu6140 inhibits entry into the m phase and arrests cells in the g2 phase . as nu6140 inhibits the activity of cdk2 by competitive binding to the atp site [ 11 , 12 ] and we found nu6140 did not rapidly downregulate cdk2 expression , cdk2 activity but not the expression level is important for cells to proceed from the g2 to m phase of the cell cycle . another broad - spectrum cdk inhibitor ( roscovitine ) has also been shown to decrease the expression of mitotic control genes and prevent the entry of ht29 human colon cancer cells into mitosis . targeting tf expression in cancer cells is a promising and attractive method for finding new treatment options . it has been shown that knockdown of oct4 and nanog in pancreatic cancer cells reduced their proliferation , migration , invasion , chemoresistance , and tumorigenesis . according to the results of our study the expression of sox2 could be the target of choice to eliminate cancer cells with stem - cell - like properties . recently it has been shown that inhibition of sox2 reduced the proliferation and migration of gastric cancer cells , increased their apoptosis , induced changes in cell cycle in vitro , and reduced their in vivo tumorigenic potential . for the effective elimination of cancer cells with stem - cell - like properties a combined treatment of agents may be more effective , as successfully shown with nocodazole and nu6140 in our study . we also produced a screening method to detect expression patterns of three tfs by using multicolor flow cytometric analysis . multicolor flow cytometric analysis could be a useful tool for screening different potential medical agents and their combinations for the treatment of he s cells and cancer cells in vitro . by applying the methods described in this study knowledge critical to create novel approaches to eliminate cancer cells with stem - cell - properties the results of this study point to a connection between cdk2-cyclin a complex activity and self - renewal and pluripotency maintenance in he s and in hec cells . modulating the activity of this interaction with nu6140 decreased the expression of transcription factors nanog , oct4 , and sox2 . he s cells were more sensitive than hec cells to inhibiting agent nu6140 . in embryoid bodies formed from nu6140 treated he s cells commitment to ectodermal and mesodermal lineages was affected compared to embryoid bodies formed from untreated cells . a combined treatment ( cells arrested with nocodazole and then treated with nu6140 ) was more efficient in decreasing the expression of all three transcription factors than treatment with nocodazole or nu6140 individually in he s and hec cells . in addition , applied multiparameter flow cytometric methods for simultaneous detection of pluripotency and differentiation markers in single cells could be a useful tool for in vitro screening in order to design novel medical agents to modulate stem - cell - like properties of cancer cells .

as cyclin - dependent kinases ( cdks ) regulate cell cycle progression and rna transcription , cdks are attractive targets for creating cancer cell treatments . in this study we investigated the effects of the small molecular agent nu6140 ( inhibits cdk2 and cyclin a interaction ) on human embryonic stem ( he s ) cells and embryonal carcinoma - derived ( hec ) cells via the expression of transcription factors responsible for pluripotency . a multiparameter flow cytometric method was used to follow changes in the expression of nanog , oct4 , and sox2 together in single cells . both he s and hec cells responded to nu6140 treatment by induced apoptosis and a decreased expression of nanog , oct4 , and sox2 in surviving cells . a higher sensitivity to nu6140 application in he s than hec cells was detected . nu6140 treatment arrested he s and hec cells in the g2 phase and inhibited entry into the m phase as evidenced by no significant increase in histone 3 phosphorylation . when embryoid bodies ( ebs ) formed from nu6104 treated he s cells were compared to ebs from untreated he s cells differences in ectodermal , endodermal , and mesodermal lineages were found . the results of this study highlight the importance of cdk2 activity in maintaining pluripotency of he s and hec cells and in differentiation of he s cells .

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

cyclin - dependent kinases ( cdks ) regulate cell cycle progression and rna transcription in different cell types . cdk2 regulates cell cycle progression by forming ( a ) cyclin e - cdk2 complexes at the boundary of g1 to s transition and ( b ) cyclin a - cdk2 complexes for orderly s phase progression and g2 to m phase transition . several specific features of human embryonic stem ( he s ) cells are of special interest in studying the effect of cdk2 inhibition . expression of p21 , a natural inhibitor of cdk2 , is associated with g1 arrest in he s cells . previous studies indicate the important role of cdk2 activity in modulating the he s cell cycle . it is important therefore to clarify how the cdk2 inhibitor nu6140 changes cell cycle and pluripotency of he s cells . pluripotency of he s cells is generally maintained by a transcriptional network coordinated by the three transcription factors oct4 , nanog , and sox2 . transcription factors ( tfs ) have been shown to function as cellular rheostats ; that is , small changes in tf expression result in big changes in self - renewal and pluripotency of he s cells . expression of transcription factors characteristic to he s cells has been found in several cancers and is often associated with a poor clinical outcome . embryonal carcinoma - derived cells ( hec , ntera-2 , bg01v , and 2102ep ) have several advantages as a tool to study stem - cell - like signatures in carcinoma cells as they generally show expressions of proteins and mrnas specific to he s cells and possess low rates of spontaneous differentiation . the effect of cdk2 inhibitors on the proliferation of various cancer cells has been extensively studied , but less attention has been paid to the ability of cdk2 inhibitors to modulate the expression of transcription factors responsible for pluripotency . in this study we aimed to characterise the effect of the cdk2 inhibitor nu6140 on the cell cycle in carcinoma - derived cells compared to he s cells . we applied multicolor flow cytometric methods to a single cell analysis of the expression of cdk2 and pluripotency markers in different cell cycle phases . as a consequence of our first experiment another important question we wanted to answer was whether the modulation of transcription factors responsible for pluripotency ( nanog , oct4 , and sox2 ) was reversible and long lasting . the human embryonal carcinoma - derived ( hec ) cell line 2102ep ( globalstem , usa ) was maintained in a dmem medium ( containing glucose 4.5 g / l ) ( gibco life technologies ) containing 10% fetal bovine serum ( paa laboratories , linz , austria ) and mem nonessential - amino - acids - solution ( 0.1 mm , invitrogen , usa ) . for more accurate analysis we used sequential selection of cell populations only single cells were selected for detecting cells in different cell cycle phases and only these cells were used to analyse for the expression of various parameters ( see figures 16 ) , allowing the minimisation of nonspecific signals . embryoid bodies ( ebs ) were formed by growing he s cells in suspension in a low attachment culture plate with knockout dmem / f12 media ( gibco , life technologies ) supplemented by 20% knockout serum replacement , nonessential amino acids ( 0.1 mm ) , glutamax ( 2 mm ) , and beta - mercaptoethanol ( 0.1 mm ) . first we assessed the ability of the cdk2 inhibitor to influence the expression of pluripotency markers in he s cells . using flow cytometric analysis the detection of the expression of transcription factors ( nanog , oct4 , and sox2 ) was combined with the analysis of cell cycle profiles . in untreated and dmso treated he s cells the expression of nanog and oct4 was high ( 95.6% and 98.1% nanog+oct4 + cells in dmso treated or untreated cells population ) . nu6140 treatment had a minimal effect on reducing sox2 expression ( 2 - 3% of cells were without sox2 expression ) and the number of surviving cells expressing all three markers ( sox2 , nanog , and oct4 ) was higher compared to cells treated with nocodazole ( 7786% and 47% with nu6140 and nocodazole , resp . ) the number of he s cells decreasing after treatment neared significance with nu6140 ( p = 0.06 ) and was significant for nocodazole ( p < 0.01 ) when they were compared to dmso treated cells , indicating that both agents were able to inhibit cell proliferation ( supplementary material figure 1(a ) ) . to compare the effects of nu6140 on he s and hec cells we carried out a similar experiment on 2012ep cells to the previous experiment on he s cells . a decreased expression of nanog and oct4 in surviving cells ( 51% of nanog+oct4 + cells , figure 2(a ) ) was detected in nocodazole treated cells compared to those treated with dmso ( 94% of nanog+oct4 + cells ) . the expression of nanog and oct4 was high in surviving cells after nu6140 treatment ( 81% compared to 94% nanog+oct4 + cells using dmso , figure 2(a ) ) . the effects of nocodazole and nu6140 were similar in he s and hec cells , but he s cells were more sensitive than hec cells . we investigated whether the expression of transcription factors is restored after the washout of nocodazole or nu6140 and a further culturing of cells in a fresh medium for 24 h. after release from the nocodazole - block an increase in nanog and oct4 expression was detected ( from 4% to 77% nanog+oct4 + cells , figure 1(a ) ) in he s cells , accompanied by a decrease in the subpopulation without nanog and oct4 expression ( from 91% to 14% , figure 1(a ) ) . since he s cells in this study were cultured on matrigel feeder surface but in previous experiments we used he s cells first cultured on mef feeder and then plated on matrigel coated plates , these differences in culturing could affect the growing rate , colony formation , and recovery of cells . similar changes in sox2 expression were also detectable in he s cells both with treatment of nocodazole or nu6140 and after their release from these agents . in treatments using nocodazole or nu6140 we detected amongst the released cells a high number of he s cells expressing sox2 and a smaller subpopulation expressing only sox2 without nanog and oct4 expression . when 2102ep ( hec ) cells were used instead of he s cells , the removal of nocodazole resulted in similar effects ; however removal of nu6140 had a different effect on the expression of all three tfs in hec cells . he s cells were able to restore pluripotency marker expression after the removal of nocodazole and nu6140 , with the number of cells remaining unchanged and no significant increase in proliferation of cells detected ( supplementary material figure 1 ) . after washout of nocodazole or nu6140 , he s cells recovered more efficiently than hec cells , indicating that different mechanisms regulate the expression of pluripotency markers in he s and hec cells . as a result of finding the differences in he s and hec recovery after treatment with nu6140 , we investigated the correlation between the levels of pluripotency markers and cdk2 expression in he s and hec cells . nocodazole treatment of he s cells resulted in ( i ) a decrease in cdk2 and nanog coexpressing cells ( from 99% to 11% ) ; ( ii ) an increase in cells expressing cdk2 without nanog expression ( from 0.4% to 81% , figure 1(b ) ) ; ( iii ) an increase in the number of cells without nanog and cdk2 expression ( from 0.2% to 7% , figure 1(b ) ) . after release from nocodazole arrest and further culturing in fresh medium , all he s cells restored the expression of cdk2 , 79% of cells expressed cdk2 and nanog , and 15% of cells expressed cdk2 without nanog expression ( figure 1(b ) ) . nocodazole and nu6140 both affected cdk2 expression in hec cells , which was different from nu6140 treatment of he s cells where no change in cdk2 expression was detected . as a consequence of our results showing the effect of nu6140 on cdk2 expression , we analysed the effect of nu6140 on the distribution of cells within the different cell cycle phases in he s and hec cells , which possess different cell cycle profiles . higher concentrations ( 5 and 10 m ) of nu6140 increased the number of he s cells in the g2/m phase and caused a decrease of cells in the g1 phase ( figure 3(a ) ) . similar effects in cell cycle distribution in he s cells were also found for 2102ep cells with nu6140 and nocodazole treatment ( figure 3(a ) ) . nocodazole increased the number of he s cells within the m phase ( 17% of cells possessed phosphorylated h3 at serine 10 after nocodazole treatment compared to 2% after dmso treatment , figure 3(b ) ) . there was no increase in phosphorylation of h3 at ser10 ( 1.92.5% ) after nu6140 treatment , showing these cells accumulate preferably in the g2 , not the m phase ; this effect was similar in hec cells ( data not shown ) . in he s cells treated with nocodazole the expression level of cdk2 was lower in m phase cells ( which expressed both cdk2 and phospho - ser10-histone ) and significantly higher in cells within other cell cycle phases ( figure 3(b ) ) . a similar low level of cdk2 in the m phase was detected by applying increasing concentrations of nu6140 . when the mean fluorescence values of the two cell populations ( cdk2 + cells with and without phosphorylated h3 ) were compared , a lower expression of cdk2 in cells within the m phase compared to cells in other cell cycle phases was detected after nu6140 application ( figure 3(c ) ) . since nu6140 treatment decreased the number of adherent he s cells and downregulated expression of pluripotency markers nanog , oct4 , and sox2 , we asked whether these effects could be due to apoptosis of he s cells . nu6140 treatment induced cleavage of caspase 3 in 4.3% of he s cells without nanog expression and in 2.2% of cells that still expressed nanog ( figure 4(a ) ) . since cleaved caspase 3 has been reported to mediate differentiation of he s cells , we analyzed nu6140 treated he s cells for the expression of gata4 as a marker of early differentiation [ 34 , 35 ] . in our study a small number of gata4 expressing cells was detected in dmso treated and untreated he s cells . in both he s and hec cells the number of cells coexpressing nanog and oct4 decreased slightly as a result of nocodazole arrest ( figure 5 ) . combined treatment with nocodazole followed by nu6140 was more effective in decreasing expression of all three tfs in he s cells than hec cells , though the tendencies were similar . when he s cells were analysed for expression of pluripotency factors , 42% of cells expressed nanog , oct4 , and sox2 3 days after treatment with nu6140 . next we applied common embryoid bodies ( ebs ) formation protocols in order to characterize the differentiation ability of nu6140 treated and released he s cells . after 4 days , embryoid bodies were analysed for the expression of ectoderm ( sox1 ) , mesoderm ( brachyury ) , and endoderm ( sox17 ) markers and pluripotency marker oct4 simultaneously by using flow cytometric method . we could detect lower number of ectodermal marker sox1 expressing cells from ebs formed from nu6140 treated cells compared to ebs formed from untreated he s cells ( 39% versus 78% , figure 6(c ) ) . endodermal marker sox17 expression was detected in 4.5% of ebs formed from nu6140 treated he s cells , but in untreated ebs sox17 expression was detected only in 2.7% of cells . the number of oct4 expressing cells was higher in ebs formed from nu6140 treated he s cells ( 1721% ) than from untreated he s cells ( 1.92.8% ) . the distribution of cells within cell cycle phases was similar in both ebs formed from nu6140 treated he s cells or from untreated cells ( 62% , 23% , and 15% versus 61% , 18% , and 21% in g0g1 , s , and g2/m phases , resp . ) in this study we focused on the small molecule nu6140 , which competes with atp for binding to the complex of cdk2 and our experiments using nu6140 treatment focused on changes in the expression patterns of cell pluripotency markers for human embryonic h9 stem cells ( he s ) and embryonal carcinoma - derived 2102ep cells ( hec ) . we found that both he s and hec cells were sensitive in suppressing the expression of nanog and oct4 with nocodazole or nu6140 , whereas the effect on sox2 expression was more pronounced after nocodazole treatment . after the removal of nocodazole or nu6140 , the expression of nanog , oct4 , and sox2 was restored in he s cells ; a similar tendency was detected in hec cells after nocodazole treatment and release . analysing embryoid bodies ( ebs ) revealed that differentiation into ectodermal and mesodermal lineages was affected in ebs formed from nu6140 treated he s cells compared to ebs from untreated he s cells . our finding that application of nu6140 also increased apoptosis and inhibited growth of he s and hec cells was therefore expected . the effect of nu6140 on the expression of pluripotency markers sox2 , nanog , and oct4 had not however been reported before this study . the differences in sensitivity to nu6140 in he s and hec cells found in this study point to the different roles of cdk2-cyclin a complex formation and its importance in regulating the expression of the three transcription factors . cells of all three germ layers were detected in embryoid bodies ( ebs ) formed from nu6140 treated he s cells ; however commitment into the endodermal lineage was somewhat higher and ectoderm and mesoderm lineages lower when compared to ebs from untreated cells . we found in this study that up to 20% of ebs formed from nu6140 treated cells expressed oct4 by day 4 , whilst only 2 - 3% ebs from untreated cells expressed oct4 . the differences we found in commitment of cells into endodermal , ectodermal , and mesodermal lineages between untreated he s cells and those treated with nu6140 indicates that inhibition of cdk2 with nu6140 may have long term consequences . results of our study confirm that he s cells have a longer s phase and are more sensitive to treatment with nu6140 than hec cells . a combined treatment of nocodazole and then nu6140 was more efficient in decreasing expression of nanog , oct4 , and sox2 in both he s and hec cells than either treatment alone . in hec cells [ 41 , 42 ] , overexpression of sox2 in some hec cell lines , and a low differentiation ability indicating that the control of pluripotency factors is more complicated in hec cells than in he s cells . taken together all these differences at various levels of regulating pluripotency indicate that the expression of tfs in hec cells is not as tightly controlled as it is in he s cells . in both he s and hec cells a correlation between the expressions of cdk2 and sox2 was found in surviving cells after treatment with nu6140 and most of cells expressing sox2 also expressed cdk2 . a gradual downregulation of cdk2 and sox2 was detected in nocodazole or nu6140 treated he s and hec cells . additionally only a few gata4 expressing cells were detected after nu6140 treatment of he s cells , which could indicate that cdk2 activity is needed for cell survival and proper differentiation . has shown that high expression of transcription factors and normal cell cycle profiles are crucial for initiation of he s cell differentiation . in this study we have shown that the expression of cdk2 in m phase arrested cells ( nocodazole treatment ) was lower than in normally cycling cells . in untreated cells and in dmso treated cells we detected similar expression levels of cdk2 independently of the cell cycle phases . in our study m phase arrested cells ( application of nocodazole ) decreased expression of nanog , oct4 , and sox2 in both he s and hec cells . the effects of cdk2 inhibitor nu6140 to increase apoptosis and accumulate cells in the g2/m phase has been reported earlier for hela cervical carcinoma cells . a novel finding of our study was that histone 3 phosphorylation decreased after treatment with nu6140 in both he s cells and hec cells , indicating that nu6140 inhibits entry into the m phase and arrests cells in the g2 phase . as nu6140 inhibits the activity of cdk2 by competitive binding to the atp site [ 11 , 12 ] and we found nu6140 did not rapidly downregulate cdk2 expression , cdk2 activity but not the expression level is important for cells to proceed from the g2 to m phase of the cell cycle . multicolor flow cytometric analysis could be a useful tool for screening different potential medical agents and their combinations for the treatment of he s cells and cancer cells in vitro . by applying the methods described in this study knowledge critical to create novel approaches to eliminate cancer cells with stem - cell - properties the results of this study point to a connection between cdk2-cyclin a complex activity and self - renewal and pluripotency maintenance in he s and in hec cells . modulating the activity of this interaction with nu6140 decreased the expression of transcription factors nanog , oct4 , and sox2 . he s cells were more sensitive than hec cells to inhibiting agent nu6140 . in embryoid bodies formed from nu6140 treated he s cells commitment to ectodermal and mesodermal lineages was affected compared to embryoid bodies formed from untreated cells . a combined treatment ( cells arrested with nocodazole and then treated with nu6140 ) was more efficient in decreasing the expression of all three transcription factors than treatment with nocodazole or nu6140 individually in he s and hec cells .

despite significant progress in head and neck cancer treatment , relapse still occurs in 17 - 30% patients with this type of neoplasm . the basic method of local treatment of relapse in these patients is surgery or repeated radiotherapy . salvage surgery , due to the risk of complications and the necessity of conducting extensive , often maiming , surgery , is only possible in 20% cases . the possibility of repeated irradiation is also limited due to high risk of radiotherapy related morbidity . in case of brachytherapy , the relapsed lesion location creates a limitation , which in many cases prevents correct placement of brachytherapy applicators in tumor . traditionally , brachytherapy is reserved for application in surface lesions where the risk of mechanical damages and surgery related complications is small . in case of deep lesions , due to high risk of complications in course of application , this article presents a technique of computed tomography ( ct ) image - guided transdermal application of interstitial brachytherapy possible in patients with relapsed tumor in hardly accessible areas . the utilization of ct for imaging of the application site allows to decrease the risk of damaging important anatomical structures and accurate distribution of applicators within the tumor . patients with neoplastic lesions located deep under the skin surface in hardly accessible areas were qualified for treatment using image - guided brachytherapy . the lesions were located in close vicinity to critical organs such as spinal cord , trachea , eyeball , lens , or directly adjacent to large vessels or structures , whose mechanical damage could lead to hemorrhage , paralysis , or organ dysfunction . this distinguished our image - guided brachytherapy of visually controlled procedure applied on the basis of tumor extent , determined in physical examination or formerly conducted imaging examinations . the study involves analysis of data concerning 4 patients with local relapse of head and neck cancer treated in the department of brachytherapy , lublin region cancer center in 2013 - 2015 . an isolated inoperable local relapse occurred , for which brachytherapy was applied assumed as palliative treatment . the indications for application of image guided brachytherapy were above all close vicinity of large blood vessels ( all patients ) , the organ of vision ( patient 4 ) , the extent of the neoplastic process ( patient 2 ) , and small relapsed tumor size requiring precise application ( patient 1 ) . clinical characteristics of patients df fractionated dose , dc complete dose , pr partial remission , sd stable disease , ebrt external beam radiation therapy , rtog radiation therapy oncology group , ctcae common terminology criteria for adverse events , recist response evaluation criteria in solid tumors the procedure was conducted under general ( patients 2 , 3 and 4 ) or local anesthesia ( patient 1 ) . under local anesthesia steel or titanium needle applicators , as well as elastic interstitial applicators were used in brachytherapy . in sites , where it was impossible to drive the applicators through the lesion , needle applicators were used . a disadvantage was the necessity of conducting the treatment in several stages , which was associated with the need for performing several application procedures , as well as several steps of treatment planning . elastic applicators were used in cases when it was possible to form an applicator outlet on the other side of the tumor . due to poor visibility of the placed applicators , it was necessary to apply markers just after the application . after applicators were in place , they remained in the lesion throughout the treatment process . the application was conducted under guidance of ct device located in the operating room and moving on rails . both , the ct device and the operating table movement were controlled using a console located in the operating room . somatom sensation open ct device ( siemens ag , munich , germany ) was used including optional fluoroscopy accessory from siemens . before the commencement of the treatment , ct examination was conducted with or without contrast agent , and then , the image was fused with the previously performed ct or magnetic resonance imaging ( mri ) examination . the extent of the tumor was determined on the basis of the conducted examinations and projected on patient s skin using laser beams . then , under continuous ct guidance , applicator needle or elastic applicator lead was percutaneously ( transdermally ) inserted into the tumor in a way facilitating irradiation of the whole tumor area , and simultaneously controlling the position of the needle against the adjacent structures ( figure 1 ) . if it was possible , the applicators were inserted in such a way that their layout in the lesion was possibly most parallel , and their distance from one another as well as from the edge of the tumor was not greater than 2 cm . technical difficulties were encountered in cases requiring the insertion of an applicator under different angles towards the tomographic image plane . it was associated with the necessity of constant control of needle position at the time of insertion on several tomographic scans simultaneously . due to little respiratory movement of anatomic structures in head and neck region , ct guided insertion of the needle was precise and relatively safe , despite close vicinity of many important organs . only the region directly under the needle was not sufficiently visible due to artifacts related to the material , of which the needle was made . needle applicators were located in the lesion only during each fraction of treatment but elastic applicators , used for their stabilization , remained in the tumor from the beginning of the first to the end of the last fraction ( figure 2 ) . computed tomography - guided applicators , next steps ( patient 4 ) patient 4 with applicators on the completion of a single treatment session , ct scan was performed to plan further treatment , and then , after drawing the clinical target volume ( ctv ) and organs at risk ( oar ) , time and place of ionizing radiation source stopping points were planned . the condition for treatment plan acceptance was encompassing the total ctv with a dose constituting 90% of the planned dose ( d90 ) . additionally , the minimum significant dose for local control of the tumor was reported ( d100 ) . these doses and total doses were converted according to linear quadratic model of biologic effective dose ( bed ) , using the following formula bed = nd ( 1 + d/(/ ) ) , where d fractional dose , n number of fractions , / alpha / beta ratio , in case of squamous cell carcinoma of head and neck it is 10 . in order to compare them with conventional fractioning of 2 gy , the doses were converted to dose equivalent 2 gy ( deq2 ) using the following formula : deq2 = bed/(1 + 2/(/ ) ) . in order to facilitate the evaluation of correlation between the dose and possible future damages , the volume receiving 100% of the prescribed dose was reported , as well as high dose areas receiving 150% of the prescribed dose ( v150 ) and 200% of the prescribed dose ( v200 ) . the image - guided high - dose - rate ( ig - hdr ) plan for patients . the color code for the isodose color wash is as follows : red 200% , orange 150% , yellow 110% , green 100% , blue 90% ( transverse view left , sagittal view right ) due to various locations , doses received by critical organs were also reported . depending on the type of the organ , either maximum dose ( dmax ) or dose per 1 cm of the organ located in the maximum dose area ( d1 cm ) were reported . the doses were converted to bed and to deq2 according to the linear quadratic model , adopting / ratio value of 3 for future radiation induced complications . the treatment was conducted using 24 channel gammamed plus device from varian ( varian medical systems , inc . , if it was necessary , the removal was conducted after premedication with opioid analgesics . because of the risk of hemorrhage , the surgical procedure was conducted in an operating room equipped with ge oec 9900 elite system ( ge health care , milwaukee , wi , usa ) , and offering the possibility of interventional embolization of a damaged vessel by a surgical radiology specialist . after applicators removal , ct scan with contrast was performed in order to rule out potential complications , especially fresh bleeding in the applicator area . patients with neoplastic lesions located deep under the skin surface in hardly accessible areas were qualified for treatment using image - guided brachytherapy . the lesions were located in close vicinity to critical organs such as spinal cord , trachea , eyeball , lens , or directly adjacent to large vessels or structures , whose mechanical damage could lead to hemorrhage , paralysis , or organ dysfunction . this distinguished our image - guided brachytherapy of visually controlled procedure applied on the basis of tumor extent , determined in physical examination or formerly conducted imaging examinations . the study involves analysis of data concerning 4 patients with local relapse of head and neck cancer treated in the department of brachytherapy , lublin region cancer center in 2013 - 2015 . an isolated inoperable local relapse occurred , for which brachytherapy was applied assumed as palliative treatment . the indications for application of image guided brachytherapy were above all close vicinity of large blood vessels ( all patients ) , the organ of vision ( patient 4 ) , the extent of the neoplastic process ( patient 2 ) , and small relapsed tumor size requiring precise application ( patient 1 ) . clinical characteristics of patients df fractionated dose , dc complete dose , pr partial remission , sd stable disease , ebrt external beam radiation therapy , rtog radiation therapy oncology group , ctcae common terminology criteria for adverse events , recist response evaluation criteria in solid tumors the procedure was conducted under general ( patients 2 , 3 and 4 ) or local anesthesia ( patient 1 ) . under local anesthesia steel or titanium needle applicators , as well as elastic interstitial applicators were used in brachytherapy . in sites , where it was impossible to drive the applicators through the lesion , needle applicators were used . a disadvantage was the necessity of conducting the treatment in several stages , which was associated with the need for performing several application procedures , as well as several steps of treatment planning . elastic applicators were used in cases when it was possible to form an applicator outlet on the other side of the tumor . due to poor visibility of the placed applicators , it was necessary to apply markers just after the application . after applicators were in place , they remained in the lesion throughout the treatment process . the application was conducted under guidance of ct device located in the operating room and moving on rails . both , the ct device and the operating table movement were controlled using a console located in the operating room . somatom sensation open ct device ( siemens ag , munich , germany ) was used including optional fluoroscopy accessory from siemens . before the commencement of the treatment , ct examination was conducted with or without contrast agent , and then , the image was fused with the previously performed ct or magnetic resonance imaging ( mri ) examination . the extent of the tumor was determined on the basis of the conducted examinations and projected on patient s skin using laser beams . then , under continuous ct guidance , applicator needle or elastic applicator lead was percutaneously ( transdermally ) inserted into the tumor in a way facilitating irradiation of the whole tumor area , and simultaneously controlling the position of the needle against the adjacent structures ( figure 1 ) . if it was possible , the applicators were inserted in such a way that their layout in the lesion was possibly most parallel , and their distance from one another as well as from the edge of the tumor was not greater than 2 cm . technical difficulties were encountered in cases requiring the insertion of an applicator under different angles towards the tomographic image plane . it was associated with the necessity of constant control of needle position at the time of insertion on several tomographic scans simultaneously . due to little respiratory movement of anatomic structures in head and neck region , ct guided insertion of the needle was precise and relatively safe , despite close vicinity of many important organs . only the region directly under the needle was not sufficiently visible due to artifacts related to the material , of which the needle was made . needle applicators were located in the lesion only during each fraction of treatment but elastic applicators , used for their stabilization , remained in the tumor from the beginning of the first to the end of the last fraction ( figure 2 ) . computed tomography - guided applicators , next steps ( patient 4 ) patient 4 with applicators on the completion of a single treatment session , ct scan was performed to plan further treatment , and then , after drawing the clinical target volume ( ctv ) and organs at risk ( oar ) , time and place of ionizing radiation source stopping points were planned . the condition for treatment plan acceptance was encompassing the total ctv with a dose constituting 90% of the planned dose ( d90 ) . additionally , the minimum significant dose for local control of the tumor was reported ( d100 ) . these doses and total doses were converted according to linear quadratic model of biologic effective dose ( bed ) , using the following formula bed = nd ( 1 + d/(/ ) ) , where d fractional dose , n number of fractions , / alpha / beta ratio , in case of squamous cell carcinoma of head and neck it is 10 . in order to compare them with conventional fractioning of 2 gy , the doses were converted to dose equivalent 2 gy ( deq2 ) using the following formula : deq2 = bed/(1 + 2/(/ ) ) . in order to facilitate the evaluation of correlation between the dose and possible future damages , the volume receiving 100% of the prescribed dose was reported , as well as high dose areas receiving 150% of the prescribed dose ( v150 ) and 200% of the prescribed dose ( v200 ) . the image - guided high - dose - rate ( ig - hdr ) plan for patients . the color code for the isodose color wash is as follows : red 200% , orange 150% , yellow 110% , green 100% , blue 90% ( transverse view left , sagittal view right ) due to various locations , doses received by critical organs were also reported . depending on the type of the organ , either maximum dose ( dmax ) or dose per 1 cm of the organ located in the maximum dose area ( d1 cm ) were reported . the doses were converted to bed and to deq2 according to the linear quadratic model , adopting / ratio value of 3 for future radiation induced complications . the treatment was conducted using 24 channel gammamed plus device from varian ( varian medical systems , inc . , if it was necessary , the removal was conducted after premedication with opioid analgesics . because of the risk of hemorrhage , the surgical procedure was conducted in an operating room equipped with ge oec 9900 elite system ( ge health care , milwaukee , wi , usa ) , and offering the possibility of interventional embolization of a damaged vessel by a surgical radiology specialist . after applicators removal , ct scan with contrast was performed in order to rule out potential complications , especially fresh bleeding in the applicator area . various fractioning schemes were used depending on the possibility of retaining the applicators within the lesion . in 2 patients ( patient 2 and 3 ) brachytherapy was performed using elastic applicators . five fractions of 3 gy or 4 gy were used in 1 day intervals . this required a single application procedure and a single treatment planning session . the applicator insertion was performed in general anesthesia and after completion of the whole treatment process , the applicators were removed . in the other patients , needle applicators were used and the insertion and treatment planning were performed before each treatment session . due to this fact , fractional doses were higher ( 5 gy / fraction ) and the interval between sessions was longer ( from 3 to 7 days ) . in one patient , the insertion procedure was performed in general anesthesia and in the others , in local anesthesia after premedication . d100 median of a single fraction was 3.8 gy ( range 3 - 4.2 gy ) , and d90 median of a single fraction was 5.2 gy ( range 3.7 - 5.8 ) . d100 total dose median was 15.4 gy ( range 10 - 19.5 gy ) , and d90 total dose median was 20.6 gy ( range 16.1 - 26 gy ) . biologic effective dose median in isodose encompassing the whole irradiated volume ( d100 ) of all treatment fractions was 20.7 gy ( range 13.4 - 27.1 gy ) and deq2 was 17.2 gy ( range 11.1 - 22.6 gy ) . biologic effective dose median in isodose encompassing 90% of the irradiated volume ( d90 ) was 30.4 gy ( range 24.7 - 39.5 gy ) and deq2 was 25.3 gy ( range 20.6 - 32.9 gy ) . clinical target volume , which received 100% dose , as well as 150% and 200% dose was characterized in table 2 . dose homogeneity index median was 0.31 ( range 0.18 - 0.68 ) ( table 2 ) . depending on the location of irradiated area , doses received by various critical organs were reported ( tables 3 , 4 and 5 ) . % of prescription dose covering 100% of target volume ; d90% % of prescription dose covering 90% of target volume ; v100% , v150% , v200% % of target volume receiving prescription dose , 150% of prescription dose and 200% of prescription dose ; dhi dose homogeneity index ; bed d100% biologic effective dose in d100% ; bed d90% biologic effective dose in d90% ; deq2 d100% dose equivalent 2 gy in d100% ; deq2 d90% dose equivalent 2 gy in d90% ; fr fractions doses in critical organs : brain , spinal cord , and eyeballs in individual patients spinal cord max . , eye l. max . , eye r. max . maximum dose in spinal cord and left and right eyeballs ; brain 1 cm dose in 1 cm of brain tissue ; bed spinal cord , bed eye l. , bed eye r. biologic effective dose of maximum dose in spinal cord and left and right eyeballs ; bed brain biologic effective dose of the dose in 1 cm of brain tissue ; deq2 spinal cord , deq2 eye l. , deq2 eye r. dose equivalent 2 gy of maximum dose in spinal cord and left and right eyeballs ; deq2 brain dose equivalent 2 gy of the dose in 1 cm of brain tissue doses in critical organs : lens , otical nerves and optic chiasma in individual patients ( only patients 2 and 4 ) lens l. max . , optic n. l. max . , optic n. r. max . , optic chiasm max . maximum dose in left and right lens , left and right otical nerves and optic chiasma ; bed lens l. , bed lens r. , bed optic n. l. , bed optic n. r. , bed optic chiasm . biologic effective dose of maximum dose in maximum dose in left and right lens , left and right otical nerves and optic chiasma ; deq2 lens l. , deq2 lens r. , deq2 optic n. l. , deq2 optic n. r. , deq2 optic chiasm dose equivalent 2 gy of maximum dose in left and right lens , left and right otical nerves and optic chiasma doses in critical organs : salivary glands , larynx , thyroid , and bone tissue in individual patients parotid l. 1 cm , parotid r. 1 cm , larynx 1 cm , thyroid gland 1 cm , bone 1 cm dose in 1 cm of salivary glands , larynx , thyroid , and bone tissue ; bed parotid l. , bed parotid r. , bed larynx , bed thyroid gland , bed bone biologic effective dose of the dose in 1 cm of salivary glands , larynx , thyroid , and bone tissue ; deq2 parotid l. , deq2 parotid r. , deq2 larynx , deq2 thyroid gland , deq2 bone dose equivalent 2 gy of the dose in 1 cm of salivary glands , larynx , thyroid , and bone tissue treatment toxicity was evaluated using common terminology criteria for adverse events ( ctcae ) v.4.0 , and the radiotherapy related morbidity was measured using radiation therapy oncology group ( rtog ) scale . despite close vicinity of many important structures in the head and neck area , no mechanical injury of these organs was observed either in course of the insertion procedure or while the applicators were remaining in the treatment site between subsequent treatment sessions . none of the patients suffered from paralysis in result of mechanical damage of the nerves . due to abundant vascularity of head and neck organs , there was a small degree of bleeding at the time of applicator removal , which was resolved spontaneously . no hemorrhagic foci were found on ct scans taken after the insertion and removal of applicators . no clinical features of internal hemorrhage were observed in these patients . in 3 patients ( patients 2 , 3 , 4 ) , due to location of the irradiated area and acute radiotherapy induced morbidity of the skin , rtog scale grade 1 occurred . in one patient ( patient 2 ) , a few days after completion of brachytherapy , an infection of the irradiated area requiring antibiotic therapy , ctcae scale grade 2 occurred . we focused only on acute reactions because of short follow - up . in the period of observation lasting for 3 to 7 months ( median 3.5 months ) , partial regression ( pr ) was observed in 2 patients and stable disease in other 2 patients . in patient 1 , within 3 months of observation , clinically confirmed partial regression occurred . because of the general condition of the patient , no imaging examination was performed in this case . the patient died due to lung metastases 4 months after treatment completion . in patient 2 , disease regression was being confirmed in ct examination throughout the 7 months observation period . in patients 3 and 4 , the ct examination revealed disease stabilization after treatment completion . after the treatment , in 3 patients partial relief of some symptoms was observed . in patient number 4 , trismus status improved and pain symptoms decreased . in patient 2 , despite intensified debilitation related to skin , an infection in the area of applicator insertion , dyspnea caused by tumor mass was reduced . in patient number 1 , dysphagia related to tumor mass was reduced . treatment toxicity was evaluated using common terminology criteria for adverse events ( ctcae ) v.4.0 , and the radiotherapy related morbidity was measured using radiation therapy oncology group ( rtog ) scale . despite close vicinity of many important structures in the head and neck area , no mechanical injury of these organs was observed either in course of the insertion procedure or while the applicators were remaining in the treatment site between subsequent treatment sessions . none of the patients suffered from paralysis in result of mechanical damage of the nerves . due to abundant vascularity of head and neck organs , there was a small degree of bleeding at the time of applicator removal , which was resolved spontaneously . no hemorrhagic foci were found on ct scans taken after the insertion and removal of applicators . no clinical features of internal hemorrhage were observed in these patients . in 3 patients ( patients 2 , 3 , 4 ) , due to location of the irradiated area and acute radiotherapy induced morbidity of the skin , rtog scale grade 1 occurred . in one patient ( patient 2 ) , a few days after completion of brachytherapy , an infection of the irradiated area requiring antibiotic therapy , ctcae scale grade 2 occurred . in the period of observation lasting for 3 to 7 months ( median 3.5 months ) , partial regression ( pr ) was observed in 2 patients and stable disease in other 2 patients . in patient 1 , within 3 months of observation , clinically confirmed partial regression occurred . because of the general condition of the patient , no imaging examination was performed in this case . the patient died due to lung metastases 4 months after treatment completion . in patient 2 , disease regression was being confirmed in ct examination throughout the 7 months observation period . in patients 3 and 4 , after the treatment , in 3 patients partial relief of some symptoms was observed . in patient number 4 , trismus status improved and pain symptoms decreased . in patient 2 , despite intensified debilitation related to skin , an infection in the area of applicator insertion , dyspnea caused by tumor mass was reduced . in patient number 1 , dysphagia related to tumor mass was reduced . the main factor limiting the application of hdr brachytherapy in the head and neck area is the tumor location . due to the necessity of performing visually controlled application , brachytherapy in this area is only possible in case of lip cancer , oral cavity cancer , selected neoplasms of nasopharynx , nasal cavity and nasopharynx , as well as in case of repeated radiotherapy of nodular relapses in the head and neck area [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ] . reports describing image guided brachytherapy predominantly concern liver , rectal , and lung cancers [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ] . studies concerning image guided brachytherapy in patients with head and neck cancer are singular . in analysis by kolotas et al . , 49 patients with nodular relapse in the neck area were subjected to palliative interstitial brachytherapy . the patients were irradiated twice a day with 3 gy fractions up to the total dose of 30 - 36 gy . no patient experienced massive bleeding in course of the procedure . while , in a study by jiang et al . , 25 patients with squamous cell carcinoma relapse in the area of head and neck were treated with brachytherapy using permanent implants . the procedure was performed under ct or usg guidance . the total dose specified in isodose encompassing no nerve paralysis or serious hemorrhage was observed . in a study by kishan et al . data were retrospectively analyzed from 18 patients with cancer , located in various areas including 2 in the head and neck area , who had undergone hdr brachytherapy performed under guidance of different imaging methods . various dose fractioning schemes were used : 6 gy in 6 fractions and 10 gy in one fraction ( as a boost after external beam radiation therapy ) . in one patient , the lesion was located directly close to the left internal carotid artery , and in other , one in soft tissues of the neck bordering the chest . serious complications related to brachytherapy were not observed in any of the patients . due to short observation time and small number of patients in the described study , the treatment results and late toxicity were not analyzed . in our study , for four patients with relapsed neoplastic lesion located in a site preventing visually controlled brachytherapy , the application of ct image guided hdr brachytherapy facilitated the treatment in hardly accessible locations , too close to large nerves or blood vessels . due to the use of different dose fractioning schemes than kolotas et al . and kishan et al . , it is only possible to compare biological equivalent dose ( bed ) of the total dose . biological equivalent dose of the total dose in kolotas et al . was similar ( 32.5 - 39 gy , / = 10 ) , and in kishan et al . it was a little higher ( 48 - 56.7 gy , / = 10 ) . similarly , to other studies , no complications occurred during application and no early toxicity was observed [ 22 , 23 , 24 ] . moreover , positive effects were achieved in case of both treatment efficiency and quality of life . due to the necessity of having appropriate technical facilities and difficulty of its application , thus , there are no studies based on large group of patients evaluating safety of the procedure and treatment results . there are also no studies comparing different application techniques as well as different types of applicators used in the head and neck region . the intention of image guided hdr brachytherapy is to provide local lesion control in sites , which , due to their extent or lack of technical facilities , do not qualify for using other radiotherapy techniques , especially stereotactic radiotherapy . image guided brachytherapy has the potential to be a valuable treatment method for patients with local and regional relapse of head and neck cancer located in sites preventing standard brachytherapy . it is a safe method facilitating good therapeutic effect and improving the quality of patient s life . it seems justified to conduct prospective studies to evaluate the efficiency and safety of ct image guided brachytherapy .

purposethe aim of the study was the evaluation of image guided transdermal application of interstitial brachytherapy in patients undergoing repeated irradiation for relapsed local tumor of the head and neck area.material and methodsthe article describes transdermal application of interstitial high - dose - rate ( hdr ) brachytherapy in 4 patients treated due to relapsed local tumor in soft palate , submandibular area , laryngopharynx , as well as pterygoid muscles and maxillary sinus . the application was conducted under continuous computed tomography ( ct)-image guidance ( ct fluoroscopy ) . patients qualified for this type of treatment had neoplastic lesions located deep under the skin surface . because of their location , access to the lesions was limited , and the risk of damaging the adjacent tissues such as vessels and nerves was high . the following parameters have been evaluated : clinical response using recist 1.1 , incidence of perisurgical complications using ctcae 4.0 and the frequency of occurrence of radiotherapy related early morbidity using rtog.resultsvarious radiation schemes were used , from 3 to 5 fractions of 3.5 - 5 gy . the median total dose ( d90 ) was 20.6 gy . biologic effective dose ( bed ) and equivalent 2 gy ( deq2 ) median doses were 30.4 gy and 25.3 gy , respectively . in the follow - up period of 3 - 7 months ( the median value of 3.5 months ) , 2 patients had partial regression of the disease and in 2 others the neoplastic process was stabilized . none of the patients had serious complications of treatment ( of 3rd degree or higher).conclusionscomputed tomography - image guided brachytherapy proved to be a safe method of treatment in patients with local relapse in sites , in which traditional visually controlled application was impossible due to risk of complications . despite short observation period and small study group , it seems justified to conduct prospective studies for the evaluation of efficacy and safety of ct - image guided brachytherapy .

Purpose Material and methods Treatment qualification Application technique Results Early toxicity Treatment efficiency Quality of life Discussion Conclusions Disclosure

despite significant progress in head and neck cancer treatment , relapse still occurs in 17 - 30% patients with this type of neoplasm . salvage surgery , due to the risk of complications and the necessity of conducting extensive , often maiming , surgery , is only possible in 20% cases . the possibility of repeated irradiation is also limited due to high risk of radiotherapy related morbidity . in case of deep lesions , due to high risk of complications in course of application , this article presents a technique of computed tomography ( ct ) image - guided transdermal application of interstitial brachytherapy possible in patients with relapsed tumor in hardly accessible areas . the utilization of ct for imaging of the application site allows to decrease the risk of damaging important anatomical structures and accurate distribution of applicators within the tumor . patients with neoplastic lesions located deep under the skin surface in hardly accessible areas were qualified for treatment using image - guided brachytherapy . the study involves analysis of data concerning 4 patients with local relapse of head and neck cancer treated in the department of brachytherapy , lublin region cancer center in 2013 - 2015 . the indications for application of image guided brachytherapy were above all close vicinity of large blood vessels ( all patients ) , the organ of vision ( patient 4 ) , the extent of the neoplastic process ( patient 2 ) , and small relapsed tumor size requiring precise application ( patient 1 ) . under local anesthesia steel or titanium needle applicators , as well as elastic interstitial applicators were used in brachytherapy . in sites , where it was impossible to drive the applicators through the lesion , needle applicators were used . a disadvantage was the necessity of conducting the treatment in several stages , which was associated with the need for performing several application procedures , as well as several steps of treatment planning . due to poor visibility of the placed applicators , it was necessary to apply markers just after the application . the application was conducted under guidance of ct device located in the operating room and moving on rails . before the commencement of the treatment , ct examination was conducted with or without contrast agent , and then , the image was fused with the previously performed ct or magnetic resonance imaging ( mri ) examination . then , under continuous ct guidance , applicator needle or elastic applicator lead was percutaneously ( transdermally ) inserted into the tumor in a way facilitating irradiation of the whole tumor area , and simultaneously controlling the position of the needle against the adjacent structures ( figure 1 ) . if it was possible , the applicators were inserted in such a way that their layout in the lesion was possibly most parallel , and their distance from one another as well as from the edge of the tumor was not greater than 2 cm . due to little respiratory movement of anatomic structures in head and neck region , ct guided insertion of the needle was precise and relatively safe , despite close vicinity of many important organs . only the region directly under the needle was not sufficiently visible due to artifacts related to the material , of which the needle was made . needle applicators were located in the lesion only during each fraction of treatment but elastic applicators , used for their stabilization , remained in the tumor from the beginning of the first to the end of the last fraction ( figure 2 ) . computed tomography - guided applicators , next steps ( patient 4 ) patient 4 with applicators on the completion of a single treatment session , ct scan was performed to plan further treatment , and then , after drawing the clinical target volume ( ctv ) and organs at risk ( oar ) , time and place of ionizing radiation source stopping points were planned . the condition for treatment plan acceptance was encompassing the total ctv with a dose constituting 90% of the planned dose ( d90 ) . these doses and total doses were converted according to linear quadratic model of biologic effective dose ( bed ) , using the following formula bed = nd ( 1 + d/(/ ) ) , where d fractional dose , n number of fractions , / alpha / beta ratio , in case of squamous cell carcinoma of head and neck it is 10 . in order to compare them with conventional fractioning of 2 gy , the doses were converted to dose equivalent 2 gy ( deq2 ) using the following formula : deq2 = bed/(1 + 2/(/ ) ) . in order to facilitate the evaluation of correlation between the dose and possible future damages , the volume receiving 100% of the prescribed dose was reported , as well as high dose areas receiving 150% of the prescribed dose ( v150 ) and 200% of the prescribed dose ( v200 ) . the image - guided high - dose - rate ( ig - hdr ) plan for patients . depending on the type of the organ , either maximum dose ( dmax ) or dose per 1 cm of the organ located in the maximum dose area ( d1 cm ) were reported . the doses were converted to bed and to deq2 according to the linear quadratic model , adopting / ratio value of 3 for future radiation induced complications . because of the risk of hemorrhage , the surgical procedure was conducted in an operating room equipped with ge oec 9900 elite system ( ge health care , milwaukee , wi , usa ) , and offering the possibility of interventional embolization of a damaged vessel by a surgical radiology specialist . patients with neoplastic lesions located deep under the skin surface in hardly accessible areas were qualified for treatment using image - guided brachytherapy . the study involves analysis of data concerning 4 patients with local relapse of head and neck cancer treated in the department of brachytherapy , lublin region cancer center in 2013 - 2015 . the indications for application of image guided brachytherapy were above all close vicinity of large blood vessels ( all patients ) , the organ of vision ( patient 4 ) , the extent of the neoplastic process ( patient 2 ) , and small relapsed tumor size requiring precise application ( patient 1 ) . clinical characteristics of patients df fractionated dose , dc complete dose , pr partial remission , sd stable disease , ebrt external beam radiation therapy , rtog radiation therapy oncology group , ctcae common terminology criteria for adverse events , recist response evaluation criteria in solid tumors the procedure was conducted under general ( patients 2 , 3 and 4 ) or local anesthesia ( patient 1 ) . under local anesthesia steel or titanium needle applicators , as well as elastic interstitial applicators were used in brachytherapy . in sites , where it was impossible to drive the applicators through the lesion , needle applicators were used . a disadvantage was the necessity of conducting the treatment in several stages , which was associated with the need for performing several application procedures , as well as several steps of treatment planning . due to poor visibility of the placed applicators , it was necessary to apply markers just after the application . the application was conducted under guidance of ct device located in the operating room and moving on rails . before the commencement of the treatment , ct examination was conducted with or without contrast agent , and then , the image was fused with the previously performed ct or magnetic resonance imaging ( mri ) examination . then , under continuous ct guidance , applicator needle or elastic applicator lead was percutaneously ( transdermally ) inserted into the tumor in a way facilitating irradiation of the whole tumor area , and simultaneously controlling the position of the needle against the adjacent structures ( figure 1 ) . if it was possible , the applicators were inserted in such a way that their layout in the lesion was possibly most parallel , and their distance from one another as well as from the edge of the tumor was not greater than 2 cm . due to little respiratory movement of anatomic structures in head and neck region , ct guided insertion of the needle was precise and relatively safe , despite close vicinity of many important organs . needle applicators were located in the lesion only during each fraction of treatment but elastic applicators , used for their stabilization , remained in the tumor from the beginning of the first to the end of the last fraction ( figure 2 ) . computed tomography - guided applicators , next steps ( patient 4 ) patient 4 with applicators on the completion of a single treatment session , ct scan was performed to plan further treatment , and then , after drawing the clinical target volume ( ctv ) and organs at risk ( oar ) , time and place of ionizing radiation source stopping points were planned . the condition for treatment plan acceptance was encompassing the total ctv with a dose constituting 90% of the planned dose ( d90 ) . these doses and total doses were converted according to linear quadratic model of biologic effective dose ( bed ) , using the following formula bed = nd ( 1 + d/(/ ) ) , where d fractional dose , n number of fractions , / alpha / beta ratio , in case of squamous cell carcinoma of head and neck it is 10 . in order to compare them with conventional fractioning of 2 gy , the doses were converted to dose equivalent 2 gy ( deq2 ) using the following formula : deq2 = bed/(1 + 2/(/ ) ) . in order to facilitate the evaluation of correlation between the dose and possible future damages , the volume receiving 100% of the prescribed dose was reported , as well as high dose areas receiving 150% of the prescribed dose ( v150 ) and 200% of the prescribed dose ( v200 ) . the image - guided high - dose - rate ( ig - hdr ) plan for patients . depending on the type of the organ , either maximum dose ( dmax ) or dose per 1 cm of the organ located in the maximum dose area ( d1 cm ) were reported . the doses were converted to bed and to deq2 according to the linear quadratic model , adopting / ratio value of 3 for future radiation induced complications . because of the risk of hemorrhage , the surgical procedure was conducted in an operating room equipped with ge oec 9900 elite system ( ge health care , milwaukee , wi , usa ) , and offering the possibility of interventional embolization of a damaged vessel by a surgical radiology specialist . five fractions of 3 gy or 4 gy were used in 1 day intervals . due to this fact , fractional doses were higher ( 5 gy / fraction ) and the interval between sessions was longer ( from 3 to 7 days ) . in one patient , the insertion procedure was performed in general anesthesia and in the others , in local anesthesia after premedication . d100 median of a single fraction was 3.8 gy ( range 3 - 4.2 gy ) , and d90 median of a single fraction was 5.2 gy ( range 3.7 - 5.8 ) . d100 total dose median was 15.4 gy ( range 10 - 19.5 gy ) , and d90 total dose median was 20.6 gy ( range 16.1 - 26 gy ) . biologic effective dose median in isodose encompassing the whole irradiated volume ( d100 ) of all treatment fractions was 20.7 gy ( range 13.4 - 27.1 gy ) and deq2 was 17.2 gy ( range 11.1 - 22.6 gy ) . biologic effective dose median in isodose encompassing 90% of the irradiated volume ( d90 ) was 30.4 gy ( range 24.7 - 39.5 gy ) and deq2 was 25.3 gy ( range 20.6 - 32.9 gy ) . clinical target volume , which received 100% dose , as well as 150% and 200% dose was characterized in table 2 . % of prescription dose covering 100% of target volume ; d90% % of prescription dose covering 90% of target volume ; v100% , v150% , v200% % of target volume receiving prescription dose , 150% of prescription dose and 200% of prescription dose ; dhi dose homogeneity index ; bed d100% biologic effective dose in d100% ; bed d90% biologic effective dose in d90% ; deq2 d100% dose equivalent 2 gy in d100% ; deq2 d90% dose equivalent 2 gy in d90% ; fr fractions doses in critical organs : brain , spinal cord , and eyeballs in individual patients spinal cord max . maximum dose in spinal cord and left and right eyeballs ; brain 1 cm dose in 1 cm of brain tissue ; bed spinal cord , bed eye l. , bed eye r. biologic effective dose of maximum dose in spinal cord and left and right eyeballs ; bed brain biologic effective dose of the dose in 1 cm of brain tissue ; deq2 spinal cord , deq2 eye l. , deq2 eye r. dose equivalent 2 gy of maximum dose in spinal cord and left and right eyeballs ; deq2 brain dose equivalent 2 gy of the dose in 1 cm of brain tissue doses in critical organs : lens , otical nerves and optic chiasma in individual patients ( only patients 2 and 4 ) lens l. max . biologic effective dose of maximum dose in maximum dose in left and right lens , left and right otical nerves and optic chiasma ; deq2 lens l. , deq2 lens r. , deq2 optic n. l. , deq2 optic n. r. , deq2 optic chiasm dose equivalent 2 gy of maximum dose in left and right lens , left and right otical nerves and optic chiasma doses in critical organs : salivary glands , larynx , thyroid , and bone tissue in individual patients parotid l. 1 cm , parotid r. 1 cm , larynx 1 cm , thyroid gland 1 cm , bone 1 cm dose in 1 cm of salivary glands , larynx , thyroid , and bone tissue ; bed parotid l. , bed parotid r. , bed larynx , bed thyroid gland , bed bone biologic effective dose of the dose in 1 cm of salivary glands , larynx , thyroid , and bone tissue ; deq2 parotid l. , deq2 parotid r. , deq2 larynx , deq2 thyroid gland , deq2 bone dose equivalent 2 gy of the dose in 1 cm of salivary glands , larynx , thyroid , and bone tissue treatment toxicity was evaluated using common terminology criteria for adverse events ( ctcae ) v.4.0 , and the radiotherapy related morbidity was measured using radiation therapy oncology group ( rtog ) scale . despite close vicinity of many important structures in the head and neck area , no mechanical injury of these organs was observed either in course of the insertion procedure or while the applicators were remaining in the treatment site between subsequent treatment sessions . none of the patients suffered from paralysis in result of mechanical damage of the nerves . due to abundant vascularity of head and neck organs , there was a small degree of bleeding at the time of applicator removal , which was resolved spontaneously . in 3 patients ( patients 2 , 3 , 4 ) , due to location of the irradiated area and acute radiotherapy induced morbidity of the skin , rtog scale grade 1 occurred . in the period of observation lasting for 3 to 7 months ( median 3.5 months ) , partial regression ( pr ) was observed in 2 patients and stable disease in other 2 patients . treatment toxicity was evaluated using common terminology criteria for adverse events ( ctcae ) v.4.0 , and the radiotherapy related morbidity was measured using radiation therapy oncology group ( rtog ) scale . despite close vicinity of many important structures in the head and neck area , no mechanical injury of these organs was observed either in course of the insertion procedure or while the applicators were remaining in the treatment site between subsequent treatment sessions . none of the patients suffered from paralysis in result of mechanical damage of the nerves . due to abundant vascularity of head and neck organs , there was a small degree of bleeding at the time of applicator removal , which was resolved spontaneously . in 3 patients ( patients 2 , 3 , 4 ) , due to location of the irradiated area and acute radiotherapy induced morbidity of the skin , rtog scale grade 1 occurred . in the period of observation lasting for 3 to 7 months ( median 3.5 months ) , partial regression ( pr ) was observed in 2 patients and stable disease in other 2 patients . the main factor limiting the application of hdr brachytherapy in the head and neck area is the tumor location . due to the necessity of performing visually controlled application , brachytherapy in this area is only possible in case of lip cancer , oral cavity cancer , selected neoplasms of nasopharynx , nasal cavity and nasopharynx , as well as in case of repeated radiotherapy of nodular relapses in the head and neck area [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ] . reports describing image guided brachytherapy predominantly concern liver , rectal , and lung cancers [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ] . studies concerning image guided brachytherapy in patients with head and neck cancer are singular . , 49 patients with nodular relapse in the neck area were subjected to palliative interstitial brachytherapy . the patients were irradiated twice a day with 3 gy fractions up to the total dose of 30 - 36 gy . , 25 patients with squamous cell carcinoma relapse in the area of head and neck were treated with brachytherapy using permanent implants . data were retrospectively analyzed from 18 patients with cancer , located in various areas including 2 in the head and neck area , who had undergone hdr brachytherapy performed under guidance of different imaging methods . various dose fractioning schemes were used : 6 gy in 6 fractions and 10 gy in one fraction ( as a boost after external beam radiation therapy ) . in one patient , the lesion was located directly close to the left internal carotid artery , and in other , one in soft tissues of the neck bordering the chest . serious complications related to brachytherapy were not observed in any of the patients . due to short observation time and small number of patients in the described study , the treatment results and late toxicity were not analyzed . in our study , for four patients with relapsed neoplastic lesion located in a site preventing visually controlled brachytherapy , the application of ct image guided hdr brachytherapy facilitated the treatment in hardly accessible locations , too close to large nerves or blood vessels . , it is only possible to compare biological equivalent dose ( bed ) of the total dose . was similar ( 32.5 - 39 gy , / = 10 ) , and in kishan et al . due to the necessity of having appropriate technical facilities and difficulty of its application , thus , there are no studies based on large group of patients evaluating safety of the procedure and treatment results . there are also no studies comparing different application techniques as well as different types of applicators used in the head and neck region . the intention of image guided hdr brachytherapy is to provide local lesion control in sites , which , due to their extent or lack of technical facilities , do not qualify for using other radiotherapy techniques , especially stereotactic radiotherapy . image guided brachytherapy has the potential to be a valuable treatment method for patients with local and regional relapse of head and neck cancer located in sites preventing standard brachytherapy . it seems justified to conduct prospective studies to evaluate the efficiency and safety of ct image guided brachytherapy .

fenretinide ( n-4-(hydroxyphenyl)-retinamide ; 4-hpr ) is a synthetic analog of vitamin a that has shown promise as both a chemotherapeutic and chemopreventive agent in solid tumors and hematologic malignancies [ 16 ] . while fenretinide binding to retinoic acid receptors ( rars ) can promote apoptosis in some cell types , the agent can induce death in a rar - independent manner . rar - independent mechanisms of cell death likely involve the production of reactive oxygen species ( ros ) and the generation of sphingolipid second messenger molecules [ 711 ] . while there have been numerous studies on fenretinide in recent years , the identification of diverse potential mechanisms for fenretinide antineoplastic activity suggests that the agent may function differently in different cell types [ 1 , 11 ] . fenretinide has been shown to activate jnk [ 6 , 12 ] , promote ros generation , activate endoplasmic reticulum ( er ) stress pathways [ 13 , 14 ] as well as activate the intrinsic apoptotic pathway with various bcl2 family members as targets [ 1517 ] . recent studies have suggested that fenretinide may be an effective agent in the treatment of acute lymphoblastic leukemia ( all ) since the drug effectively kills all cell lines but not nonmalignant lymphoid cell types . a recent study from the reynolds group has demonstrated that fenretinide can synergize with abt-737 to effectively kill all cells . abt-737 is a small molecule inhibitor of many antiapoptotic bcl2 family members ( but not mcl-1 ) that is currently in clinical trials for a variety of cancers [ 1921 ] . mcl-1 has been found to promote resistance to abt-737-induced apoptosis and suppression of mcl-1 promotes sensitivity to the drug [ 1921 ] . fenretinide was found to promote mcl-1 degradation by a jnk - mediated mechanism and thus has promise in overcoming mcl-1 mediated chemoresistance in all and other leukemias . kim and colleagues have discovered that fenretinide can promote apoptosis via ros activation of jnk , p38 , erk , and pkc though the potential pkc isoforms involved were not identified . the protein kinase c ( pkc ) family is composed of at least 11 members with distinct functions and tissue distributions [ 2225 ] . pkc isoforms are divided into three groups based on structural features : classical ( cpkc ) , novel ( npkc ) , and atypical ( apkc ) . the cpkcs include pkc and pkc and require calcium and diacylglycerol ( dag ) for activation . the npkcs include pkc and pkc and require dag but not calcium for activation . both cpkc and npkc members have been implicated in hematopoietic malignancies [ 24 , 25 ] . pkc has been linked to leukemogenesis in b - cell chronic lymphocytic leukemia ( b - cll ) . pkc promotes chemoresistance in all and acute myeloid leukemia ( aml ) cell lines [ 27 , 28 ] and may be a negative prognostic factor in aml [ 29 , 30 ] . pkc may support b - cll cells by cd5-mediated signaling . pkc has been implicated in hairy cell leukemia as an activator of erk and rac1 . the role of pkc in leukemia is more complicated . unlike pkc , pkc , and pkc which generally regulate survival signaling pathways , pkc cells derived from pkc null mice are resistant to apoptosis in response to chemotherapy drug or irradiation . the mechanism how pkc supports apoptosis is complex . in response to a stress challenge , tyrosine phosphorylation of pkc promotes its translocation to the nucleus where it is cleaved by caspase 3 [ 3336 ] . the cleaved pkc is active and targets a number of nuclear substrates that may be essential for the induction of cell death including lamin , dna dependent protein kinase ( dna - pk ) , and p53 [ 3336 ] . cleaved pkc has been suggested to target mcl-1 for degradation . considering that fenretinide promotes mcl-1 degradation in all cell lines , the possibility arises that the fenretinide - induced apoptosis may involve pkc . in the present study , we examined pkc expression and cleavage and mcl-1 expression in response to fenretinide in three all cell lines ( reh , ccrf - cem , and molt4 ) and in a mixed lineage leukemia ( mll ) cell line ( rs4;11 ) . fenretinide promoted cleavage of pkc and suppression of mcl-1 in cell lines sensitive to the drug ( rs4;11 and ccrf - cem ) but not in cell lines that were more resistant ( reh and molt4 ) . in ccrf - cem cells , fenretinide promoted translocation of jnk from the cytosol to the nucleus . consistent with the findings of the reynolds group , fenretinide likely promotes mcl-1 protein degradation since fenretinide did not inhibit mcl-1 gene expression in three of the four leukemia lines and the drug actually promoted gene expression in reh cells ( perhaps as part of an initial sos response for these cells ) . the antioxidant vitamin c protected all cell lines from the drug and suppressed pkc cleavage suggesting that the mechanism by which fenretinide promotes death involves ros and pkc . suppression of pkc in ccrf - cem cells by shrna sensitized the cells to fenretinide suggesting that loss of the kinase may also be important in the drug 's mode of action . basal ros production was increased in cells with reduced pkc suggesting that the increase in sensitivity to the drug may be due to enhanced ros generation . the findings in this study suggest that pkc plays an important role in fenretinide - induced apoptosis in lymphoid leukemia cell lines . hek-293 t cells were obtained from the atcc ( manassas , va ) and maintained in dmem supplemented with 10% fetal bovine serum at 37c in 5% co2 . reh , ccrf - cem , molt4 , and rs4;11 cells were obtained from the atcc and maintained in rpmi 1640 + 10% fetal bovine serum at 37c in 5% co2 . a set of four shrnas targeting pkc from the lentiviral shrna library of the rnai consortium ( designated trcn0000010193 , trcn0000010194 , trcn0000010202 , and trcn0000010203 ) was obtained from open biosystems ( huntsville , al ) . lentiviral packaging plasmids md2.g ( addgene plasmid 12259 ) and pspax2 ( addgene plasmid 12260 ) were constructed by the laboratory of didier trono and obtained from addgene ( cambridge , ma ) . a control lentiviral transfer plasmid , plko.1 trc control ( addgene plasmid 10879 ) , was constructed by the laboratory of david root and obtained from addgene . each trc lentiviral shrna plasmid was transiently cotransfected in an equimolar mix with the lentiviral packaging plasmids into hek 293 t cells using lipofectamine 2000 ( invitrogen , carlsbad , ca ) as directed by the manufacturer . lentiviral supernatants were harvested at 48 hours post transfection first by centrifugation for ten minutes at room temperature at 800 g and then by filtration through 0.45 m surfactant - free cellulose acetate to assure complete removal of producing cells . polybrene ( chemicon , temecula , ca ) was then added to a concentration of 8 g / ml , and the resulting virus stock was used at once to spinoculate ccrf - cem cells . ccrf - cem cells were resuspended at a concentration of 0.5 million cells per ml of virus stock , transferred to the wells of a 12 well tissue culture cluster , and centrifuged for 45 minutes at 30c at 1300 g . after the addition of one volume of fresh virus stock , the cells were subject to a second round of centrifugation , followed by incubation at 37c in 5% co2 for 60 minutes . the infected cells were then washed twice with growth medium to remove the polybrene and allowed to grow for two doubling times ( 4244 hours ) , after which time they were subject to selection with 1.0 g / ml puromycin ( invivogen , san diego , ca ) . puromycin - resistant pools of infected cells were assessed for pkc knockdown by western analysis . cells were pretreated with 0.4 m vitamin c for 2 hours prior to treatment with either vehicle ( 0.1% dmso ) or 1 m fenretinide for 24 hours . cells were centrifuged and resuspended in warm ( 37c ) medium containing 10 m 5-(and-6)-carboxy-2 , 7-dichlorodihydrofluorescein diacetate ( carboxy - h2dcfda ; invitrogen ) and incubated for 20 minutes at 37c . cells were then centrifuged and washed with phosphate - buffered saline , and then analyzed on a becton dickinson facscalibur flow cytometer ( bd biosciences , san jose , ca ) . cells were treated with varying doses of fenretinide ( calbiochem , san diego , ca ) for 24 hours . where appropriate , cells were pretreated 2 hours with vitamin c ( sigma , st . louis , mo ) , caspase 8 inhibitor ( calbiochem ) , or caspase 9 inhibitor ( calbiochem ) or 1 hour with bryostatin-1 ( calbiochem ) prior to the addition of fenretinide . cell viability was measured by trypan blue dye exclusion and apoptosis was evaluated using the annexin v - fitc apoptosis detection kit ( mbl international , woburn , ma ) . the cells were washed twice with pbs and resuspended in 450 l 1 x annexin binding buffer . 5 l of annexin v - fitc and 5 l of pi were added to each tube . the cells were mixed gently and incubated for 5 minutes in the dark at room temperature . cells were analyzed on a becton dickinson facscalibur flow cytometer ( bd biosciences , san jose , ca ) , placing the fitc signal in fl1 and the pi signal in fl2 . intact cells were gated in the fsc / ssc plot to exclude small debris . cells in the lower right quadrant of the fl1/fl2 dot plot ( labeled with annexin v - fitc only ) are considered to be in early apoptosis , and cells in the upper right quadrant ( labeled with annexin v - fitc and pi ) are in late apoptosis / necrosis . total protein ( 2 10 cell equivalents ) was subjected to sds - page using antibodies specific for the analyzed proteins . antibodies used were caspase 3 ( santa cruz biotechnology , santa cruz , ca ) , caspase 8 ( santa cruz biotechnology ) , caspase 9 ( santa cruz biotechnology ) , parp ( santa cruz biotechnology ) , pkc ( santa cruz biotechnology ) , pkc i ( santa cruz biotechnology ) , pkc ii ( santa cruz biotechnology ) , pkc ( santa cruz biotechnology ) , pkc ( santa cruz biotechnology ) , p - jnk ( cell signaling , danvers , ma ) , jnk ( cell signaling ) , bcl2 ( dako , carpinteria , ca ) , bcl - xl ( santa cruz biotechnology ) , mcl-1 ( santa cruz biotechnology ) , and tubulin ( sigma ) . cells were suspended in rpmi media and sedimented onto round poly - l - lysine coated coverslips ( biocoat , bd biosciences , bedford , ma ) , using a sorval rc-6 high speed centrifuge ( thermo fischer , pittsburgh , pa ) , with an hb-6 swinging bucket rotor and custom made tube inserts at 1200 g for 15 minutes . louis mo ) , and a polyclonal antibody against pkc-. secondary antibodies goat antimouse alexa fluor 488 and goat antirabbit alexa fluor 594 , and counter stained with dapi . cells were imaged with a leica dm rxa-2 microscope ( leica microsystems , bannockburn , il ) , with fluorescence optics using a leica 63 1.4 na apochromat cs objective and a hamamatsu orca - er cooled ccd camera ( hamamatsu , e. bridgewater , nj ) . images were collected as a z series of 0.5 m , and were deconvolved by constrained iterative , blind deconvolution using simple pci imaging software ( hamamatsu imaging , sewickley , pa ) . z series are presented as a maximal projection and final images were cropped and adjusted for contrast in photoshop ( adobe , mountain view , ca ) . total rna was extracted from cells using trireagent ( sigma ) as directed by the manufacturer . to ensure complete removal of trace genomic dna or other factors that could interfere with downstream enzymatic processes , all rna samples were subjected to final purification using rneasy mini columns ( qiagen , valencia , ca ) with on - column treatment by dnase i as directed by the manufacturer . cdna was prepared from 1.0 g of total rna per 20 l mix containing 0.07 g/l random - sequence hexamer primers , 1 mm dntps , 5 mm dtt , 0.2 u/l superasin rnase inhibitor ( ambion , austin , tx ) , and 10 u/l superscript iii reverse transcriptase ( invitrogen ) . all components except enzyme were added and the mixture was incubated at room temperature for 2 minutes to allow nucleic acids to anneal . after addition of reverse transcriptase , the mixture was incubated for ten minutes at 25c , then one hour at 50c , followed by heat - inactivation of the enzyme for 15 minutes at 72c . all cdnas were stored at 80c when not in use . to verify the complete removal of any residual genomic material in the real - time pcr assays , we incubated in parallel 1.0 g of total rna per 20 l of a mix containing all components except reverse transcriptase . real - time pcr was carried out using an abi model 7500 sequence detection system ( applied biosystems , foster city , ca ) . duplicate 25 l reactions containing the cdna equivalent of 50 pg total rna were run and repeated if the ct values were more than 0.25 cycles apart . as primers and probes we used the following taqman gene expression assays ( applied biosystems ) as directed by the manufacturer : mcl1 assay i d hs00172036_m1 , bcl2 assay i d hs00236808_s1 , b2 m assay i d hs99999907_m1 , and 18srrna hs99999901_s1 . we used 18s rrna to normalize gene expression . to calculate the relative abundance ( ra ) of each transcript of interest relative to that of 18s , the following formula was employed : ra = 1000000 2 , where ct is the mean ct of the transcript of interest minus the mean ct of the transcript for 18s . statistical analysis was performed using standard t - test analysis with sigma stat computer software ( ssps , chicago , il ) . sensitivity to fenretinide was investigated in the t - all derived cell lines ccrf - cem and molt4 cells , in the pre - b all derived cell line reh , and in the mll derived cell line rs4;11 . cells were treated with vehicle ( 0.1% dmso ) or with 1 m , 5 m , or 10 m fenretinide for 24 hours and programmed cell death was assessed by annexin v staining using facscan . as shown in figure 1 , ccrf - cem and rs4;11 cells were the most sensitive to fenretinide . at 1 m fenretinide , > 30% of ccrf - cem and rs4;11 cells were positive for annexin v staining while higher doses of the drug induce apoptosis in the majority of cells from both cell lines ( i.e. , > 80% at 5 m and > 89% at 10 m ; figure 1 ) . compared to ccrf - cem and rs4;11 cells , molt 4 cells were more resistant to fenretinide . as shown in figure 1 , reh cells displayed the greatest resistance to fenretinide - induced apoptosis . since rs4;11 cells are p53 + and ccrf - cem cells contain mutant p53 , p53 does not appear to be required for fenretinide - induced cell death . this finding is consistent with published reports indicating that fenretinide acts to kill cells via a p53 independent mechanism . as shown in figure 2 , treatment of cells with 1 m fenretinide for 24 hours promoted potent ros generation in rs4;11 cells ( i.e. , > 3.8 fold increase , p < .001 ) and ccrf - cem cells ( i.e. , ~9 fold increase , p < .001 ) . molt4 cells treated similarly with fenretinide exhibited a slight but statistically significant increase in ros produced ( i.e. , 45% , p < .003 ) . treatment of reh cells with 1 m fenretinide for 24 hours had no effect on ros generation . however , reh cells treated with 5 m drug did show a > -4 fold increase in ros production ( p < .002 ) after 24 hours ( data not shown ) . these results suggest that there is a positive correlation between ros generation and sensitivity to fenretinide . if ros generation is critical to fenretinide - induced apoptosis , it would be expected that an antioxidant such as vitamin c ( l - abscorbic acid ) would protect cells from the cytotoxic effects of the drug . reh , molt4 , rs4;11 , and ccrf - cem cells were treated with vehicle ( 0.2% dmso ) , 0.4 m vitamin c , 5 m fenretinide , or a combination of vitamin c and drug at the specified concentrations for 24 hours . induction of apoptosis was then measured by facscan analysis of cells to identify annexin v positive cells . vitamin c alone had no effect on rs4;11 or molt4 cells but did have a slight effect on the other two cells lines ( figure 3 ) . vitamin c promoted an increase of apoptotic ccrf - cem cells compared to cells treated with vehicle ( i.e. , 9.4% versus 5.7% , resp . ; vitamin c alone reduced the population of apoptotic ccrf - cem cells compared to cells treated with vehicle ( i.e. , 5.3% versus 3.2% , resp . ; p < .002 ) . as shown in figure 3 , vitamin c protected all four cell lines from fenretinide - induced apoptosis . this result strongly suggests that ros production is a key element in the mechanism by which fenretinide kills leukemia cells . it has been previously established that fenretinide can promote apoptosis in leukemia cell lines such as ccrf - cem by induction of mitochondrial - mediated oxidative stress [ 7 , 8 , 18 ] . however , a recent study has suggested that caspase activity is not required for fenretinide - induced cell death in lymphoid leukemia cell lines . cells were treated with varying doses of drug for 24 hours and caspase activation was observed by western analysis of parp cleavage . as shown in figure 4 , parp cleavage was observed in rs4;11 and ccrf - cem cells treated with 1 m fenretinide while complete parp cleavage is seen with 5 m of the drug . reh cells and molt4 cells , which are relatively more resistant to fenretinide - induced cell death compared to rs4;11 and ccrf - cem cells , displayed less parp cleavage in response to fenretinide . in fact , parp is only partially cleaved in reh cells even when treated with 10 m fenretinide ( figure 4 ) . rs4;11 cells display the greatest activation of executioner caspase 3 ( as observed by loss of pro - caspase ) with fenretinide treatment . interestingly , activation of caspase 9 ( which mediates the intrinsic / mitochondrial apoptotic pathway ) was not very pronounced in rs4;11 or reh cells and was not detected in ccrf - cem or molt4 cells ( data not shown ) . as shown in figure 4 , fenretinide activated caspase 8 in the rs4;11 and ccrf - cem cell lines ( as observed by cleavage of the caspase ) but not in molt4 cells . there was limited activation of caspase 8 in reh cells compared to rs4;11 and ccrf - cem cells . a dose of 10 m fenretinide is required to observe some caspase 8 cleavage in reh cells while 5 m drug results in near complete cleavage of the caspase in both rs4;11 and ccrf - cem cells ( figure 4 ) . however , fenretinide - induced apoptosis in rs4;11 or ccrf - cem cells does not require either caspase 8 or caspase 9 . as shown in figure 5 , inhibition of caspase 9 actually increased sensitivity to fenretinide in both cell lines . while inhibition of caspase 8 did result in statistically significant protection from fenretinide in rs4;11 and ccrf - cem cells ( p = .001 and p = .003 , resp . ) , the protection accorded was only partial indicating that caspase 8 may be necessary but not sufficient for fenretinide - induced apoptosis in these cell lines . these results are similar to findings reported recently from the reynolds group that demonstrated that fenretinide - induced apoptosis in the t - all - derived cell line cog - ll-317 involves a caspase independent mechanism . a number of signaling pathways that may be activated by fenretinide have been identified ( e.g. , jnk ) [ 6 , 8 , 1214 ] ; however , a role for pkc in fenretinide - induced apoptosis has yet to be established . a role for pkc in fenretinide - induced apoptosis in head and neck squamous carcinoma cells has been suggested but an analysis of which pkc isoforms might participate was not performed . reh and rs4;11 cells were treated with varying doses of fenretinide for 24 hours and then lysed for western blot analysis . as shown in figure 6 , fenretinide had little effect on the classical pkc isoforms ( i.e. , pkc , pkc i , and pkc ii ) in reh cells but the expression of each of these was suppressed in rs4;11 cells . fenretinide potently induced cleavage of novel pkc isoforms pkc and pkc in rs4;11 cells and to a lesser extent in reh cells ( figure 4 ) . while the significance of pkc cleavage is not clear , cleavage of pkc has been implicated as a positive regulator of apoptosis [ 3336 ] . ccrf - cem and molt4 cells were similarly treated with fenretinide and expression of pkc , pkc , and pkc was examined ( figure 7 ) . as was the case for rs4;11 cells , fenretinide suppressed pkc expression and promoted cleavage of pkc in ccrf - cem cells . while the drug did suppress expression of pkc , no cleavage product was detected ( figure 5 ) . as was the case for reh cells , fenretinide treatment of molt4 cells had little effect on pkc expression and only promoted pkc cleavage with higher concentrations . considering that cleaved pkc has been implicated as a positive regulator of apoptosis , it is not surprising to find that the leukemia cells that are more sensitive to fenretinide ( i.e. , rs4;11 and ccrf - cem cells ) exhibit greater production of cleaved pkc compared to the leukemia cells that are more resistant ( i.e. , reh and molt4 ) . nuclear translocation of pkc has been shown to be an important event in apoptotic stress signaling pathways involving the kinase [ 3336 ] . to determine if fenretinide promoted nuclear translocation of pkc , subcellular localization of the kinase was examined by immunofluorescence microscopy in vehicle ( 0.1% dmso ) treated ccrf - cem cells and cells treated with 1 m fenretinide for 24 hours . as shown in figure 8 , pkc is mainly found in the cytoplasm and in association with centrosomes in cells treated with vehicle . however , in cells treated with fenretinide , antibody against pkc labels the nuclear region , and is not present in the cytoplasm ( figure 8) . pkc continues to localize to the centrosome in cells treated with fenretinide ( figure 8 , arrow ) . the promotion of pkc localization to the nucleus is consistent with proapoptotic signaling described by others [ 3336 ] . a recent study from the reynolds group suggests that fenretinide suppresses expression of the antiapoptotic bcl2 family member mcl-1 in all cells . cleaved pkc has been suggested to regulate the degradation of mcl-1 during apoptosis in u.v . it is not surprising that fenretinide sensitive cell lines rs4;11 and ccrf - cem display reduced expression of mcl-1 with concomitant cleavage of pkc when treated with higher doses of the drug ( figures 6 and 7 ) . fenretinide had no effect on the protein expression of the other antiapoptotic bcl2 family members expressed in the reh and rs4;11 cells ( i.e. , bcl2 and bcl - xl ; figure 6 ) . in the reynolds group study , jnk was implicated as regulating mcl-1 degradation by observations made using ccrf - cem cells . as shown in figure 6 , rs4;11 cells exhibit suppression of mcl-1 protein expression in response to fenretinide but do not display significant fenretinide - induced activation of jnk ( as indicated by phosphorylation of the kinase ) . in fact , both jnk1 and jnk2 appear to be downregulated by fenretinide in rs4;11 cells at higher concentrations ( figure 6 ) . thus at least in rs4;11 cells , suppression of mcl-1 expression is independent of jnk . to examine if suppression of mcl-1 expression by fenretinide might be mediated by a transcriptional mechanism , gene expression of mcl-1 as well as fellow antiapoptotic family member bcl2 was assessed in cells treated with the drug . reh , molt4 , ccrf - cem , and rs4;11 cells were treated with vehicle ( 0.1% dmso ) or 10 m fenretinide for 6 hours or 24 hours . rna was isolated and transcription of mcl-1 and bcl2 was measured by real - time pcr ( rt - pcr ) . expression of 18s rna was also measured so expression of each gene is reported as transcript per million copies of 18s . to control the effect of fenretinide on transcription overall , b2 m was also measured by rt - pcr . fenretinide does appear to have differential effects on transcription overall among the various cell lines as determined by the changes in the level of b2 m . there was a slight reduction of b2 m transcript observed in molt4 and rs4;11 cells treated with fenretinide for 24 hours ( ~15% reduction each ; figure 9 ) . however , fenretinide suppressed b2 m transcription by nearly 2-fold after 24 hours in reh cells and by nearly 3-fold after 24 hours in ccrf - cem cells . as shown in figure 9 , fenretinide did not suppress mcl-1 transcription by more than 20% in any of the cells after 24 hours . in fact , there was a slight ( i.e. , ~35% ) increase in mcl-1 transcript in rs4;11 cells when treated with the drug for 24 hours . thus loss of transcript can not account for the suppression of mcl-1 protein by 10 m fenretinide in these cells that was demonstrated in figure 6 . ccrf - cem cells , which also exhibit reduced mcl-1 protein in response to the drug ( see figure 7 ) , likewise do not exhibit much decrease ( i.e. , ~18% ) in mcl-1 transcription . actually relative to b2 m transcription , there is a > 2-fold increase in mcl-1 transcript in the fenretinide treated ccrf - cem cells when compared to control . interestingly , there was a > 2-fold increase in mcl-1 gene expression in reh cells treated with 10 m fenretinide for 6 hours compared to vehicle treated control cells ( figure 9 ) . it remains to be determined if fenretinide promotes mcl-1 protein expression in reh cells by a transcriptional mechanism . also , it is possible that fenretinide regulates mcl-1 gene expression by other means such as a mechanism involving a micro - rna . still , the data presented here suggest that downregulation of mcl-1 in the fenretinide sensitive cell lines ( i.e. , rs4;11 and ccrf - cem ) does not occur via a transcriptional mechanism . interestingly , reh and rs4;11 cells treated with 10 m fenretinide for 24 hours exhibited > 3-fold reduction and > 8-fold reduction , respectively , in bcl2 gene expression compared to vehicle control cells treated for 24 hours ( figure 9 ) . fenretinide induced loss of pkc in these cells ( figures 6 and 7 ) . pkc has been implicated as a regulator of bcl2 gene expression in hematopoietic cells . it should be noted that fenretinide did not affect bcl2 protein levels after 24 hours in either reh or rs4;11 ; however , bcl2 protein has a relatively long half life compared to mcl-1 . overexpression of exogenous bcl2 in ccrf - cem cells has been shown to protect the cells from the cytotoxic effects of fenretinide . the effect of fenretinide to suppress bcl2 gene expression thus may be key to the cell death mechanism as the antiapoptotic protein interferes with fenretinide drug action when bcl2 gene expression is regulated by an artificial promoter ( i.e. , cmv promoter ) and thus not subject to regulation by the drug . while this potential mechanism needs to be investigated further , the ability of fenretinide to suppress bcl2 expression would have positive benefits in antileukemia strategies that would utilize fenretinide . a plausible mechanism how fenretinide promotes pkc cleavage might involve ros activation of caspase 3 since the protease mediates cleavage of this pkc isoform [ 3336 ] . nuclear translocation of pkc precedes caspase 3 cleavage and indeed fenretinide promotes pkc nuclear translocation ( figure 8) . as shown in figure 4 , caspase 3 is potently activated and parp cleaved in rs4;11 and ccrf - cem cells treated with fenretinide even when cells are treated with 1 m drug . vitamin c was shown to effectively protect the leukemia cells from fenretinide - induced apoptosis likely via a mechanism that suppresses ros ( figure 3 ) . if pkc participates in the death process , it would be predicted that vitamin c would prevent fenretinide - induced cleavage of pkc . rs4;11 cells were treated with vehicle ( 0.1% dmso ) , 0.4 m vitamin c , 5 m fenretinide , or a combination of vitamin c and drug at the specified concentrations for 24 hours . consistent with the protective effects of vitamin c against drug - induced apoptosis , vitamin c blocked fenretinide - induced activation of caspase 3 ( i.e. , inhibited pro - caspase cleavage ) and suppressed cleavage of parp in the rs4;11 cells ( figure 10 ) . as shown in figure 10 , the antioxidant was effective at preventing pkc cleavage in cells treated with fenretinide . this result supports a mechanism where fenretinide promotes ros formation to activate caspase 3 resulting in pkc cleavage . it has been previously demonstrated that the pkc agonist bryostatin-1 protects reh cells from apoptosis induced by a number of clinically relevant chemotherapeutic drugs including etoposide , ara c , and adriamycin . the mechanism how bryostatin-1 protects reh cells from chemotherapeutic drugs involves , at least in part , the activation of pkc resulting in the phosphorylation of bcl2 . bryostatin-1 mimics diacylglycerol ( dag ) and binds to the dag binding site of pkc resulting in activation ; however , prolonged exposure to the drug can result in downregulation of particular pkc isoforms likely due to feedback mechanisms . reh cells were treated with vehicle ( 0.2% dmso ) or 10 m fenretinide for 24 hours . where appropriate , cells were pretreated with 10 nm bryostatin-1 1 hour prior to addition of vehicle or fenretinide . cell viability was determined by trypan blue dye exclusion dye assay . as shown in figure 11(a ) , 10 m fenretinide kills roughly 20% of reh cells . while 10 nm bryostatin-1 alone has no toxic effect on the reh cells , nearly half of cells treated with both 10 nm bryostatin-1 and 10 m fenretinide were killed after 24 hours ( figure 11(a ) ) . the increase in fenretinide - induced cell death in the reh cells due to bryostatin-1 was statistically significant ( p = .0006 ) . thus while bryoststain-1 protects reh cells from chemotherapeutic drugs , the pkc agonist promotes cell death in response to fenretinide . to determine if bryostatin-1 acted similarly on rs4;11 cells treated with fenretinide , rs4;11 cells were treated with fenretinide in the absence or presence of 10 nm bryostatin-1 . since rs4;11 cells are much more sensitive to fenretinide compared to reh cells ( see figure 1 ) , rs4;11 cells were treated with 1 m fenretinide . bryoststin-1 actually protected rs4;11 cells from fenretinide - induced apoptosis . as shown in figure 11(b ) , rs4;11 cells pretreated with 10 nm bryostatin-1 demonstrated a significant decrease in response to 1 m fenretinide after 24 hours compared to cells treated with fenretinide alone ( 46% cell death versus 33% cell death resp . ; p = .02 ) . thus unlike reh cells , bryostatin-1 can protect rs4;11 cells from the cytotoxic effects of fenretinide . differential effects of bryostatin-1 on pkc in reh cells and rs4;11 cells may provide a possible explanation for the difference in effect of bryostatin-1 on fenretinide - induced apoptosis in the two cell lines . reh and rs4;11 cells were treated with vehicle ( 0.2% dmso ) , 10 nm bryostatin-1 , fenretinide , or a combination of bryostatin-1 and drug at the specified concentrations for 24 hours . reh cells were treated with 10 m fenretinide and rs4;11 cells were treated with 1 m fenretinide . consistent with the protective effects of bryostatin-1 against fenretinide - induced apoptosis in the rs4;11 cells , the pkc agonist blocked fenretinide - induced cleavage of both pkc and pkc ( figure 12 ) . bryostatin-1 alone had no effect on pkc in reh cells though it did prevent fenretinide - induced cleavage of pkc ( figure 12 ) . since bryostatin-1 augments fenretinide - induced cell death but blocks cleavage of pkc in reh cells , it is likely that cleaved pkc is not critical in the death process . meanwhile , bryostatin-1 potently suppressed pkc expression in reh cells and there was a near complete inhibition of pkc in reh cells treated with the combination of bryostatin-1 and fenretinide ( figure 12 ) . this result suggests that loss of pkc sensitizes reh cells to fenretinide - induced cell death . to determine if cleaved pkc was required for fenretinide - induced apoptosis , expression of the kinase was suppressed in ccrf - cem cells by shrna . as shown in figure 13 , shrna targeting pkc resulted in significant loss of protein when compared to the cells transduced with the negative control . control shrna ccrf - cem cells and cells expressing pkc shrna were treated with vehicle ( 0.1% dmso ) or with 1 m , 5 m , or 10 m fenretinide for 24 hours and cell death was measured by trypan blue dye exclusion . as shown in figure 14 , ccrf - cem cells expressing pkc shrna were more sensitive to fenretinide compared to control ( i.e. , ~60% cell death versus ~36% cell death with 5 m drug , resp . ) . the increase in fenretinide - induced death in the ccrf - cem cells expressing pkc shrna compared to control cells was significant at drug concentrations of 1 m and 5 m ( i.e. , p = .03 and p = .002 , resp . ) . as shown in figure 13 , ccrf - cem cells expressing pkc shrna displayed more complete parp cleavage with higher doses of fenretinide compared to control cells though there was not an observed increase in caspase 3 cleavage . ccrf - cem cells expressing pkc shrna displayed a complete loss of both full length and cleaved pkc when treated with fenretinide ( figure 13 ) . suppression of pkc did lead to an increase in basal ros levels in ccrf - cem cells . as shown in figure 15 , cells with pkc shrna exhibited a nearly 2 fold increase in ros production compared to control ( p < .001 ) . the increased sensitivity to fenretinide in the cells with pkc shrna is reminiscent of observations with reh cells treated with a combination of bryostatin-1 and fenretinide . bryostatin-1 in combination with fenretinide resulted in complete loss of pkc in reh cells ( figure 12 ) and the pkc agonist promoted sensitivity to fenretinide ( figure 11(a ) ) . these results suggest that the role for pkc in fenretinide - induced cell death in the leukemia cells is complex . the findings of this study suggest that pkc may play an important role in fenretinide - induced apoptosis in lymphoid leukemia cells . previous studies have established that the synthetic vitamin a analog acts via diverse mechanisms but a role for any particular pkc isoform has remained elusive [ 16 ] . an understanding of how fenretinide kills malignant cells is critical since the drug has promise as both a chemotherapeutic agent and a chemoprevention agent [ 1 , 2 , 43 ] . fenretinide is well tolerated by patients and has shown promise for treating children with neuroblastoma [ 4345 ] . the effectiveness of fenretinide in killing all derived cells while sparing nonmalignant lymphoid cells suggests that it may be an effective agent in the therapy of all , especially as it is so well tolerated in children [ 5 , 9 , 10 , 18 , 45 ] . furthermore , the ability of fenretinide to overcome abt-737 resistance in all cell lines suggests that fenretinide may be useful in combination with bh3 mimetic drugs like abt-737 . a role for pkc in fenretinide - induced apoptosis in the leukemia cell lines seemed logical as many of the reported effects of the drug would be expected to impact pkc mediated signaling ( e.g. , stimulation of ros production , stimulation of sphingolipid production , activation of jnk ) [ 6 , 810 ] . in the leukemia cells treated with fenretinide , there was a positive correlation between drug - induced apoptosis , drug - induced ros production , and drug - induced pkc cleavage ( figures 1 , 2 , 6 , and 7 ) . fenretinide was shown to promote nuclear translocation of pkc ( figure 8) which is an important event in apoptotic signaling involving the kinase [ 3336 ] . the antioxidant vitamin c protected all the leukemia cells from fenretinide - induced cell death ( figure 3 ) and prevented cleavage of the kinase ( figure 10 ) . these findings would suggest that ros might promote pkc cleavage to initiate proapoptotic signaling . while this notion might prove true , a recent study has suggested that pkc regulates ros production as primary fibroblasts from pkc knockout mice or mice expressing a dominant negative mutant of pkc exhibit increased production of ros in response to uv irradiation . consistent with such a notion , the suppression of pkc by shrna in ccrf - cem cells promoted basal ros production ( figure 15 ) and sensitized the cells to fenretinide - induced apoptosis ( figure 14 ) . the findings that inhibition of ros blocks pkc cleavage ( thus suppressing activation of prostress signaling mediated by the kinase ) while loss of pkc enhances ros production suggest that pkc may regulate ros and in turn , ros may regulate the kinase . such a mechanism is not too different from the relationship between pkc and caspase 3 ; the protease cleaves and activates pkc but the kinase in turn activates caspase 3 [ 3336 ] . the production of sphingolipids has been shown to be an important event in fenretinide - induced apoptosis [ 1 , 3 , 9 , 10 ] . pkc on the one hand has been shown to be activated by the sphingolipid ceramide , and on the other hand , promotes ceramide production [ 47 , 48 ] . in response to etoposide , pkc is translocated to the mitochondria and promotes ceramide production in prostate cancer cells . interestingly , whereas bryostatin-1 protects reh cells from etoposide , the pkc agonist promotes cell death in response to fenretinide ( figure 11(a ) ) . while it was shown that activation of pkc appears to be critical in protection of the reh cells from etoposide , bryostatin-1 suppressed pkc expression ( figure 12 ) , and potentiated fenretinide - induced cell death in the reh cells ( figure 11(a ) ) . perhaps the differences may be due to different effects of ceramide and dihydroceramide on pkc mediated signaling pathways and for that matter pathways regulated by other pkc isoforms . however , a role for dihydroceramide as a regulator of cell death is only just emerging , so much work is needed to establish how dihydroceramide might promote cell death . it will be helpful to understand how pkc may regulate and be regulated by dihydroceramide . pkc has been implicated as a positive regulator of jnk , particularly during stress signaling leading to apoptosis . activation of jnk in ccrf - cem cells leading to mcl-1 degradation observed by kang and colleagues could be the result of pkc activation of jnk . however , the loss of mcl-1 observed in rs4;11 cells in the present study occurs in the apparent absence of jnk activation ( figure 6 ) . the correlation between fenretinide - induced pkc cleavage and loss of mcl-1 in the drug sensitive rs4;11 and ccrf - cem cells ( figures 6 and 7 ) is consistent with reports demonstrating that pkc cleavage product phosphorylates mcl-1 resulting in its proteasomal degradation . however , fenretinide - induced degradation of mcl-1 is not required for drug - induced apoptosis since reh cells actually express increasingly higher amounts of the antiapoptotic protein with higher concentrations of fenretinide ( figure 6 ) but high concentrations of the drug are still toxic to the cells ( figure 1 ) . fenretinide ( 10 m ) does promote mcl-1 gene expression after 6 hours in reh cells ( figure 9 ) , so the increased expression of mcl-1 protein may reflect an increase in gene expression . the cleaved pkc produced in the reh cells may not be sufficient to promote degradation of mcl-1 . these findings would suggest that pkc may not be as important in fenretinide - induced apoptosis in the reh cells . still , the finding that bryostatin-1 potently suppresses pkc expression and potentiates fenretinide - induced cell death suggests that pkc would suggest otherwise . identification of pkc targets other than mcl-1 will provide a better understanding of how the kinase regulates fenretinide - induced apoptosis . one possible mechanism may involve regulation of ros production . while we may not fully understand how pkc regulates fenretinide - mediated apoptosis in lymphoid leukemia cells , it is clear that the kinase may be a useful target for optimizing antileukemia strategies that make use of fenretinide .

the synthetic vitamin a analog fenretinide is a promising chemotherapeutic agent . in the current paper , the role of pkc was examined in fenretinide - induced apoptosis in lymphoid leukemia cells . levels of proapoptotic cleaved pkc positively correlated with drug sensitivity . fenretinide promoted reactive oxygen species ( ros ) generation . the antioxidant vitamin c prevented fenretinide - induced pkc cleavage and protected cells from fenretinide . suppression of pkc expression by shrna sensitized cells to fenretinide - induced apoptosis possibly by a mechanism involving ros production . a previous study demonstrated that fenretinide promotes degradation of antiapoptotic mcl-1 in all cells via jnk . now we have found that fenretinide - induced mcl-1 degradation may involve pkc as cleavage of the kinase correlated with loss of mcl-1 even in cells when jnk was not activated . these results suggest that pkc may play a complex role in fenretinide - induced apoptosis and may be targeted in antileukemia strategies that utilize fenretinide .

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

fenretinide ( n-4-(hydroxyphenyl)-retinamide ; 4-hpr ) is a synthetic analog of vitamin a that has shown promise as both a chemotherapeutic and chemopreventive agent in solid tumors and hematologic malignancies [ 16 ] . while fenretinide binding to retinoic acid receptors ( rars ) can promote apoptosis in some cell types , the agent can induce death in a rar - independent manner . rar - independent mechanisms of cell death likely involve the production of reactive oxygen species ( ros ) and the generation of sphingolipid second messenger molecules [ 711 ] . recent studies have suggested that fenretinide may be an effective agent in the treatment of acute lymphoblastic leukemia ( all ) since the drug effectively kills all cell lines but not nonmalignant lymphoid cell types . a recent study from the reynolds group has demonstrated that fenretinide can synergize with abt-737 to effectively kill all cells . mcl-1 has been found to promote resistance to abt-737-induced apoptosis and suppression of mcl-1 promotes sensitivity to the drug [ 1921 ] . fenretinide was found to promote mcl-1 degradation by a jnk - mediated mechanism and thus has promise in overcoming mcl-1 mediated chemoresistance in all and other leukemias . pkc promotes chemoresistance in all and acute myeloid leukemia ( aml ) cell lines [ 27 , 28 ] and may be a negative prognostic factor in aml [ 29 , 30 ] . the role of pkc in leukemia is more complicated . considering that fenretinide promotes mcl-1 degradation in all cell lines , the possibility arises that the fenretinide - induced apoptosis may involve pkc . in the present study , we examined pkc expression and cleavage and mcl-1 expression in response to fenretinide in three all cell lines ( reh , ccrf - cem , and molt4 ) and in a mixed lineage leukemia ( mll ) cell line ( rs4;11 ) . fenretinide promoted cleavage of pkc and suppression of mcl-1 in cell lines sensitive to the drug ( rs4;11 and ccrf - cem ) but not in cell lines that were more resistant ( reh and molt4 ) . the antioxidant vitamin c protected all cell lines from the drug and suppressed pkc cleavage suggesting that the mechanism by which fenretinide promotes death involves ros and pkc . suppression of pkc in ccrf - cem cells by shrna sensitized the cells to fenretinide suggesting that loss of the kinase may also be important in the drug 's mode of action . basal ros production was increased in cells with reduced pkc suggesting that the increase in sensitivity to the drug may be due to enhanced ros generation . the findings in this study suggest that pkc plays an important role in fenretinide - induced apoptosis in lymphoid leukemia cell lines . sensitivity to fenretinide was investigated in the t - all derived cell lines ccrf - cem and molt4 cells , in the pre - b all derived cell line reh , and in the mll derived cell line rs4;11 . at 1 m fenretinide , > 30% of ccrf - cem and rs4;11 cells were positive for annexin v staining while higher doses of the drug induce apoptosis in the majority of cells from both cell lines ( i.e. as shown in figure 1 , reh cells displayed the greatest resistance to fenretinide - induced apoptosis . these results suggest that there is a positive correlation between ros generation and sensitivity to fenretinide . if ros generation is critical to fenretinide - induced apoptosis , it would be expected that an antioxidant such as vitamin c ( l - abscorbic acid ) would protect cells from the cytotoxic effects of the drug . as shown in figure 3 , vitamin c protected all four cell lines from fenretinide - induced apoptosis . this result strongly suggests that ros production is a key element in the mechanism by which fenretinide kills leukemia cells . however , a recent study has suggested that caspase activity is not required for fenretinide - induced cell death in lymphoid leukemia cell lines . reh cells and molt4 cells , which are relatively more resistant to fenretinide - induced cell death compared to rs4;11 and ccrf - cem cells , displayed less parp cleavage in response to fenretinide . as shown in figure 4 , fenretinide activated caspase 8 in the rs4;11 and ccrf - cem cell lines ( as observed by cleavage of the caspase ) but not in molt4 cells . a dose of 10 m fenretinide is required to observe some caspase 8 cleavage in reh cells while 5 m drug results in near complete cleavage of the caspase in both rs4;11 and ccrf - cem cells ( figure 4 ) . however , fenretinide - induced apoptosis in rs4;11 or ccrf - cem cells does not require either caspase 8 or caspase 9 . , the protection accorded was only partial indicating that caspase 8 may be necessary but not sufficient for fenretinide - induced apoptosis in these cell lines . these results are similar to findings reported recently from the reynolds group that demonstrated that fenretinide - induced apoptosis in the t - all - derived cell line cog - ll-317 involves a caspase independent mechanism . , jnk ) [ 6 , 8 , 1214 ] ; however , a role for pkc in fenretinide - induced apoptosis has yet to be established . a role for pkc in fenretinide - induced apoptosis in head and neck squamous carcinoma cells has been suggested but an analysis of which pkc isoforms might participate was not performed . while the significance of pkc cleavage is not clear , cleavage of pkc has been implicated as a positive regulator of apoptosis [ 3336 ] . ccrf - cem and molt4 cells were similarly treated with fenretinide and expression of pkc , pkc , and pkc was examined ( figure 7 ) . as was the case for rs4;11 cells , fenretinide suppressed pkc expression and promoted cleavage of pkc in ccrf - cem cells . considering that cleaved pkc has been implicated as a positive regulator of apoptosis , it is not surprising to find that the leukemia cells that are more sensitive to fenretinide ( i.e. nuclear translocation of pkc has been shown to be an important event in apoptotic stress signaling pathways involving the kinase [ 3336 ] . to determine if fenretinide promoted nuclear translocation of pkc , subcellular localization of the kinase was examined by immunofluorescence microscopy in vehicle ( 0.1% dmso ) treated ccrf - cem cells and cells treated with 1 m fenretinide for 24 hours . as shown in figure 8 , pkc is mainly found in the cytoplasm and in association with centrosomes in cells treated with vehicle . a recent study from the reynolds group suggests that fenretinide suppresses expression of the antiapoptotic bcl2 family member mcl-1 in all cells . cleaved pkc has been suggested to regulate the degradation of mcl-1 during apoptosis in u.v . it is not surprising that fenretinide sensitive cell lines rs4;11 and ccrf - cem display reduced expression of mcl-1 with concomitant cleavage of pkc when treated with higher doses of the drug ( figures 6 and 7 ) . in the reynolds group study , jnk was implicated as regulating mcl-1 degradation by observations made using ccrf - cem cells . as shown in figure 6 , rs4;11 cells exhibit suppression of mcl-1 protein expression in response to fenretinide but do not display significant fenretinide - induced activation of jnk ( as indicated by phosphorylation of the kinase ) . to examine if suppression of mcl-1 expression by fenretinide might be mediated by a transcriptional mechanism , gene expression of mcl-1 as well as fellow antiapoptotic family member bcl2 was assessed in cells treated with the drug . thus loss of transcript can not account for the suppression of mcl-1 protein by 10 m fenretinide in these cells that was demonstrated in figure 6 . actually relative to b2 m transcription , there is a > 2-fold increase in mcl-1 transcript in the fenretinide treated ccrf - cem cells when compared to control . also , it is possible that fenretinide regulates mcl-1 gene expression by other means such as a mechanism involving a micro - rna . still , the data presented here suggest that downregulation of mcl-1 in the fenretinide sensitive cell lines ( i.e. fenretinide induced loss of pkc in these cells ( figures 6 and 7 ) . while this potential mechanism needs to be investigated further , the ability of fenretinide to suppress bcl2 expression would have positive benefits in antileukemia strategies that would utilize fenretinide . a plausible mechanism how fenretinide promotes pkc cleavage might involve ros activation of caspase 3 since the protease mediates cleavage of this pkc isoform [ 3336 ] . nuclear translocation of pkc precedes caspase 3 cleavage and indeed fenretinide promotes pkc nuclear translocation ( figure 8) . vitamin c was shown to effectively protect the leukemia cells from fenretinide - induced apoptosis likely via a mechanism that suppresses ros ( figure 3 ) . if pkc participates in the death process , it would be predicted that vitamin c would prevent fenretinide - induced cleavage of pkc . consistent with the protective effects of vitamin c against drug - induced apoptosis , vitamin c blocked fenretinide - induced activation of caspase 3 ( i.e. as shown in figure 10 , the antioxidant was effective at preventing pkc cleavage in cells treated with fenretinide . this result supports a mechanism where fenretinide promotes ros formation to activate caspase 3 resulting in pkc cleavage . it has been previously demonstrated that the pkc agonist bryostatin-1 protects reh cells from apoptosis induced by a number of clinically relevant chemotherapeutic drugs including etoposide , ara c , and adriamycin . the mechanism how bryostatin-1 protects reh cells from chemotherapeutic drugs involves , at least in part , the activation of pkc resulting in the phosphorylation of bcl2 . the increase in fenretinide - induced cell death in the reh cells due to bryostatin-1 was statistically significant ( p = .0006 ) . thus while bryoststain-1 protects reh cells from chemotherapeutic drugs , the pkc agonist promotes cell death in response to fenretinide . bryoststin-1 actually protected rs4;11 cells from fenretinide - induced apoptosis . differential effects of bryostatin-1 on pkc in reh cells and rs4;11 cells may provide a possible explanation for the difference in effect of bryostatin-1 on fenretinide - induced apoptosis in the two cell lines . consistent with the protective effects of bryostatin-1 against fenretinide - induced apoptosis in the rs4;11 cells , the pkc agonist blocked fenretinide - induced cleavage of both pkc and pkc ( figure 12 ) . bryostatin-1 alone had no effect on pkc in reh cells though it did prevent fenretinide - induced cleavage of pkc ( figure 12 ) . since bryostatin-1 augments fenretinide - induced cell death but blocks cleavage of pkc in reh cells , it is likely that cleaved pkc is not critical in the death process . this result suggests that loss of pkc sensitizes reh cells to fenretinide - induced cell death . to determine if cleaved pkc was required for fenretinide - induced apoptosis , expression of the kinase was suppressed in ccrf - cem cells by shrna . the increase in fenretinide - induced death in the ccrf - cem cells expressing pkc shrna compared to control cells was significant at drug concentrations of 1 m and 5 m ( i.e. the increased sensitivity to fenretinide in the cells with pkc shrna is reminiscent of observations with reh cells treated with a combination of bryostatin-1 and fenretinide . bryostatin-1 in combination with fenretinide resulted in complete loss of pkc in reh cells ( figure 12 ) and the pkc agonist promoted sensitivity to fenretinide ( figure 11(a ) ) . these results suggest that the role for pkc in fenretinide - induced cell death in the leukemia cells is complex . the findings of this study suggest that pkc may play an important role in fenretinide - induced apoptosis in lymphoid leukemia cells . previous studies have established that the synthetic vitamin a analog acts via diverse mechanisms but a role for any particular pkc isoform has remained elusive [ 16 ] . the effectiveness of fenretinide in killing all derived cells while sparing nonmalignant lymphoid cells suggests that it may be an effective agent in the therapy of all , especially as it is so well tolerated in children [ 5 , 9 , 10 , 18 , 45 ] . furthermore , the ability of fenretinide to overcome abt-737 resistance in all cell lines suggests that fenretinide may be useful in combination with bh3 mimetic drugs like abt-737 . a role for pkc in fenretinide - induced apoptosis in the leukemia cell lines seemed logical as many of the reported effects of the drug would be expected to impact pkc mediated signaling ( e.g. in the leukemia cells treated with fenretinide , there was a positive correlation between drug - induced apoptosis , drug - induced ros production , and drug - induced pkc cleavage ( figures 1 , 2 , 6 , and 7 ) . fenretinide was shown to promote nuclear translocation of pkc ( figure 8) which is an important event in apoptotic signaling involving the kinase [ 3336 ] . the antioxidant vitamin c protected all the leukemia cells from fenretinide - induced cell death ( figure 3 ) and prevented cleavage of the kinase ( figure 10 ) . while this notion might prove true , a recent study has suggested that pkc regulates ros production as primary fibroblasts from pkc knockout mice or mice expressing a dominant negative mutant of pkc exhibit increased production of ros in response to uv irradiation . consistent with such a notion , the suppression of pkc by shrna in ccrf - cem cells promoted basal ros production ( figure 15 ) and sensitized the cells to fenretinide - induced apoptosis ( figure 14 ) . the findings that inhibition of ros blocks pkc cleavage ( thus suppressing activation of prostress signaling mediated by the kinase ) while loss of pkc enhances ros production suggest that pkc may regulate ros and in turn , ros may regulate the kinase . such a mechanism is not too different from the relationship between pkc and caspase 3 ; the protease cleaves and activates pkc but the kinase in turn activates caspase 3 [ 3336 ] . the production of sphingolipids has been shown to be an important event in fenretinide - induced apoptosis [ 1 , 3 , 9 , 10 ] . interestingly , whereas bryostatin-1 protects reh cells from etoposide , the pkc agonist promotes cell death in response to fenretinide ( figure 11(a ) ) . while it was shown that activation of pkc appears to be critical in protection of the reh cells from etoposide , bryostatin-1 suppressed pkc expression ( figure 12 ) , and potentiated fenretinide - induced cell death in the reh cells ( figure 11(a ) ) . however , the loss of mcl-1 observed in rs4;11 cells in the present study occurs in the apparent absence of jnk activation ( figure 6 ) . the correlation between fenretinide - induced pkc cleavage and loss of mcl-1 in the drug sensitive rs4;11 and ccrf - cem cells ( figures 6 and 7 ) is consistent with reports demonstrating that pkc cleavage product phosphorylates mcl-1 resulting in its proteasomal degradation . however , fenretinide - induced degradation of mcl-1 is not required for drug - induced apoptosis since reh cells actually express increasingly higher amounts of the antiapoptotic protein with higher concentrations of fenretinide ( figure 6 ) but high concentrations of the drug are still toxic to the cells ( figure 1 ) . the cleaved pkc produced in the reh cells may not be sufficient to promote degradation of mcl-1 . these findings would suggest that pkc may not be as important in fenretinide - induced apoptosis in the reh cells . still , the finding that bryostatin-1 potently suppresses pkc expression and potentiates fenretinide - induced cell death suggests that pkc would suggest otherwise . identification of pkc targets other than mcl-1 will provide a better understanding of how the kinase regulates fenretinide - induced apoptosis . one possible mechanism may involve regulation of ros production . while we may not fully understand how pkc regulates fenretinide - mediated apoptosis in lymphoid leukemia cells , it is clear that the kinase may be a useful target for optimizing antileukemia strategies that make use of fenretinide .

the function of the metalloenzyme sod1 is to convert superoxide , a toxic by - product of mitochondrial oxidative phosphorylation , to water or hydrogen peroxide . however , alteration in wild type sod1 expression or mutations in the gene have been held responsible for the activation of catabolic pathways associated with degenerative diseases , including amyotrophic lateral sclerosis ( als ) . als is a disorder involving the degeneration of motor neurons , muscle atrophy , and paralysis . in few familiar forms of als , mutations in sod1 gene initially it has been suggested that mutation in sod1 gene led to a decrease in the protein enzymatic activity ( loss of function hypothesis ) . however , subsequent studies have clarified that mutant sod1 possesses a neurotoxic property ( gain of function hypothesis ) responsible for the pathogenic mechanism of the disease . indeed , the finding that overexpression of mutant sod1 in transgenic mice recapitulates several clinical features of als disease even in the presence of endogenous mouse sod1 has led to the conclusion that the disease results from a toxic gain of function . mutations in sod1 that impair its functions may lead to increased oxidative damage , promoting the activation of apoptotic pathways . the effects of the reaction oxidative species ( ros ) are dose - dependent , and low ros concentration is necessary to guarantee cellular homeostasis while high ros dose exerts toxic effects on the cells and may contribute to cellular dysfunction . indeed , oxidative stress is a hallmark of aging and several chronic diseases such as alzheimer 's disease , duchenne dystrophy , and als . how such an oxidative insult plays a direct role in the disease - related decrease of muscle performance and mass remains largely unknown . in addition , the discrepancy among different studies has further complicated the achievement of a conclusive link between altered balance of ros generation and altered homeostasis - associated diseases . in a previous work we demonstrated that muscle specific expression of the mutant isoform of sod1 gene ( sod1 ) induces muscle atrophy associated with a significant reduction in muscle strength and alterations in the contractile apparatus . we provided evidences that muscle - restricted expression of sod1 gene is sufficient to increase oxidative stress and to induce a reduction in protein synthesis and the activation of proteolytic pathway . it has been demonstrated that lactate - induced oxidative stress delays c2c12 differentiation while treatment of the same cell line with resveratrol , that confers resistance against oxidative stress , promotes myogenesis and hypertrophy . interestingly , high glucose - induced oxidative stress has been correlated with lipid deposition in muscle derived stem cells leading to their adipogenic differentiation . in this study , we address the role of the toxic effect of mutant sod1 gene ( sod1 ) on in vitro myogenic program and we demonstrate that sod1 expression prevents myoblasts differentiation and retains c2c12 cells in an undifferentiated state that show features common to fibro / adipogenic cells . c2c12 cells were stably transfected with ppuro and pmexmlc / sod1 plasmids ( ratio 1 : 10 ) by using superfect transfection reagent ( qiagen ) according to the manufacturer 's instructions , as control c2c12 cells were also transfected with ppuro and pmex empty vector . after 1 day from transfection the medium was replaced with fresh medium containing puromycin 3 g / ml ( sigma aldrich ) . after 48 hours the cells were split 1 : 10 into the selective medium . c2c12 and c2c12 mlc / sod1 cells were maintained in dulbecco 's modified eagle 's medium ( dmem ) with 4.5 g / l glucose , l - glutamine ( sigma ) , supplemented with 15% fetal bovine serum ( gibco ) , 100 u / ml penicillin ( sigma aldrich ) , 100 g / ml streptomycin ( sigma aldrich ) , and 2,5 g / ml of puromycin ( sigma aldrich ) for c2c12 mlc / sod1 cells . to induce differentiation , cells were shifted to differentiation medium ( dm ) , dmem with 2% horse serum ( gibco ) . cells were harvested at d0 , d2 ( 48 h after shift in dm ) , and d5 ( 120 h after shift in dm ) . c2c12 and c2c12 mlc / sod1 cells grown in 60 mm culture dishes were washed twice with cold phosphate - buffered saline , pelleted , resuspended in 100 ul of modified lysis buffer ( tris - hcl , ph 7.5/20 mm , edta/2 mm , egta/2 mm , sucrose/250 mm , dtt/5 mm , triton - x/0.1% , pmsf/1 mm , naf/10 mm , sov4/0.2 mm , and cocktail protease inhibitors/1x ( sigma aldrich ) ) , and processed for western blot analysis . filters were blotted with antibodies against hsod1 ( santa cruz ) , gp91phox ( bd ) anti - perilipin 2 ( lifespan biosciences ) , phospho - p42/p44 mapk ( millipore ) , p42/p44 mapk ( cell signaling ) phospho - akt ( thr 308 ) ( sigma aldrich ) , akt ( cell signaling ) , phospho - p70 ( thr389 ) ( cell signaling ) , p70 ( cell signaling ) , and hdac4 ( cell signaling ) . signals were acquired by chemidoc - it imaging system ( uvp , llc ) and the analysis was performed using visionworks ls image acquisition analysis software . total rna extraction was performed using trizol reagent method ( sigma aldrich ) as described by the manufacturers . microrna ( mirna ) was reverse - transcribed using the taqman microrna reverse transcription kit ( life technologies ) , and mrna was reverse - transcribed using quantitect reverse transcription kit ( qiagen ) . quantitative pcr was performed on an abi prism 7500 sds ( life technologies ) , using premade 6-carboxyfluorescein- ( fam- ) labeled taqman assays for beta actin , pax7 , pax3 myod , myogenin , mrf4 , and smarcd3 ( life technologies ) . fam - labeled taqman microrna assays for mir1 , mir133a , mir206 , and u6 snrna ( applied biosystems , usa ) were performed as described . quantitative rt - pcr sample values were normalized to the expression of beta - actin or u6 snrna for mrna and microrna , respectively . the relative level for each gene and mirna was calculated using the 2-ddct method and reported as mean fold change in gene expression . cells were fixed in 4% paraformaldehyde for 1 h. after being washed with ddh2o , cells were treated with 100% propylene glycol for 5 min and stained with a filtered oil red o solution ( 0.5% oil red o in propylene glycol ) for 8 min at 60c . the cells were treated with 85% propylene glycol solution for 5 min , washed twice with ddh2o , and mounted with glycerol . samples were visualized using an inverted microscope ( axioskop 2 plus ; carl zeiss microimaging inc . ) . c2c12 and c2c12 mlc / sod1 cells were exposed to 100 nm ( sigma aldrich ) trichostatin a ( tsa ) in gm ( growth medium ) for 24 h. tsa was removed and the cells were analyzed for myosin expression ( at day 5 in dm ) or by cytofluorimetric profile ( at day 2 in dm ) . cells were fixed in 4% paraformaldehyde and incubated overnight at 4c with primary antibody against mhc ( mf-20 hybridoma bank ) ; nuclei were visualized using hoechst staining . samples were viewed under an inverted microscope ( axioskop 2 plus ; carl zeiss microimaging inc . ) . to quantify the differentiation and fusion of control and tsa treated we calculated the differentiation index as the percentage of mhc - positive cells above total nuclei and the fusion index as the average number of nuclei in mhc - positive cells with at least three nuclei above total number of nuclei , respectively . c2c12 mlc / sod1 were detached from culture with 0.25% trypsin , 2 mm edta ( sigma aldrich ) at indicated times . the isolated cells were then filtered through a 40 m cell strainer ( falcon ) and incubated with the following antibodies ( 10 ng / ml ) : cd31-pecy7 , cd45-efluor 450 ( ebioscience ) , sca-1-fitc ( macs ) , and 7 integrin - pe ( r&d systems inc . ) . a subsequent incubation with 7-aminoactinomycin d ( 1/1000 ; sigma ) , data were collected from 10,000 cells and analyzed on cyan adp ( dako ) flow cytometer using the summit 4.3 software ( dako ) . groups were compared using nonparametric tests ( mann whitney rank sum test ) and student 's t - test . a p value of < 0.05 was considered statistically significant . to investigate the role of mutant sod1 gene in myoblast differentiation we stably transfected the c2c12 cells line with the mlc / sod1 expression cassette ( figure 1(a ) ) that allows the expression of the mutated isoform of sod1 gene under the control of the myosin light chain promoter . as expected , sod1 transgene expression was accumulated in c2c12 mlc / sod1 cells induced to differentiate ( figure 1(b ) ) . the postmitotic expression of sod1 transgene induced the accumulation of gp91 protein , a marker of mitochondria oxidative damage ( figure 1(c ) ) that might mediate the toxic properties of mutant sod1 on muscle differentiation and homeostasis . to explore if sod1 transgene expression directly interferes with muscle differentiation , we stimulated differentiation by shifting the cells from growth to the differentiation medium ( dm ) ( figure 2(a ) ) . morphological and immunofluorescence analysis revealed a dramatic inhibition of muscle differentiation in c2c12 mlc / sod1 cells , with significant reduction in the number and size of the myosin positive cells , compared with control c2c12 myotubes ( figures 2(a)-2(b ) ) . morphometric analysis revealed a complete inhibition of fusion index , quantified as the percentage of hoechst - stained nuclei located within multinucleated cells , positive to sarcomeric myosin ( figure 2(b ) ) . the altered differentiated muscle phenotype was also confirmed by western blot analysis , revealing a drastic downmodulation of the sarcomeric myosin heavy chain expression , a specific marker of myogenic differentiation ( figure 2(c ) ) . of note , c2c12 cells stably transfected with wild type sod1 cassette ( mlc / sod1 ) did not show any morphological differences compared to control c2c12 cell lines ( data not shown ) . a key myogenic factor that triggers myoblast differentiation is myod [ 12 , 13 ] , which resulted in significant downregulation throughout the time course of differentiation in c2c12 mlc / sod1 cells ( figure 3(a ) ) . myogenin is the myogenic factor that functions downstream of myod and plays a critical role in triggering terminal differentiation process of myoblasts [ 12 , 14 ] . myogenin expression resulted in significant downregulation in c2c12 mlc / sod1 cells during differentiation ( figure 3(b ) ) . the final stage of skeletal muscle differentiation and maturation program is also dependent on the concerted action of another myogenic factors , namely , mrf4 , which promotes the activation of myosin heavy chain expression . real time pcr analysis revealed a significant reduction of mrf4 transcripts in c2c12 mlc / sod1 cells during differentiation , compared to control c2c12 myotubes ( figure 3(c ) ) . recent works have shown that among genes which are important for proper muscle differentiation and function , micrornas ( mirnas ) play a crucial role [ 1517 ] . among them mir133 , mir206 , and mir1 are abundantly expressed in muscle tissue and specifically induced during myogenesis and c2c12 differentiation . it has been reported that mir1 and mir133 are involved in a complex molecular mechanism by which mir1 induces the downmodulation of an inhibitor of muscle differentiation , namely , histone deacetylase ( hdac ) 4 and mir133 , which is clustered on the same chromosomal loci of mir1 and enhances myoblast proliferation inhibiting the serum response factor ( srf ) . in addition , mir206 facilitates satellite cell differentiation [ 20 , 21 ] by restricting its proliferative potential through the repression of pax-7 expression . these findings implicate these myomirnas in a complex regulatory loop to control cell proliferation , commitment , and differentiation . real time pcr analysis revealed a significant downmodulation of mir133 , mir206 , and mir1 during the differentiation time course of c2c12 mlc / sod1 cells compared to control c2c12 cells ( figures 3(d ) , 3(e ) , and 3(f ) ) . the activation of a specific developmental program requires the integration of multiple extrinsic signals from the cell membrane that culminate in changes of nuclear gene expression patterns . among the known signal transduction intermediates in muscle cells , the serine / threonine kinase akt and the mitogen - activated protein kinases ( mapk ) have been shown to modulate myogenic differentiation . in a previous study , we have demonstrated that muscle specific expression of sod1 gene in transgenic animals promotes a reduction of the phosphatidylinositol 3-kinase ( pi3k)/akt pathway and leads to muscle atrophy . in this study , we explored whether the inhibitory effects of mutant sod1 gene on myogenic program perturb the relevant signaling pathways of the myogenic program . figure 4 shows that the absolute ratio of pakt / akt was negatively regulated ( figure 4(a ) ) and pp70/p70 , the downstream effector of akt , was significantly reduced in c2c12 mlc / sod1 cultures compared with control c2c12 cells ( figure 4(b ) ) . in addition we observed a significant downmodulation of the phosphorylated active form of a factor associated with mapk differentiation cascade , namely , erk1/2 ( figure 4(c ) ) . overall these results demonstrate that the postmitotic expression of sod1 mutant gene prevents c2c12 differentiation , affecting the activation of the muscle regulatory factors , muscle mirna , and the signal transduction cascades responsible for myogenic differentiation , and might impinge the maintenance of the muscle phenotype . to validate this hypothesis , we analyzed the expression of pax-7 , which is a key factor that triggers the specification of uncommitted skeletal muscle progenitors to myogenic cells . of note , we observed a complete inhibition of pax-7 expression in c2c12 mlc / sod1 cells compared to c2c12 myoblasts , suggesting that sod1 expression not only inhibits muscle differentiation but also confers to transfected cells an immature state ( figure 5(a ) ) . recently , it has been demonstrated that satellite cells in adult muscle are bipotential stem cells that can give rise to brown adipogenic as well as myogenic progenitors . the lineage switch between myogenic and brown adipogenic commitment is controlled by the muscle specific mir133a , which is highly expressed in satellite cells and can repress the expression of adipogenic markers to enforce myogenic commitment in satellite cells . further , recent evidences revealed that pax-3 transcription factor , whose ectopic expression in c2c12 myoblasts efficiently inhibits myogenic specification , plays a pivotal role during differentiation into adipocytes cells of human - induced pluripotent stem cells . in addition , the exclusive expression of pax-3 or myod gene in stem cells allows a clear and distinct choice between myogenic and fibro / adipogenic potential cell lineage . based on these evidences , we performed real time pcr and histochemical analysis in both c2c12 and c2c12 mlc / sod1 cells to evaluate the potential adipogenic features in c2c12 mlc / sod1 transfected cell lines . as shown in figure 5(b ) the levels of pax-3 transcript were significantly upregulated in c2c12 mlc / sod1 during all stages in culture , compared to c2c12 . moreover oil red o staining revealed the accumulation of intracellular lipid droplets in c2c12 mlc / sod1 cells ( figure 5(c ) ) . these data were corroborated by western blot analysis for perilipin 2 ( plin2 ) , a marker of fatty acids uptake and storage . western blot analysis revealed that plin2 protein was accumulated in c2c12 mlc / sod1 during the differentiation process compared to control cell line ( figure 5(d ) ) . all these results together with the data of mir133a and mrfs downmodulation in c2c12 mlc / sod1 ( figure 3 ) suggest that muscle specific expression of sod1 mutant gene inhibits myoblasts differentiation and promotes adipogenic features in c2c12 cells through a mir133a and pax-3 dependent mechanism . fibro - adipogenic progenitors ( faps ) are multipotent mesenchymal cells residing in skeletal muscle interstitium [ 3032 ] . these cells are negative for cd31 , cd45 , and 7 integrin surface antigens and are characterized by the expression of the stem cell antigen 1 ( sca1 ) . faps convert environmental cues into signals that modulate muscle regeneration or turn themselves into fibro - adipocytes , inducing fat deposition and fibrosis under pathologic conditions , such as dystrophic muscles . to verify whether postmitotic expression of sod1 mutant gene promotes a faps phenotype in c2c12 cells , we performed facs analysis ( figure 6(a ) ) for sca1 , cd31 , cd45 , and 7 integrin . c2c12 and c2c12 mlc / sod1 cells were negative for cd31 and cd45 antigens ( data not shown ) . c2c12 were mainly 7 integrin and a low percentage of them was sca1 and 7 integrin ( figures 6(a)6(c ) ) . in contrast , c2c12 mlc / sod1 were mainly sca1 and 7 integrin , with a significant reduction in the number of 7 integrin cells ( figures 6(a)6(c ) ) . these results clearly evidenced the presence of fibro / adipogenic features in c2c12 mlc / sod1 . the molecular mechanism that , in concert with environmental cues , controls the identity and activity of fap cells involves a hdac - regulated network ; this network consists of muscle specific mirnas that target two alternative variants of the swi / snf chromatin remodeling complex , baf60a and baf60b , and favor the formation of a baf60c - based swi / snf complex able to confer on myod the ability to activate the myogenic program . we analyzed the protein and transcript levels , respectively , of hdac4 and baf60c in both c2c12 and c2c12 mlc / sod1 cells . we observed that hdac4 was significantly upregulated in cultures of c2c12 mlc / sod1 cells compared to control differentiated ( day 5 in dm ) c2c12 cells ( figure 7(a ) ) . in contrast , baf60c was significantly downregulated in c2c12 mlc / sod1 cells ( figure 7(b ) ) . to better correlate the induction of faps phenotype and hdac activity with sod1 expression and toxic properties , we treated c2c12 mlc / sod1 cells with the hdac class ii inhibitor trichostatin ( tsa ) and analyzed the signature profile of both faps and myogenic cells . cytofluorimetric profile revealed that tsa treatment induced a significant downmodulation of sca1 and 7 integrin cells ( figure 7(c ) ) and an increased percentage of double positive sca1 and 7 integrin cells ( figure 7(d ) ) . of note we did not observe any significant difference in the number of 7 integrin sca1 cells after treatment ( data not shown ) suggesting that tsa treatment is able to counteract the establishment of a fap phenotype but it only partially rescued the muscle phenotype . to support this evidence , we induced muscle differentiation in c2c12 mlc / sod1 cells treated with tsa and we revealed that inhibition of hdac activity partially rescued myogenic differentiation , promoting myoblast fusion and differentiation ( figures 7(e)-7(f ) ) . overall these data suggest that the postmitotic expression sod1 mutant gene promotes a faps phenotype in c2c12 cells , by upregulating hdac4 protein and preventing the baf60c - swi / snf complex myogenic commitment . in this work we defined the specific toxic effects of postmitotic expression of mutant sod1 gene on the myogenic program , demonstrating that mutant sod1 alters the identity of muscle cells , affects cell homeostasis , and inhibits muscle differentiation . the physiological activity of the metalloenzyme sod1 is to detoxify the cells from the accumulation of free radicals , converting superoxide , a toxic by - product of mitochondrial oxidative phosphorylation , to water or hydrogen peroxide . in contrast , the mutant sod1 possesses a toxic property that is responsible for the pathogenic mechanism of als , a neurodegenerative disease associated with the degeneration of motor neurons , muscle atrophy , and paralysis . different studies also support the evidence that skeletal muscle is a primary target of mutant sod1 toxicity in mice [ 5 , 35 ] , indicating that dysfunctions of affected muscle cells are not only a marginal consequence of denervation associated with motor neurons loss , but a direct consequence of cell muscle toxicity of mutant sod1 . the understanding of the mechanisms involved in mutant sod1 toxicity in muscle may facilitate the design of treatments directed toward this specific tissue to treat als or at least to delay disease progression . the aim of our work was to define the responses of myogenic cells to the toxic effects of sod1 and the signaling pathways that mediated the toxic properties of mutant sod1 product . to this purpose , we generated a stable transfected c2c12 cell line , overexpressing the mutated isoform of the sod1 gene under the control of the mlc promoter [ 11 , 37 ] . postmitotic expression of sod1 gene induced an excess of oxidative stress , as evidenced by the increased expression of gp91 protein , and impaired muscle differentiation and fusion of c2c12 cells , inducing a significant downregulation in the expression of molecular markers of myogenic differentiation , like myod , myogenin , mrf4 , and sarcomeric myosin heavy chain . epigenetic factors , including mirnas expression , play important role in muscle homeostasis and represent good molecular markers to define the stage of myogenic program and to monitor the effects of toxic factors on the modulation of muscle phenotype . mir206 , mir133a , and mir1 [ 18 , 20 , 38 ] contribute to the proper development of the myogenic program , and their alteration can impair the myogenic differentiation . based on this evidence , we investigated their expression levels in c2c12 mlc / sod1 cells and demonstrated that transfected mlc / sod1 induced a significant downmodulation of all muscle micrornas analyzed . here we have demonstrated that the inhibitory effect of the toxic sod1 protein on myogenic differentiation acts negatively on two major signaling pathways involved in muscle differentiation , such as the akt / p70 and mapk pathways . these results suggest that muscle expression of sod1 impinges muscle differentiation and might alter the identity of muscle cells in line with previous studies that demonstrate the impairment of myofiber - associated skeletal muscle satellite cells function in sod1-g93a mice and the altered expression of myogenic regulatory factors in the mouse model of amyotrophic lateral sclerosis . aguiari and colleagues have shown that high glucose growth medium induces an increase in ros production and promotes the adipocyte differentiation of muscle derived stem cells . since c2c12 mlc / sod1 cells show higher levels of oxidative stress and impaired myogenic differentiation process , modulating relevant genes and myomirna of the maintenance of the muscle phenotype , we supposed a link between toxic properties of sod1 and activation of adipogenic differentiation . in particular , we demonstrated that transfected mlc / sod1 cells show low levels of mir133a , pax-7 , and myod expression and high levels of pax-3 and perilipin 2 , a marker of lipid droplets . our data support the evidences by yin and colleagues who showed that mir133a controls satellite cells commitment to the adipocyte lineage ; in this work the authors demonstrated that mir133a inhibition promotes proadipogenic differentiation of satellite cells and that the new - formed preadipocytes completely lose the expression of both pax-7 and myod . moreover , mohsen - kanson et al . demonstrated that pax-3 plays a pivotal role during stem cell differentiation into adipocytes and that the exclusive expression of pax-3 or myod allows cells to choose between myogenic and fibro / adipogenic cell lineage . our data indicate that muscle expression of sod1 induced deregulation of myogenic process , led to the impairment of myoblast differentiation , and promoted adipogenic commitment . the fibro - adipogenic precursors or faps cells are bipotent cells positive for sca1 and negative for 7 integrin , cd31 , and cd45 , markers of satellite cells , endothelial cells , and hematopoietic cells , respectively . faps are mesenchymal cells residing in skeletal muscle interstitium and quiescent in physiological conditions and are efficiently activated to proliferate after muscle injury . it has been shown that faps may exacerbate the dystrophic phenotype turning into fibro - adipocytes , mediating fat deposition and fibrosis and thereby disrupting the environment conducive for muscle regeneration . interestingly , it has been demonstrated that , during ischemia - induced regeneration , oxidative stress negatively modulates myogenic differentiation and it is well known that oxidative stress is a hallmark of chronic disease including the duchenne muscle dystrophy where faps have been deeply studied . here we demonstrate , by histochemical assay and facs analysis , the presence of lipid droplets in c2c12 mlc / sod1 cells and their significant and exclusive positivity for sca1 , demonstrating that c2c12 mlc / sod1 cells share common features with faps . a recent study has revealed a link between hdacs , myomirs , and chromatin remodeling that underlies faps commitment to the promyogenic phenotype . the authors demonstrated that the signaling pathway that mediates the transition from myogenic to fibro / adipogenic phenotype requires the action of mir1 , mir206 , and mir133a , which favor the incorporation of specific core - protein , baf60c , into the chromatin remodeling swi / snf complex . moreover , the authors also demonstrated that in a murine model of duchenne dystrophy treatments with the hdacs inhibitor ( tsa ) address faps to a proper myogenic fate , blocking the fibro - adipogenic one . since hdac4 regulates faps fate and since our transfected cell line shares common features with faps , including higher levels of hdac4 protein , lower levels of baf60c , and deregulation of myomirna expression , we hypothesized that hdac4 is responsible for c2c12 mlc / sod1 adipogenic choice . to prove our hypothesis we treated c2c12 mlc / sod1 cells with tsa and interestingly this suggests that inhibition of hdac activity interferes with the establishment of a fap phenotype and partially rescues muscle differentiation . three main conclusions can be drawn from the present work ; the postmitotic expression of sod1 mutant gene ( 1 ) induces the impairment of the myogenic differentiation process of c2c12 cells ; ( 2 ) triggers the myoblast towards an adipogenic phenotype ; ( 3 ) promotes faps features in c2c12 cells by epigenetic changes that involve hdacs proteins . further studies will clarify the different molecular mechanisms that are modulated by multiple toxic effects of mutant sod1 protein in skeletal muscle and whether oxidative stress can represent a determinant for myoblasts choice toward a fibro / adipogenic fate .

to determine the role of mutant sod1 gene ( sod1g93a ) on muscle cell differentiation , we derived c2c12 muscle cell lines carrying a stably transfected sod1g93a gene under the control of a myosin light chain ( mlc ) promoter - enhancer cassette . expression of mlc / sod1g93a in c2c12 cells resulted in dramatic inhibition of myoblast differentiation . transfected sod1g93a gene expression in postmitotic skeletal myocytes downregulated the expression of relevant markers of committed and differentiated myoblasts such as myod , myogenin , mrf4 , and the muscle specific mirna expression . the inhibitory effects of sod1g93a gene on myogenic program perturbed akt / p70 and mapk signaling pathways which promote differentiation cascade . of note , the inhibition of the myogenic program , by transfected sod1g93a gene expression , impinged also the identity of myogenic cells . expression of mlc / sod1g93a in c2c12 myogenic cells promoted a fibro - adipogenic progenitors ( faps ) phenotype , upregulating hdac4 protein and preventing the myogenic commitment complex baf60c - swi / snf . we thus identified potential molecular mediators of the inhibitory effects of sod1g93a on myogenic program and disclosed potential signaling , activated by sod1g93a , that affect the identity of the myogenic cell population .

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

however , subsequent studies have clarified that mutant sod1 possesses a neurotoxic property ( gain of function hypothesis ) responsible for the pathogenic mechanism of the disease . indeed , the finding that overexpression of mutant sod1 in transgenic mice recapitulates several clinical features of als disease even in the presence of endogenous mouse sod1 has led to the conclusion that the disease results from a toxic gain of function . the effects of the reaction oxidative species ( ros ) are dose - dependent , and low ros concentration is necessary to guarantee cellular homeostasis while high ros dose exerts toxic effects on the cells and may contribute to cellular dysfunction . indeed , oxidative stress is a hallmark of aging and several chronic diseases such as alzheimer 's disease , duchenne dystrophy , and als . in addition , the discrepancy among different studies has further complicated the achievement of a conclusive link between altered balance of ros generation and altered homeostasis - associated diseases . in a previous work we demonstrated that muscle specific expression of the mutant isoform of sod1 gene ( sod1 ) induces muscle atrophy associated with a significant reduction in muscle strength and alterations in the contractile apparatus . we provided evidences that muscle - restricted expression of sod1 gene is sufficient to increase oxidative stress and to induce a reduction in protein synthesis and the activation of proteolytic pathway . it has been demonstrated that lactate - induced oxidative stress delays c2c12 differentiation while treatment of the same cell line with resveratrol , that confers resistance against oxidative stress , promotes myogenesis and hypertrophy . in this study , we address the role of the toxic effect of mutant sod1 gene ( sod1 ) on in vitro myogenic program and we demonstrate that sod1 expression prevents myoblasts differentiation and retains c2c12 cells in an undifferentiated state that show features common to fibro / adipogenic cells . c2c12 cells were stably transfected with ppuro and pmexmlc / sod1 plasmids ( ratio 1 : 10 ) by using superfect transfection reagent ( qiagen ) according to the manufacturer 's instructions , as control c2c12 cells were also transfected with ppuro and pmex empty vector . c2c12 and c2c12 mlc / sod1 cells were maintained in dulbecco 's modified eagle 's medium ( dmem ) with 4.5 g / l glucose , l - glutamine ( sigma ) , supplemented with 15% fetal bovine serum ( gibco ) , 100 u / ml penicillin ( sigma aldrich ) , 100 g / ml streptomycin ( sigma aldrich ) , and 2,5 g / ml of puromycin ( sigma aldrich ) for c2c12 mlc / sod1 cells . c2c12 and c2c12 mlc / sod1 cells grown in 60 mm culture dishes were washed twice with cold phosphate - buffered saline , pelleted , resuspended in 100 ul of modified lysis buffer ( tris - hcl , ph 7.5/20 mm , edta/2 mm , egta/2 mm , sucrose/250 mm , dtt/5 mm , triton - x/0.1% , pmsf/1 mm , naf/10 mm , sov4/0.2 mm , and cocktail protease inhibitors/1x ( sigma aldrich ) ) , and processed for western blot analysis . quantitative pcr was performed on an abi prism 7500 sds ( life technologies ) , using premade 6-carboxyfluorescein- ( fam- ) labeled taqman assays for beta actin , pax7 , pax3 myod , myogenin , mrf4 , and smarcd3 ( life technologies ) . quantitative rt - pcr sample values were normalized to the expression of beta - actin or u6 snrna for mrna and microrna , respectively . c2c12 and c2c12 mlc / sod1 cells were exposed to 100 nm ( sigma aldrich ) trichostatin a ( tsa ) in gm ( growth medium ) for 24 h. tsa was removed and the cells were analyzed for myosin expression ( at day 5 in dm ) or by cytofluorimetric profile ( at day 2 in dm ) . to investigate the role of mutant sod1 gene in myoblast differentiation we stably transfected the c2c12 cells line with the mlc / sod1 expression cassette ( figure 1(a ) ) that allows the expression of the mutated isoform of sod1 gene under the control of the myosin light chain promoter . the postmitotic expression of sod1 transgene induced the accumulation of gp91 protein , a marker of mitochondria oxidative damage ( figure 1(c ) ) that might mediate the toxic properties of mutant sod1 on muscle differentiation and homeostasis . to explore if sod1 transgene expression directly interferes with muscle differentiation , we stimulated differentiation by shifting the cells from growth to the differentiation medium ( dm ) ( figure 2(a ) ) . morphological and immunofluorescence analysis revealed a dramatic inhibition of muscle differentiation in c2c12 mlc / sod1 cells , with significant reduction in the number and size of the myosin positive cells , compared with control c2c12 myotubes ( figures 2(a)-2(b ) ) . the altered differentiated muscle phenotype was also confirmed by western blot analysis , revealing a drastic downmodulation of the sarcomeric myosin heavy chain expression , a specific marker of myogenic differentiation ( figure 2(c ) ) . of note , c2c12 cells stably transfected with wild type sod1 cassette ( mlc / sod1 ) did not show any morphological differences compared to control c2c12 cell lines ( data not shown ) . a key myogenic factor that triggers myoblast differentiation is myod [ 12 , 13 ] , which resulted in significant downregulation throughout the time course of differentiation in c2c12 mlc / sod1 cells ( figure 3(a ) ) . myogenin expression resulted in significant downregulation in c2c12 mlc / sod1 cells during differentiation ( figure 3(b ) ) . the final stage of skeletal muscle differentiation and maturation program is also dependent on the concerted action of another myogenic factors , namely , mrf4 , which promotes the activation of myosin heavy chain expression . real time pcr analysis revealed a significant reduction of mrf4 transcripts in c2c12 mlc / sod1 cells during differentiation , compared to control c2c12 myotubes ( figure 3(c ) ) . real time pcr analysis revealed a significant downmodulation of mir133 , mir206 , and mir1 during the differentiation time course of c2c12 mlc / sod1 cells compared to control c2c12 cells ( figures 3(d ) , 3(e ) , and 3(f ) ) . the activation of a specific developmental program requires the integration of multiple extrinsic signals from the cell membrane that culminate in changes of nuclear gene expression patterns . among the known signal transduction intermediates in muscle cells , the serine / threonine kinase akt and the mitogen - activated protein kinases ( mapk ) have been shown to modulate myogenic differentiation . in a previous study , we have demonstrated that muscle specific expression of sod1 gene in transgenic animals promotes a reduction of the phosphatidylinositol 3-kinase ( pi3k)/akt pathway and leads to muscle atrophy . in this study , we explored whether the inhibitory effects of mutant sod1 gene on myogenic program perturb the relevant signaling pathways of the myogenic program . figure 4 shows that the absolute ratio of pakt / akt was negatively regulated ( figure 4(a ) ) and pp70/p70 , the downstream effector of akt , was significantly reduced in c2c12 mlc / sod1 cultures compared with control c2c12 cells ( figure 4(b ) ) . in addition we observed a significant downmodulation of the phosphorylated active form of a factor associated with mapk differentiation cascade , namely , erk1/2 ( figure 4(c ) ) . overall these results demonstrate that the postmitotic expression of sod1 mutant gene prevents c2c12 differentiation , affecting the activation of the muscle regulatory factors , muscle mirna , and the signal transduction cascades responsible for myogenic differentiation , and might impinge the maintenance of the muscle phenotype . to validate this hypothesis , we analyzed the expression of pax-7 , which is a key factor that triggers the specification of uncommitted skeletal muscle progenitors to myogenic cells . of note , we observed a complete inhibition of pax-7 expression in c2c12 mlc / sod1 cells compared to c2c12 myoblasts , suggesting that sod1 expression not only inhibits muscle differentiation but also confers to transfected cells an immature state ( figure 5(a ) ) . the lineage switch between myogenic and brown adipogenic commitment is controlled by the muscle specific mir133a , which is highly expressed in satellite cells and can repress the expression of adipogenic markers to enforce myogenic commitment in satellite cells . further , recent evidences revealed that pax-3 transcription factor , whose ectopic expression in c2c12 myoblasts efficiently inhibits myogenic specification , plays a pivotal role during differentiation into adipocytes cells of human - induced pluripotent stem cells . in addition , the exclusive expression of pax-3 or myod gene in stem cells allows a clear and distinct choice between myogenic and fibro / adipogenic potential cell lineage . based on these evidences , we performed real time pcr and histochemical analysis in both c2c12 and c2c12 mlc / sod1 cells to evaluate the potential adipogenic features in c2c12 mlc / sod1 transfected cell lines . as shown in figure 5(b ) the levels of pax-3 transcript were significantly upregulated in c2c12 mlc / sod1 during all stages in culture , compared to c2c12 . moreover oil red o staining revealed the accumulation of intracellular lipid droplets in c2c12 mlc / sod1 cells ( figure 5(c ) ) . western blot analysis revealed that plin2 protein was accumulated in c2c12 mlc / sod1 during the differentiation process compared to control cell line ( figure 5(d ) ) . all these results together with the data of mir133a and mrfs downmodulation in c2c12 mlc / sod1 ( figure 3 ) suggest that muscle specific expression of sod1 mutant gene inhibits myoblasts differentiation and promotes adipogenic features in c2c12 cells through a mir133a and pax-3 dependent mechanism . fibro - adipogenic progenitors ( faps ) are multipotent mesenchymal cells residing in skeletal muscle interstitium [ 3032 ] . these cells are negative for cd31 , cd45 , and 7 integrin surface antigens and are characterized by the expression of the stem cell antigen 1 ( sca1 ) . faps convert environmental cues into signals that modulate muscle regeneration or turn themselves into fibro - adipocytes , inducing fat deposition and fibrosis under pathologic conditions , such as dystrophic muscles . to verify whether postmitotic expression of sod1 mutant gene promotes a faps phenotype in c2c12 cells , we performed facs analysis ( figure 6(a ) ) for sca1 , cd31 , cd45 , and 7 integrin . these results clearly evidenced the presence of fibro / adipogenic features in c2c12 mlc / sod1 . the molecular mechanism that , in concert with environmental cues , controls the identity and activity of fap cells involves a hdac - regulated network ; this network consists of muscle specific mirnas that target two alternative variants of the swi / snf chromatin remodeling complex , baf60a and baf60b , and favor the formation of a baf60c - based swi / snf complex able to confer on myod the ability to activate the myogenic program . we analyzed the protein and transcript levels , respectively , of hdac4 and baf60c in both c2c12 and c2c12 mlc / sod1 cells . we observed that hdac4 was significantly upregulated in cultures of c2c12 mlc / sod1 cells compared to control differentiated ( day 5 in dm ) c2c12 cells ( figure 7(a ) ) . in contrast , baf60c was significantly downregulated in c2c12 mlc / sod1 cells ( figure 7(b ) ) . to better correlate the induction of faps phenotype and hdac activity with sod1 expression and toxic properties , we treated c2c12 mlc / sod1 cells with the hdac class ii inhibitor trichostatin ( tsa ) and analyzed the signature profile of both faps and myogenic cells . of note we did not observe any significant difference in the number of 7 integrin sca1 cells after treatment ( data not shown ) suggesting that tsa treatment is able to counteract the establishment of a fap phenotype but it only partially rescued the muscle phenotype . to support this evidence , we induced muscle differentiation in c2c12 mlc / sod1 cells treated with tsa and we revealed that inhibition of hdac activity partially rescued myogenic differentiation , promoting myoblast fusion and differentiation ( figures 7(e)-7(f ) ) . overall these data suggest that the postmitotic expression sod1 mutant gene promotes a faps phenotype in c2c12 cells , by upregulating hdac4 protein and preventing the baf60c - swi / snf complex myogenic commitment . in this work we defined the specific toxic effects of postmitotic expression of mutant sod1 gene on the myogenic program , demonstrating that mutant sod1 alters the identity of muscle cells , affects cell homeostasis , and inhibits muscle differentiation . in contrast , the mutant sod1 possesses a toxic property that is responsible for the pathogenic mechanism of als , a neurodegenerative disease associated with the degeneration of motor neurons , muscle atrophy , and paralysis . different studies also support the evidence that skeletal muscle is a primary target of mutant sod1 toxicity in mice [ 5 , 35 ] , indicating that dysfunctions of affected muscle cells are not only a marginal consequence of denervation associated with motor neurons loss , but a direct consequence of cell muscle toxicity of mutant sod1 . the aim of our work was to define the responses of myogenic cells to the toxic effects of sod1 and the signaling pathways that mediated the toxic properties of mutant sod1 product . to this purpose , we generated a stable transfected c2c12 cell line , overexpressing the mutated isoform of the sod1 gene under the control of the mlc promoter [ 11 , 37 ] . postmitotic expression of sod1 gene induced an excess of oxidative stress , as evidenced by the increased expression of gp91 protein , and impaired muscle differentiation and fusion of c2c12 cells , inducing a significant downregulation in the expression of molecular markers of myogenic differentiation , like myod , myogenin , mrf4 , and sarcomeric myosin heavy chain . epigenetic factors , including mirnas expression , play important role in muscle homeostasis and represent good molecular markers to define the stage of myogenic program and to monitor the effects of toxic factors on the modulation of muscle phenotype . mir206 , mir133a , and mir1 [ 18 , 20 , 38 ] contribute to the proper development of the myogenic program , and their alteration can impair the myogenic differentiation . based on this evidence , we investigated their expression levels in c2c12 mlc / sod1 cells and demonstrated that transfected mlc / sod1 induced a significant downmodulation of all muscle micrornas analyzed . here we have demonstrated that the inhibitory effect of the toxic sod1 protein on myogenic differentiation acts negatively on two major signaling pathways involved in muscle differentiation , such as the akt / p70 and mapk pathways . these results suggest that muscle expression of sod1 impinges muscle differentiation and might alter the identity of muscle cells in line with previous studies that demonstrate the impairment of myofiber - associated skeletal muscle satellite cells function in sod1-g93a mice and the altered expression of myogenic regulatory factors in the mouse model of amyotrophic lateral sclerosis . since c2c12 mlc / sod1 cells show higher levels of oxidative stress and impaired myogenic differentiation process , modulating relevant genes and myomirna of the maintenance of the muscle phenotype , we supposed a link between toxic properties of sod1 and activation of adipogenic differentiation . in particular , we demonstrated that transfected mlc / sod1 cells show low levels of mir133a , pax-7 , and myod expression and high levels of pax-3 and perilipin 2 , a marker of lipid droplets . demonstrated that pax-3 plays a pivotal role during stem cell differentiation into adipocytes and that the exclusive expression of pax-3 or myod allows cells to choose between myogenic and fibro / adipogenic cell lineage . our data indicate that muscle expression of sod1 induced deregulation of myogenic process , led to the impairment of myoblast differentiation , and promoted adipogenic commitment . the fibro - adipogenic precursors or faps cells are bipotent cells positive for sca1 and negative for 7 integrin , cd31 , and cd45 , markers of satellite cells , endothelial cells , and hematopoietic cells , respectively . here we demonstrate , by histochemical assay and facs analysis , the presence of lipid droplets in c2c12 mlc / sod1 cells and their significant and exclusive positivity for sca1 , demonstrating that c2c12 mlc / sod1 cells share common features with faps . the authors demonstrated that the signaling pathway that mediates the transition from myogenic to fibro / adipogenic phenotype requires the action of mir1 , mir206 , and mir133a , which favor the incorporation of specific core - protein , baf60c , into the chromatin remodeling swi / snf complex . moreover , the authors also demonstrated that in a murine model of duchenne dystrophy treatments with the hdacs inhibitor ( tsa ) address faps to a proper myogenic fate , blocking the fibro - adipogenic one . since hdac4 regulates faps fate and since our transfected cell line shares common features with faps , including higher levels of hdac4 protein , lower levels of baf60c , and deregulation of myomirna expression , we hypothesized that hdac4 is responsible for c2c12 mlc / sod1 adipogenic choice . to prove our hypothesis we treated c2c12 mlc / sod1 cells with tsa and interestingly this suggests that inhibition of hdac activity interferes with the establishment of a fap phenotype and partially rescues muscle differentiation . three main conclusions can be drawn from the present work ; the postmitotic expression of sod1 mutant gene ( 1 ) induces the impairment of the myogenic differentiation process of c2c12 cells ; ( 2 ) triggers the myoblast towards an adipogenic phenotype ; ( 3 ) promotes faps features in c2c12 cells by epigenetic changes that involve hdacs proteins . further studies will clarify the different molecular mechanisms that are modulated by multiple toxic effects of mutant sod1 protein in skeletal muscle and whether oxidative stress can represent a determinant for myoblasts choice toward a fibro / adipogenic fate .

a predominant question in neuroscience is how memory functions are supported by the central nervous system and what cellular processes are necessary . one focus of this research is on protein - dependent synaptic modifications that occur as a consequence of neuronal activity . signaling cascades activated at the time of learning can induce the transcription of particular genes , ultimately leading to de novo protein synthesis and subsequent structural changes to support long - term memories . immediate - early genes ( iegs ) are induced soon after neuronal activity , and they participate in diverse functions . some iegs are regulatory transcription factors ( e.g. , zif268/egr1 ) responsible for inducing transcription of late - response genes , while others are effector iegs ( e.g. , arc / arg3.1 ) that are directly involved in cellular changes at locations such as the cytoskeleton or receptors . however , the transcripts of some iegs , such as activity - regulated cytoskeleton - associated protein ( arc ) , are transported to the dendrites and protein synthesis occurs there , thus making arc a reasonable target for researchers investigating the underlying mechanisms of postsynaptic changes supporting memory formation . arc ( also called arg3.1 ) is a plasticity - related gene whose induction occurs soon after synaptic activation [ 24 ] , mrna transcription is independent of de novo protein synthesis , and expression is primarily in excitatory neurons following behavioral experience . arc contains a synaptic activity - responsive element ( sare ) in the promoter upstream of the initiation site , which is necessary for transcription and sufficient for the induction of activity - dependent arc . arc mrna is transported to the dendrites [ 3 , 4 , 6 ] perhaps via sumoylation ( reviewed in ) , where it is intradendritically localized to activated synapses by phosphorylated erk ( extracellular signal - regulated kinase ) signaling and actin polymerization [ 6 , 811 ] , translated into protein , and becomes a part of the postsynaptic junction . the recruitment of arc to the dendrites suggests its importance for synaptic plasticity that occurs after activation . arc expression has been strongly linked to long - term potentiation ( ltp ) and learning . high frequency stimulation ( hfs ) induces both ltp and arc expression , which are dependent upon nmda receptor activation [ 3 , 4 ] but not upon the activation of ampa receptors . additionally , intrahippocampal infusions of arc antisense in vivo disrupt multiple aspects of ltp , indicating that arc protein synthesis is required for the early expression , maintenance , and consolidation of enduring ltp ( [ 13 , 14 ] , reviewed in ) . in accordance with ltp as a molecular model for learning and memory , delivery of arc antisense to the dorsal hippocampus produces long - term memory deficits in spatial water maze performance and inhibitory avoidance in rats , indicating a necessary role for arc protein in memory consolidation . furthermore , arc - knockout mice show impaired spatial learning in the morris water maze task , disrupted fear memory to context and auditory stimuli , and deficits in conditioned taste aversion and object recognition . recent findings provide evidence for the role of arc in the regulation of ampa receptors through interactions with endocytic proteins in dendrites ( [ 17 , 18 ] , reviewed in [ 19 , 20 ] ) , as well as a function in the stabilization and the expansion of the f - actin cytoskeleton at activated synapses , strengthening the argument that arc is involved in modifications that affect synaptic efficacy ( reviewed in ) . the protein product of the immediate early gene zif268 ( also termed egr1 , or early growth response gene ) is a transcription factor of the zinc finger family . expression of zif268 is regulated by synaptic activity and dependent upon nmda receptor activation . induction of ltp produces increased expression of zif268 mrna , and knockout of the zif268 gene in mice results in absent late ltp in the hippocampus and deficits in long - term memory for spatial water maze , conditioned taste aversion , socially transmitted food preference , and object recognition . additionally , infusions of zif268 antisense into the amygdala prior to contextual fear conditioning disrupt fear memory consolidation . in the present set of experiments , we used pavlovian fear conditioning to investigate the time - dependent expression of arc and zif268 . in pavlovian fear conditioning , a neutral stimulus is paired with an aversive unconditional stimulus ( ucs ) . through this pairing , the once neutral stimulus becomes able to elicit a fear response ( termed the conditional response , or cr ) . the animal also acquires fear for the context in which fear conditioning occurred . when the animal is presented with the shock - associated auditory stimulus or is placed back in the training context , it will exhibit fear behaviors indicating memory for the training experience . the amygdala is crucial for the acquisition , consolidation , and expression of classically conditioned fear , as it receives information about both conditional and unconditional stimuli ( cs and ucs , respectively ) making it a site of associative convergence [ 25 , 26 ] . amygdala lesions and protein - synthesis inhibitors delivered to the amygdala disrupt fear conditioning [ 2729 ] . the hippocampus is not necessary for conditional fear to an auditory cs in a delay paradigm , but it is essential for contextual fear . post training hippocampal lesions abolish contextual fear in rodents , but they do not affect conditioning to an auditory stimulus and protein - synthesis inhibitors given into the hippocampus block the acquisition of contextual fear memory [ 26 , 31 ] . the present study examined the time course of arc protein expression in the hippocampus following pavlovian fear conditioning . in addition , the temporal profile of zif268 , another plasticity - related gene product , was measured and compared to the pattern of expression for arc protein . immunohistochemistry followed western blot studies to show the localization of arc and zif268 in hippocampal regions with elevated protein expression post training . additional control groups for shock stimulation and simple exposure to auditory and contextual stimuli were analyzed with western blots to better determine the specific contribution of arc and zif268 protein in the hippocampus . in all experiments , male long evans rats ( n = 148 ; harlan ; madison , wi ) weighing approximately 350 g were used . the animal colony was climate - controlled and maintained on a 14 hr : 10 hr light : dark cycle with lights on at 7:00 a.m. all experimental procedures were performed during the light cycle . all procedures were approved by the institutional animal care and use committee at the university of wisconsin - milwaukee . each chamber was constructed from clear plexiglas ( front and back walls , ceiling ) and stainless steel ( side walls ) and measured 28 20.5 21 cm ( length height depth ) . stainless steel rods spaced 12 mm apart served as the floor of the chamber and were used to deliver a mild footshock from a scrambled shock generator . conditioning chambers were housed in sound - attenuating boxes that were illuminated by a 7.5 w white light bulb . there was a constant background noise of 5660 db produced by ventilation fans inside the boxes . the chambers were cleaned with 5% ammonium hydroxide solution between each rat . in all behavioral testing , the dependent measure was freezing behavior , which is operationally defined as the lack of all movement , except movement necessary for respiration . the training procedure was recorded by video cameras installed inside the sound - attenuating chambers , and freezing behavior was scored by computer software ( freezescan 1.0 ; clever sys . , rats were adapted to handling and transportation procedures for 3 min each on 6 consecutive days . rats were trained in a single 15-min session of auditory - cued fear conditioning ( figure 1(a ) ) . after an initial 6-min baseline period , the rats received four presentations of white noise ( 72 db , 10 s ) that coterminated with a footshock ( 1.3 ma , 1 s ) . the rats remained in the chamber for an additional 4 min following the last footshock before being returned to their home cages . this training protocol has previously been shown to produce both contextual and auditory - cued fear memories [ 28 , 29 , 33 ] . additional groups of animals were created to control separately for auditory and contextual experience and shock stimulation . one group experienced the same training protocol but with no shock stimuli delivered ( wn - cxt ) , and another group received footshock immediately upon placement in the chamber and removed shortly afterward ( shk - only ) . after training , animals were returned to their home cages and later euthanized with an overdose intraperitoneal injection of a sodium pentobarbital solution at varying time points post training . animals were killed at 30 min , 60 min , 90 min , 4 hr , 8 hr , 12 hr , or 24 hr after training , and nave home cage ( hc ) rats served as a control group . in a separate experiment , wn - cxt and shk - only groups were euthanized 60 min after stimulus exposure and were compared to trained rats also killed 60-min post training as well as additional hc controls . it was not possible to control the animals ' behavior ( e.g. , sleeping ) during the survival interval , however , the occurrence of any such behaviors prior to euthanasia should be controlled for by the hc group . hc animals were killed at varying times during the day across the experiment to account for circadian patterns , unsystematic animal behavior , and any variation in the animal colony . thus , the protein expression measured in the trained groups beyond that observed in the hc animals should be specific to the learning experience and not the result of unsystematic variability . euthanized rats were decapitated and the brains were quickly removed , frozen on dry ice , and stored at 80c . tissue samples were microdissected from the dorsal hippocampus ( figure 1(b ) ) . in all dissections , a rat brain atlas and a rat brain matrix ( harvard apparatus , holliston , ma ) were used to maintain consistency in tissue collection . hippocampal samples were homogenized manually with a pestle and glass tissue grinder in a buffer solution ( all in 100 ml ddh2o : 0.605 g tris - hcl , 0.25 g sodium deoxycholate , 0.876 g nacl , 0.038 g edta , 0.0042 g naf , 1 g / ml pmsf , 1 g / ml leupeptin , 1 g / ml aprotinin , 10 ml 10% sds , 1 mm sodium orthovanadate ) until there were no visible traces of solid matter . homogenates were stored in centrifuge tubes and kept frozen at 80c until time for centrifugation ( 4000 rpm for 20 min ) . the supernatant was removed , placed into small centrifuge tubes , and stored at 80c . a bradford protein assay was performed to determine the total amount of protein in the samples ( bio - rad dc protein assay kit , hercules , ca ) . sample dilutions were pipetted into 96-well plates and compared to serial dilutions of the protein standard ( bio - rad bsa 1.35 mg / ml ) using a versamax plate reader and softmax pro 4.3 ls software . samples were discarded from further analysis if they did not meet a set criterion for variance , and this standard was applied to all groups equivalently . normalized protein samples were loaded onto 7.5% sds gels for arc blots or 9.0% sds gels for zif268 blots using a mini protean holder filled with electrophoresis running buffer and a bio - rad powerpac ( 200 v , 0.04 a constant , 90 min ) . each experimental condition was represented on each gel to counterbalance any slight variation in blot development . gels were washed in transfer buffer ( 3.03 g tris , 14.4 g glycine , 200 ml methanol , 5 ml 10% sds , ddh2o up to 1 l ) before the protein was transferred to pvdf membranes using a semidry transfer cell ( bio - rad ; powerpac settings : 15 v constant , 2.00 a , 75 min ) . after protein transfer , membranes were incubated for 2 hr in blocking buffer ( 500 ml tbs , 15 g nonfat dry milk ) and then exposed to primary antibody for 90120 min . a monoclonal antibody for arc protein ( dilution 1 : 100 in antibody buffer , santa cruz ) , a polyclonal antibody for zif268 protein ( dilution 1 : 1000 , cell signaling ) , and a polyclonal antibody for -actin ( dilution 1 : 1000 , cell signaling ) were used in these experiments . after exposure to the primary antibody , membranes were washed twice for 15 min in antibody buffer ( 100 ml blocking buffer , 50 l tween-20 ) before being incubated for 90120 min in secondary antibody ( goat anti - mouse for arc blots1 : 5000 dilution , santa cruz ; goat anti - rabbit for zif268 and -actin blots1 : 2000 dilution , upstate biotechnology ) . membranes were washed twice for 15 min in wash buffer ( 100 ml tbs , 50 l tween-20 ) before exposure to chemiluminescence solution ( santa cruz ) for 3 min . washes and incubations were generally done at room temperature , however , primary incubation was sometimes performed overnight at 4c and then for 1 hr at room temperature . developments were conducted in a dark room , where membranes with chemiluminescence were exposed to autoradiographic film in a cassette . any disruptions in the signal during development of the films caused the sample to be excluded from further analysis . the bands representing arc ( molecular weight ( mw ) : 55 kda ) , zif268 ( mw : 75 kda ) , and -actin ( mw : 45 kda ) were measured using densitometry software ( nih imagej ) . optical density measures were computed for each hippocampal sample as a percentage of the home - cage animals ' ( control group ) protein expression , and these results were statistically analyzed using one - way anovas and fisher 's least significant difference ( lsd ) post - hoc comparisons when appropriate . rats ( n = 6 ) were euthanized with an overdose of isofluorane either 30 or 90 min after a single session of fear conditioning ( 15-min session with signaled shocks , see figure 1(a ) ) . the 30-min and 90-min time points were selected based on the time course for zif268 and arc expression established by the western blot analysis . the rats were perfused transcardially with 0.1 m pbs followed by 10% buffered formaldehyde prior to decapitation . brains were removed and placed in 10% buffered formaldehyde overnight , and then transferred to 30% sucrose formalin for cryoprotection for another 24 hr . prior to slice collection , brains were rinsed 3 times in 0.1 m pbs for 10 min each . using a freezing microtome , coronal slices ( 50-micron thick ) were collected throughout the rostral - caudal extent of the dorsal hippocampus and placed into 24-well plates with 500 l of 0.1 m pbs . the slices were incubated on a titer plate in 1% sodium borohydride for 15 min , 0.1 m pbs twice for 10 min each , 10% normal goat serum for 30 min , and primary antibody for 30 min . arc ( 1 : 100 dilution ) , zif268 ( 1 : 500 dilution ) , and neun ( 1 : 200 dilution ) primary antibodies were used to determine the regional localization and neuronal colocalization of arc and zif268 proteins . neun antibody ( chemicon - millipore ) binds to neuron - specific nuclear protein and is commonly used to identify neurons . slices from each treatment condition were dual - labeled for arc and zif268 protein to determine colocalization within individual neurons . , all slices were incubated with two washes of 0.1 m pbs for 10 min each before incubation in antibody solution containing anti - mouse alexa 488 and anti - rabbit alexa 594 antibodies ( 1 : 500 dilution each , invitrogen ) for 2 hr in the dark . slices were rinsed in two washes of 0.1 m pbs for 5 min each . after incubation the slices were mounted onto unsubbed slides using ultra cruz mounting medium ( santa cruz ) . finally , the slides were coverslipped and sealed with a thin coat of nail polish around the edges . photomicrographs were taken using a fluorescence microscope ( olympus ) . to determine coexpression of arc and zif268 in the same neurons , separate images were taken of the same field for each protein and then merged using the dp manager ( olympus ) . arc - expressing neurons appear green in color ( alexa 488 ) and zif268-expressing neurons fluoresce red ( alexa 594 ) , thus overlaying the images resulted in coexpressing neurons to appear yellow in color . the exposure time was the same for all the images collected , and any adjustments to the contrast or the brightness of the images were conducted exactly the same for relevant images . protein expression values obtained from western blots were found to be normally distributed , thus the results were analyzed using parametric tests : one - way analysis of variance ( anova ) followed by fisher 's lsd post - hoc comparisons when appropriate . in some cases , pearson correlations were conducted on the normalized optical density values from western blots to investigate the relationship of protein expression in the dorsal hippocampus . significance levels were set at = 0.05 , and data are presented as mean sem . seventy - six male long evans rats were trained in a single 15-min session of pavlovian fear conditioning and later killed at varying time points post training ( 30 min , 60 min , 90 min , 4 hr , 8 hr , 12 hr , and 24 hr ) . all trained groups exhibited equivalent levels of freezing averaged across the 5-min period of cs - ucs presentations ( f(6,69 ) = 1.989 , p > .05 , data not shown ) . protein levels for each experimental condition were expressed as a percentage of the untrained hc animals ' protein expression [ % optical density ( od ) of hc control ] . any animal with a % od score more than 4 standard deviations from the mean was removed from further data analysis ( arc : no outliers ; zif268 : n = 1 from 24 hr group ) . we found that induction of arc protein expression for trained rats was monophasic in the dorsal hippocampus , with a significant increase detected between 30 min and 90 min post training before returning to near basal levels at 4 hr ( figure 2(a ) ) . a one - way anova revealed that the level of arc protein expression was time - dependent following fear conditioning ( f(7,79 ) = 4.265 , p < .001 ) . lsd post hoc comparisons showed that when compared to untrained hc animals , arc protein was significantly increased in trained rats at 30 min ( md = 34.4587 , p < .01 ) , 60 min ( md = 58.0035 , p < .001 ) , and 90 min ( md = 50.8044 , p < .01 ) . in order to determine the localization of arc protein in the dorsal hippocampus , coronal brain slices were collected from rats killed at 90 min after training and prepared for immunohistochemistry . increased arc protein expression was observed in the granule cell layer of the dentate gyrus in the trained animals , whereas only sparse expression of arc protein was detected in this same region in the unstimulated hc control rats ( figures 2(b)2(f ) ) . arc - positive neurons were not evident in other regions of the dorsal hippocampus , such as the ca1 . although we did not quantify the arc - positive neurons in the photomicrographs , the images suggest that the upregulation in arc protein expression is primarily localized in the dentate gyrus . a different temporal pattern was seen in the protein expression profile for zif268 , with a single peak evident at 60 min post training ( f(7,72 ) = 3.228 , p < .01 , see figure 3(a ) ) . lsd post - hoc comparisons revealed that zif268 protein is significantly increased at 60 min when compared to hc animals ( md = 55.2942 , p < .01 ) . in fact , the protein level for zif268 measured at 60 min is significantly higher than all other time points ( 90 min : md = 60.6474 , p < .01 ; 4 hr : md = 71.1209 , p < .01 ; 8 hr : md = 86.4159 , p < .01 ; 12 hr : md = 86.9798 , p < .01 ; 24 hr : md = 69.0759 , p < .01 ) with the exception of 30 min ( md = 32.7638 , p > .05 ) . to establish the localization of zif268 protein in the dorsal hippocampus , coronal brain slices from additional rats killed 30 min post training were collected and incubated with zif268 antibody . in agreement with findings from western blot analysis , conditioned rats showed qualitatively more expression of zif268 protein in the ca1 region of the dorsal hippocampus at 30 min after training , compared to hc controls ( figures 3(b)3(f ) ) . similar increases were evident in the dentate gyrus but not in the ca3 region of the hippocampus ( data not shown ) . some transcription factors ( such as zif268 ) have relatively high basal levels of expression , and this was observed in the unstimulated hc controls ( figures 3(c ) and 3(e ) ) . however , this basal level of zif268 expression was less than the level of zif268 protein induced behaviorally , as measured by western blots ( figure 3(a ) ) and captured visually in photomicrographs ( figures 3(d ) and 3(f ) ) . these images suggest that the increase in zif268 protein measured in western blot analysis is the result of upregulation of zif268 primarily in ca1 and the dentate gyrus ( see figure 4 ) . to validate the temporal changes observed for arc and zif268 protein expression , the same hippocampal samples were assayed for -actin , a constitutively expressed protein , using western blot analysis . the levels of -actin protein in the trained groups did not show significant changes in expression compared to hc controls ( f(7,81 ) = 1.328 , p > .05 ; data not shown ) , indicating that the upregulation in arc and zif268 protein is unique and specific to the behavioral training experience . in summary , the expression profile for these proteins demonstrates an early increase in zif268 expression at 3060 min post training , followed by a gradual monophasic wave in arc induction lasting through 90 min ( figures 2 and 3 ) . the temporal dynamics of these proteins are distinctive and reflect the difference in the functions for arc and zif268 ( i.e. , synapse - specific changes and transcriptional regulation , respectively ) . the delay between the peaks for zif268 and arc proteins corroborates previous research showing that arc is a transcriptional target of zif268 and that multiple genomic responses are activated as a consequence of fear acquisition . using pearson correlation on the western blot optical density values , we investigated the relationship between expression of arc and zif268 proteins in the dorsal hippocampus . correlational analysis indicated a direct moderate relationship between the levels of these proteins in the dorsal hippocampus after fear conditioning ( r(79 ) = 0.244 , p < .05 ) . upon closer inspection , we discovered that this relationship was driven by the positive correlation between arc and zif268 proteins in the animals killed 30 min after training ( r(15 ) = 0.510 , p < .05 ) , as significant correlations between these proteins were not found for the other experimental conditions . other researchers have found arc and zif268 mrna expression in the hippocampus to be correlated following training in hippocampus - dependent tasks , such as the spatial water maze [ 36 , 37 ] . to investigate the colocalization between arc and zif268 proteins in the hippocampus , brain slices collected from trained rats ( sampled 30 and 90 min post training ) and from nave hc rats were dual labeled for these proteins and viewed using epifluorescence microscopy . in the unstimulated hc animals , only a few neurons in the dentate gyrus expressed arc protein ( figure 4(a ) ) ; however , many of these neurons were zif268-positive ( figure 4(b ) ) . the merged image revealed that the arc - positive neurons in the dentate gyrus also expressed zif268 ( figure 4(c ) ) . in the trained rats , more neurons expressed arc protein , with maximal number of arc - positive neurons reached at 90 min ( figures 4(d ) and 4(g ) ) . further , many more neurons in the dentate gyrus expressed zif268 , with qualitatively more zif268-positive neurons shown at 30 min ( figure 4(e ) ) compared to slices collected from nave and 90-min animals ( figures 4(b ) and 4(h ) ) . as was seen in the nave hc animals , arc and zif268 proteins expressed in trained animals are co - localized in the same neurons as depicted in the merged images ( figures 4(f ) and 4(i ) ) . these findings qualitatively replicate the western blot analysis results showing increased arc at 90 min and zif268 around 30 min post training in the dorsal hippocampus . they also extend those findings to suggest that after fear conditioning co - expression of zif268 and arc protein increases . to determine if the time - dependent changes in hippocampal arc and zif268 protein expression were specific to associative learning , rats were assigned to one of four conditions : trained ( t ; n = 14 ) , white noise and context exposure ( wn - cxt ; n = 9 ) , immediate shock ( shk - only ; n = 8) , or unstimulated home cage control ( hc ; trained animals received the same fear conditioning protocol used previously ( figure 1(a ) ) . wn - cxt animals experienced the same training paradigm but with the shock generator turned off , and shk - only rats received shock upon placement in the chamber and then were immediately removed . all rats were killed 60 min post - experience and dorsal hippocampal tissue was processed for western blots . protein levels for each experimental condition were expressed as a percentage of the untrained hc animals ' protein expression [ % optical density ( od ) of hc control ] . arc protein levels did change significantly due to behavioral experience in the training context ( figure 5(a ) ; ( f(3,33 ) = 3.007 , p < both the trained and wn - cxt groups had increased levels of arc protein compared to unstimulated hc controls ( trained : md = 66.3183 , p < .05 ; wn - cxt : md = 71.6449 , p < .05 ) , which was not observed in the shk - only condition ( md = 46.0808 , p > .05 ) . the expression of arc protein in the trained group was similar to arc protein levels observed in the 1-hr group measured in the original time course ( ~60% increase ; figures 2(a ) and 5(a ) ) . a different pattern of protein expression was measured for zif268 , where marked increases were seen only in the trained condition ( figure 5(b ) ) . the overall anova between hc , shk - only , wn - cxt , and trained groups did not meet our statistical criterion ( f(3,33 ) = 1.593 , p > .05 ) . however , the level of zif268 in the trained group was similar to that observed in the 1-hr time group of the original time course ( ~65% ; figures 3(a ) and 5(b ) ) . therefore , we conducted a linear planned comparison between the trained and hc groups which did indicate a significant upregulation of zif268 protein in the trained group ( f(1,33 ) = 4.693 , p < .05 ) . in contrast to both arc and zif268 protein expression , there were no significant changes in protein levels in the loading control -actin across the conditions ( f(3,37 ) = 0.112 , p > .05 ; data not shown ) . the effector ieg arc has been implicated in synaptic plasticity underlying learning and memory . our aim was to extend these earlier findings by investigating the time - dependent expression of arc protein induced by pavlovian fear conditioning and compare it to the expression profile of another ieg , zif268 . we found arc protein to be expressed soon after fear conditioning in the dorsal hippocampus . gradual increases in arc protein were detected by 30 min , and the single peak in the expression profile emerged at 1 - 2 hr post training before returning to baseline levels at 4 hr . arc protein was primarily localized in the granule cell layer of the dentate gyrus . these data indicate that arc protein expression induced in the dorsal hippocampus by fear conditioning is time - dependent and monophasic . arc protein is likely involved in the consolidation of contextual fear memories supported by the hippocampus , since auditory delay fear conditioning is not reliant on the hippocampus [ 26 , 30 ] . the levels of arc protein expression in the dorsal hippocampus were positively correlated with the regulatory transcription factor ieg zif268 . the expression profile for zif268 in the dorsal hippocampus was also monophasic ; however , maximal protein levels were measured at 60 min after fear conditioning , with increased expression seemingly localized to the dentate gyrus and ca1 . in a separate experiment , we investigated if the induction of these proteins was specific to associative learning or was the result of behavioral experience more generally . arc protein expression increased in rats that were trained as well as in those animals exposed to the context and auditory stimuli . delivery of immediate shock did not produce a significant increase in arc , so arc protein expression is not linked to ucs exposure per se . further , we would not predict a significant learning - related increase for the immediate shock condition as this procedure does not result in normal learning . since both training and exposure to the training chamber induced similar levels of arc expression , this effect likely relates to contextual processing . similar alterations in arc mrna in the hippocampus have been observed using catfish analysis for contextual fear conditioned and context - exposed animals . this selective increase in hippocampal zif268 protein only in the trained group and not in shk - only and wn - cxt conditions is similar to the upregulation of zif268 observed during retrieval of a context fear memory relative to cued - fear retrieval or reexposure to a context not paired with shock . the significance of this difference from the pattern seen with arc protein is not yet clear , but perhaps zif268 expression in the hippocampus is more specifically related to the formation of aversive memories . the single phase of arc protein upregulation we observed is similar to forskolin- and ecs- ( electroconvulsive shock ) induced expression of arc mrna [ 1 , 3 , 4 , 40 ] and protein [ 1 , 41 ] , with increased expression measured 30 min to 4 hr post activation and a return to basal levels by 8 or 24 hr . our data are also in accordance with recent ltp in vivo investigations , in which arc antisense oligodeoxynucleotide applied 2 hr , but not 4 hr , post - ltp induction resulted in the reversal of ltp , suggesting that the role of arc protein is time - limited . furthermore , our hippocampal data complement findings from other behavioral paradigms . for example , arc mrna expression in the hippocampus following spatial water maze training peaks at 30 min post training and returns to baseline at 6 hr . we do detect elevated levels of arc protein at 30 min , which may be the product of existing or newly transcribed mrna , a possibility we have not yet investigated or found answered in the literature . however , arc protein at 1 - 2 hr is likely translated from new transcripts synthesized as a consequence of the training experience . this hypothesis is congruent with data suggesting a very rapid turnover of arc , on the scale of minutes to a few hours [ 42 , 43 ] . although spatial water maze and contextual fear conditioning are two different hippocampus - dependent tasks , the mrna and protein time courses for these two studies produce a logical sequence when combined , such that a peak increase for arc mrna at 30 min is followed by maximal arc protein levels at 90 min . multiple waves of increased protein levels may follow a training experience , and the number of phases depends upon the training parameters used [ 44 , 45 ] . for instance , biphasic expression has been measured for other proteins in response to fear conditioning ; however , our data suggest that the expression of arc and zif268 protein in the hippocampus is monophasic following acquisition of conditioned fear . monophasic expression of arc is not necessarily true for all behavioral paradigms , as ramirez - amaya et al . found arc protein to be expressed in two phases in ca1 and ca3 of the hippocampus first phase at 30 min to 2 hr and the second phase at 8 and 24 hr following a single exploration session . in the same study , they found a single wave of arc protein in the dentate gyrus that lasted up to 8 hr post exploration , which is similar to the primarily monophasic expression of arc protein in the dentate gyrus reported here . although the increase was not significant , arc protein did seem to show a moderate increase 24 hr after training . since these animals were killed at varying times during the day , we can conclude that this increase is not due to set circadian patterns in ieg expression . however , memory does seem to have a time - of - day dependence , such that testing at a similar time of day as when training occurred produces better recall . recently , some attention has been given to exploring clock - genes in structures such as the hippocampus that may influence the expression of other proteins and may in effect create time - of - day dependence in memory . circadian fluctuation in protein phosphorylation has been previously observed in the hippocampus for mapk , and this signaling pathway has been implicated in arc translation . whether arc or any ieg is regulated in such a manner is purely speculation at this point , but certainly it is an important consideration for understanding time - dependent changes in protein translation . the post training expression of arc and zif268 proteins in the dorsal hippocampus corresponds to the time window for protein synthesis - dependent memory consolidation . the transient nature of arc expression after training may be related to evidence indicating that arc mrna is targeted for nonsense - mediated mrna decay ( nmd ) as this gene contains two conserved 3-utr introns ( , reviewed in [ 7 , 19 ] ) . nmd is a translation - dependent decay mechanism that likely halts protein expression to produce finite protein levels that are temporally specific to the learning event . along with other degradation pathways , such as ubiquitin - dependent degradation by proteasomes , nmd probably restricts the protein composition to local activated synapses . the notion that the burst of arc protein expression is temporally linked to the learning event is further supported by studies reporting that the level of training - induced arc expression in the hippocampus is coupled with learning performance . for example , guzowski and colleagues demonstrated that the amount of hippocampal arc mrna was positively correlated with an animal 's performance in hippocampus - dependent water maze learning . furthermore , the same study revealed hippocampal arc mrna expression to be correlated with the spatial water maze task ( hippocampus - dependent ) but not with the nonspatial water maze task ( hippocampus - independent ) , indicating that arc expression is associated specifically with learning experiences . presumably , the increased induction of arc and zif268 proteins measured in the present study is the result of contextual processing and associative fear learning , respectively , as the dorsal hippocampus is important for the acquisition and initial consolidation of contextual fear memory . recent investigations on the molecular pathways leading to the induction of arc have focused on brain - derived neurotrophic factor ( bdnf ) , erk , and camp - pka activation . converging evidence indicates that arc is a downstream effector of bdnf activation [ 14 , 43 ] , and pka - dependent arc protein expression can be stimulated either through the activation of nmda receptors or gs - coupled dopamine or -adrenergic receptors [ 41 , 51 ] . recent work by bramham and colleagues suggests that the initiation of arc translation is the result of erk - mnk ( extracellular signal - regulated kinase - mitogen - activated protein kinase - interacting kinase ) signaling in the dentate gyrus , a structure where we observed increases in arc protein . pharmacological blockade of erk with the mek inhibitor u0126 abolishes ltp and arc protein expression ; however , similar results are not observed when mtorc1 ( mammalian target of rapamycin complex 1 ) signaling is inhibited by the application of the protein - synthesis inhibitor rapamycin . comparatively , less is known about the interaction of zif268 and arc following synaptic activation . zif268 is a transcriptional regulator , and it is noteworthy that in our study the peak in zif268 expression at 60 min occurs just prior to the peak in arc at 90 min . the time course for these proteins presented here corroborates earlier evidence that arc is a transcriptional target of zif268 , as the arc promoter has a functional ere ( egr response element ) . additionally , we showed that the levels of hippocampal arc and zif268 were correlated with one another following fear conditioning , which further supports the functional relationship between these two ieg products . work by others similarly found arc and zif268 mrna to be upregulated following spatial exploration , and these iegs are often detected in the same nucleus . these mrnas are correspondingly increased in the hippocampus following spatial water maze training , and their expression profiles are positively correlated within this brain structure . however , the relationship between arc and zif268 is not perfect , as arc protein increases we measured at 30 min are likely not the result of zif268 regulation and are believed to be induced by one of the other pathways implicated in arc expression . further research is needed to determine what role zif268 has in the induction of arc that contributes to synaptic modifications underlying long - term memory . our data suggest that these two proteins interact soon after a learning experience , most likely orchestrating postsynaptic changes to increase synaptic efficacy and support memory formation . in summary , arc and zif268 proteins are transiently increased in the dorsal hippocampus in a manner that suggests that these two proteins work together to support contextual learning in fear conditioning . although we did not establish a causal relationship between associative learning and arc and zif268 protein expression , we have shown that these iegs are consistently upregulated in the hippocampus during the period when memory for context is being consolidated . the time frame of behaviorally - induced arc and zif268 protein in the dorsal hippocampus corresponds to a critical time window in which protein synthesis is required for memory consolidation . further , immunostaining revealed an increase in expression of arc and zif268 protein in the same hippocampal neurons after fear conditioning , suggesting a relationship between arc and zif268 colocalization in consolidation of a contextual fear memory .

memory consolidation requires transcription and translation of new protein . arc , an effector immediate early gene , and zif268 , a regulatory transcription factor , have been implicated in synaptic plasticity underlying learning and memory . this study explored the temporal expression profiles of these proteins in the rat hippocampus following fear conditioning . we observed a time - dependent increase of arc protein in the dorsal hippocampus 30-to-90-minute post training , returning to basal levels at 4 h. zif268 protein levels , however , gradually increased at 30-minute post training before peaking in expression at 60 minute . the timing of hippocampal arc and zif268 expression coincides with the critical period for protein synthesis - dependent memory consolidation following fear conditioning . however , the expression of arc protein appears to be driven by context exploration , whereas , zif268 expression may be more specifically related to associative learning . these findings suggest that altered arc and zif268 expression are related to neural plasticity during the formation of fear memory .

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

however , the transcripts of some iegs , such as activity - regulated cytoskeleton - associated protein ( arc ) , are transported to the dendrites and protein synthesis occurs there , thus making arc a reasonable target for researchers investigating the underlying mechanisms of postsynaptic changes supporting memory formation . arc contains a synaptic activity - responsive element ( sare ) in the promoter upstream of the initiation site , which is necessary for transcription and sufficient for the induction of activity - dependent arc . additionally , intrahippocampal infusions of arc antisense in vivo disrupt multiple aspects of ltp , indicating that arc protein synthesis is required for the early expression , maintenance , and consolidation of enduring ltp ( [ 13 , 14 ] , reviewed in ) . in accordance with ltp as a molecular model for learning and memory , delivery of arc antisense to the dorsal hippocampus produces long - term memory deficits in spatial water maze performance and inhibitory avoidance in rats , indicating a necessary role for arc protein in memory consolidation . recent findings provide evidence for the role of arc in the regulation of ampa receptors through interactions with endocytic proteins in dendrites ( [ 17 , 18 ] , reviewed in [ 19 , 20 ] ) , as well as a function in the stabilization and the expansion of the f - actin cytoskeleton at activated synapses , strengthening the argument that arc is involved in modifications that affect synaptic efficacy ( reviewed in ) . the protein product of the immediate early gene zif268 ( also termed egr1 , or early growth response gene ) is a transcription factor of the zinc finger family . additionally , infusions of zif268 antisense into the amygdala prior to contextual fear conditioning disrupt fear memory consolidation . in the present set of experiments , we used pavlovian fear conditioning to investigate the time - dependent expression of arc and zif268 . the present study examined the time course of arc protein expression in the hippocampus following pavlovian fear conditioning . in addition , the temporal profile of zif268 , another plasticity - related gene product , was measured and compared to the pattern of expression for arc protein . immunohistochemistry followed western blot studies to show the localization of arc and zif268 in hippocampal regions with elevated protein expression post training . additional control groups for shock stimulation and simple exposure to auditory and contextual stimuli were analyzed with western blots to better determine the specific contribution of arc and zif268 protein in the hippocampus . , sleeping ) during the survival interval , however , the occurrence of any such behaviors prior to euthanasia should be controlled for by the hc group . hc animals were killed at varying times during the day across the experiment to account for circadian patterns , unsystematic animal behavior , and any variation in the animal colony . a monoclonal antibody for arc protein ( dilution 1 : 100 in antibody buffer , santa cruz ) , a polyclonal antibody for zif268 protein ( dilution 1 : 1000 , cell signaling ) , and a polyclonal antibody for -actin ( dilution 1 : 1000 , cell signaling ) were used in these experiments . arc ( 1 : 100 dilution ) , zif268 ( 1 : 500 dilution ) , and neun ( 1 : 200 dilution ) primary antibodies were used to determine the regional localization and neuronal colocalization of arc and zif268 proteins . slices from each treatment condition were dual - labeled for arc and zif268 protein to determine colocalization within individual neurons . to determine coexpression of arc and zif268 in the same neurons , separate images were taken of the same field for each protein and then merged using the dp manager ( olympus ) . in some cases , pearson correlations were conducted on the normalized optical density values from western blots to investigate the relationship of protein expression in the dorsal hippocampus . we found that induction of arc protein expression for trained rats was monophasic in the dorsal hippocampus , with a significant increase detected between 30 min and 90 min post training before returning to near basal levels at 4 hr ( figure 2(a ) ) . a one - way anova revealed that the level of arc protein expression was time - dependent following fear conditioning ( f(7,79 ) = 4.265 , p < .001 ) . in order to determine the localization of arc protein in the dorsal hippocampus , coronal brain slices were collected from rats killed at 90 min after training and prepared for immunohistochemistry . increased arc protein expression was observed in the granule cell layer of the dentate gyrus in the trained animals , whereas only sparse expression of arc protein was detected in this same region in the unstimulated hc control rats ( figures 2(b)2(f ) ) . although we did not quantify the arc - positive neurons in the photomicrographs , the images suggest that the upregulation in arc protein expression is primarily localized in the dentate gyrus . a different temporal pattern was seen in the protein expression profile for zif268 , with a single peak evident at 60 min post training ( f(7,72 ) = 3.228 , p < .01 , see figure 3(a ) ) . lsd post - hoc comparisons revealed that zif268 protein is significantly increased at 60 min when compared to hc animals ( md = 55.2942 , p < .01 ) . in fact , the protein level for zif268 measured at 60 min is significantly higher than all other time points ( 90 min : md = 60.6474 , p < .01 ; 4 hr : md = 71.1209 , p < .01 ; 8 hr : md = 86.4159 , p < .01 ; 12 hr : md = 86.9798 , p < .01 ; 24 hr : md = 69.0759 , p < .01 ) with the exception of 30 min ( md = 32.7638 , p > .05 ) . to establish the localization of zif268 protein in the dorsal hippocampus , coronal brain slices from additional rats killed 30 min post training were collected and incubated with zif268 antibody . in agreement with findings from western blot analysis , conditioned rats showed qualitatively more expression of zif268 protein in the ca1 region of the dorsal hippocampus at 30 min after training , compared to hc controls ( figures 3(b)3(f ) ) . some transcription factors ( such as zif268 ) have relatively high basal levels of expression , and this was observed in the unstimulated hc controls ( figures 3(c ) and 3(e ) ) . however , this basal level of zif268 expression was less than the level of zif268 protein induced behaviorally , as measured by western blots ( figure 3(a ) ) and captured visually in photomicrographs ( figures 3(d ) and 3(f ) ) . to validate the temporal changes observed for arc and zif268 protein expression , the same hippocampal samples were assayed for -actin , a constitutively expressed protein , using western blot analysis . the levels of -actin protein in the trained groups did not show significant changes in expression compared to hc controls ( f(7,81 ) = 1.328 , p > .05 ; data not shown ) , indicating that the upregulation in arc and zif268 protein is unique and specific to the behavioral training experience . in summary , the expression profile for these proteins demonstrates an early increase in zif268 expression at 3060 min post training , followed by a gradual monophasic wave in arc induction lasting through 90 min ( figures 2 and 3 ) . the temporal dynamics of these proteins are distinctive and reflect the difference in the functions for arc and zif268 ( i.e. using pearson correlation on the western blot optical density values , we investigated the relationship between expression of arc and zif268 proteins in the dorsal hippocampus . correlational analysis indicated a direct moderate relationship between the levels of these proteins in the dorsal hippocampus after fear conditioning ( r(79 ) = 0.244 , p < .05 ) . upon closer inspection , we discovered that this relationship was driven by the positive correlation between arc and zif268 proteins in the animals killed 30 min after training ( r(15 ) = 0.510 , p < .05 ) , as significant correlations between these proteins were not found for the other experimental conditions . other researchers have found arc and zif268 mrna expression in the hippocampus to be correlated following training in hippocampus - dependent tasks , such as the spatial water maze [ 36 , 37 ] . to investigate the colocalization between arc and zif268 proteins in the hippocampus , brain slices collected from trained rats ( sampled 30 and 90 min post training ) and from nave hc rats were dual labeled for these proteins and viewed using epifluorescence microscopy . in the unstimulated hc animals , only a few neurons in the dentate gyrus expressed arc protein ( figure 4(a ) ) ; however , many of these neurons were zif268-positive ( figure 4(b ) ) . in the trained rats , more neurons expressed arc protein , with maximal number of arc - positive neurons reached at 90 min ( figures 4(d ) and 4(g ) ) . as was seen in the nave hc animals , arc and zif268 proteins expressed in trained animals are co - localized in the same neurons as depicted in the merged images ( figures 4(f ) and 4(i ) ) . these findings qualitatively replicate the western blot analysis results showing increased arc at 90 min and zif268 around 30 min post training in the dorsal hippocampus . they also extend those findings to suggest that after fear conditioning co - expression of zif268 and arc protein increases . to determine if the time - dependent changes in hippocampal arc and zif268 protein expression were specific to associative learning , rats were assigned to one of four conditions : trained ( t ; n = 14 ) , white noise and context exposure ( wn - cxt ; n = 9 ) , immediate shock ( shk - only ; n = 8) , or unstimulated home cage control ( hc ; trained animals received the same fear conditioning protocol used previously ( figure 1(a ) ) . wn - cxt animals experienced the same training paradigm but with the shock generator turned off , and shk - only rats received shock upon placement in the chamber and then were immediately removed . arc protein levels did change significantly due to behavioral experience in the training context ( figure 5(a ) ; ( f(3,33 ) = 3.007 , p < both the trained and wn - cxt groups had increased levels of arc protein compared to unstimulated hc controls ( trained : md = 66.3183 , p < .05 ; wn - cxt : md = 71.6449 , p < .05 ) , which was not observed in the shk - only condition ( md = 46.0808 , p > .05 ) . the expression of arc protein in the trained group was similar to arc protein levels observed in the 1-hr group measured in the original time course ( ~60% increase ; figures 2(a ) and 5(a ) ) . however , the level of zif268 in the trained group was similar to that observed in the 1-hr time group of the original time course ( ~65% ; figures 3(a ) and 5(b ) ) . therefore , we conducted a linear planned comparison between the trained and hc groups which did indicate a significant upregulation of zif268 protein in the trained group ( f(1,33 ) = 4.693 , p < .05 ) . in contrast to both arc and zif268 protein expression , there were no significant changes in protein levels in the loading control -actin across the conditions ( f(3,37 ) = 0.112 , p > .05 ; data not shown ) . the effector ieg arc has been implicated in synaptic plasticity underlying learning and memory . our aim was to extend these earlier findings by investigating the time - dependent expression of arc protein induced by pavlovian fear conditioning and compare it to the expression profile of another ieg , zif268 . we found arc protein to be expressed soon after fear conditioning in the dorsal hippocampus . gradual increases in arc protein were detected by 30 min , and the single peak in the expression profile emerged at 1 - 2 hr post training before returning to baseline levels at 4 hr . these data indicate that arc protein expression induced in the dorsal hippocampus by fear conditioning is time - dependent and monophasic . arc protein is likely involved in the consolidation of contextual fear memories supported by the hippocampus , since auditory delay fear conditioning is not reliant on the hippocampus [ 26 , 30 ] . the levels of arc protein expression in the dorsal hippocampus were positively correlated with the regulatory transcription factor ieg zif268 . the expression profile for zif268 in the dorsal hippocampus was also monophasic ; however , maximal protein levels were measured at 60 min after fear conditioning , with increased expression seemingly localized to the dentate gyrus and ca1 . in a separate experiment , we investigated if the induction of these proteins was specific to associative learning or was the result of behavioral experience more generally . this selective increase in hippocampal zif268 protein only in the trained group and not in shk - only and wn - cxt conditions is similar to the upregulation of zif268 observed during retrieval of a context fear memory relative to cued - fear retrieval or reexposure to a context not paired with shock . the significance of this difference from the pattern seen with arc protein is not yet clear , but perhaps zif268 expression in the hippocampus is more specifically related to the formation of aversive memories . the single phase of arc protein upregulation we observed is similar to forskolin- and ecs- ( electroconvulsive shock ) induced expression of arc mrna [ 1 , 3 , 4 , 40 ] and protein [ 1 , 41 ] , with increased expression measured 30 min to 4 hr post activation and a return to basal levels by 8 or 24 hr . our data are also in accordance with recent ltp in vivo investigations , in which arc antisense oligodeoxynucleotide applied 2 hr , but not 4 hr , post - ltp induction resulted in the reversal of ltp , suggesting that the role of arc protein is time - limited . for example , arc mrna expression in the hippocampus following spatial water maze training peaks at 30 min post training and returns to baseline at 6 hr . we do detect elevated levels of arc protein at 30 min , which may be the product of existing or newly transcribed mrna , a possibility we have not yet investigated or found answered in the literature . although spatial water maze and contextual fear conditioning are two different hippocampus - dependent tasks , the mrna and protein time courses for these two studies produce a logical sequence when combined , such that a peak increase for arc mrna at 30 min is followed by maximal arc protein levels at 90 min . for instance , biphasic expression has been measured for other proteins in response to fear conditioning ; however , our data suggest that the expression of arc and zif268 protein in the hippocampus is monophasic following acquisition of conditioned fear . in the same study , they found a single wave of arc protein in the dentate gyrus that lasted up to 8 hr post exploration , which is similar to the primarily monophasic expression of arc protein in the dentate gyrus reported here . circadian fluctuation in protein phosphorylation has been previously observed in the hippocampus for mapk , and this signaling pathway has been implicated in arc translation . the post training expression of arc and zif268 proteins in the dorsal hippocampus corresponds to the time window for protein synthesis - dependent memory consolidation . the transient nature of arc expression after training may be related to evidence indicating that arc mrna is targeted for nonsense - mediated mrna decay ( nmd ) as this gene contains two conserved 3-utr introns ( , reviewed in [ 7 , 19 ] ) . the notion that the burst of arc protein expression is temporally linked to the learning event is further supported by studies reporting that the level of training - induced arc expression in the hippocampus is coupled with learning performance . for example , guzowski and colleagues demonstrated that the amount of hippocampal arc mrna was positively correlated with an animal 's performance in hippocampus - dependent water maze learning . furthermore , the same study revealed hippocampal arc mrna expression to be correlated with the spatial water maze task ( hippocampus - dependent ) but not with the nonspatial water maze task ( hippocampus - independent ) , indicating that arc expression is associated specifically with learning experiences . presumably , the increased induction of arc and zif268 proteins measured in the present study is the result of contextual processing and associative fear learning , respectively , as the dorsal hippocampus is important for the acquisition and initial consolidation of contextual fear memory . converging evidence indicates that arc is a downstream effector of bdnf activation [ 14 , 43 ] , and pka - dependent arc protein expression can be stimulated either through the activation of nmda receptors or gs - coupled dopamine or -adrenergic receptors [ 41 , 51 ] . recent work by bramham and colleagues suggests that the initiation of arc translation is the result of erk - mnk ( extracellular signal - regulated kinase - mitogen - activated protein kinase - interacting kinase ) signaling in the dentate gyrus , a structure where we observed increases in arc protein . pharmacological blockade of erk with the mek inhibitor u0126 abolishes ltp and arc protein expression ; however , similar results are not observed when mtorc1 ( mammalian target of rapamycin complex 1 ) signaling is inhibited by the application of the protein - synthesis inhibitor rapamycin . zif268 is a transcriptional regulator , and it is noteworthy that in our study the peak in zif268 expression at 60 min occurs just prior to the peak in arc at 90 min . additionally , we showed that the levels of hippocampal arc and zif268 were correlated with one another following fear conditioning , which further supports the functional relationship between these two ieg products . work by others similarly found arc and zif268 mrna to be upregulated following spatial exploration , and these iegs are often detected in the same nucleus . these mrnas are correspondingly increased in the hippocampus following spatial water maze training , and their expression profiles are positively correlated within this brain structure . however , the relationship between arc and zif268 is not perfect , as arc protein increases we measured at 30 min are likely not the result of zif268 regulation and are believed to be induced by one of the other pathways implicated in arc expression . in summary , arc and zif268 proteins are transiently increased in the dorsal hippocampus in a manner that suggests that these two proteins work together to support contextual learning in fear conditioning . although we did not establish a causal relationship between associative learning and arc and zif268 protein expression , we have shown that these iegs are consistently upregulated in the hippocampus during the period when memory for context is being consolidated . the time frame of behaviorally - induced arc and zif268 protein in the dorsal hippocampus corresponds to a critical time window in which protein synthesis is required for memory consolidation . further , immunostaining revealed an increase in expression of arc and zif268 protein in the same hippocampal neurons after fear conditioning , suggesting a relationship between arc and zif268 colocalization in consolidation of a contextual fear memory .

after decades of neglect , the search for novel , effective antimalarials has been revived primarily due to emergence of drug resistance . widespread resistance has been reported not only for 8-aminoquinolines but recent reports indicate the appearance of increased parasite tolerance to artemisinin analogues , potentially predicating clinical resistance . the absence of highly efficacious malaria vaccines highlights the importance of developing small - molecule drugs . with support from the wellcome trust and medicines for malaria venture ( mmv ) , the ngbs consortium ( the novartis institute for tropical diseases ( nitd ) , the genomics institute of the novartis research foundation ( gnf ) , the biomedical primate research centre ( bprc ) , and the swiss tropical and public health institute ( stphi ) ) was formed and set up to discover new antimalarials which are effective against multidrug resistant parasite strains here we report our continued effort to optimize a series of imidazolopiperazine compounds in search of a preclinical candidate . as described previously , an extensive sar study on the three peripheral parts had yielded decent in vitro and in vivo antimalarial activities as well as favorable physiochemical and pharmacokinetic properties . our efforts toward the first - generation compounds led to candidates ( like compound 1 ) with double - digit nanomolar potency and moderate oral exposure in mice . however , most potent compounds with an unsubstituted piperazine had poor oral exposure which resulted in inferior in vivo efficacy when administered orally , for example , compounds 28 and 31 ( scheme 1 ) in the previous communication ( figure 1 ) . we were intrigued by the possibility of further refining the core structure of the molecule to further improve the properties . our hypothesis was further reinforced by chemical stability and mouse in vitro metabolic stability results which led to the identification of two biologically inactive metabolites ( 1a and 1b ) arising from the oxidation of piperazine ring . our plan was to substitute three possible positions ( positions 5 , 6 , and 8) to evaluate the corresponding effects on potency as well as a means to improve oral exposure . central piperazine ring oxidation is a major metabolic and chemical instability site in compound 1 . on the basis of the designed molecules , it was clear that previous synthetic routes were not amenable for the desired compounds because we could not utilize the 3 + 2 cyclization . distinct syntheses were designed and executed to obtain substitution at different positions of a piperazine ring . the synthesis started with an alkylation reaction between cbz - protected glycine derivative 2 and bromide 3 . adduct 4 was then refluxed with nh4oac in toluene to give imidazole 5 with removal of water using a dean stark apparatus . regioselective n - alkylation with ethyl 2-bromoacetate followed by a one - pot deprotection / cyclization sequence furnished the lactam core , which was reduced by borane to furnish amine 8 . the quaternary -carbon did not pose a significant amount of steric hindrance for the hatu mediated amidation reaction with n - boc - glycine . amide 9 was obtained at room temperature in decent yield and further was brominated to provide intermediate 10 . then a standard buchwald hartwig amination reaction with substituted aniline followed by deprotection finished the synthesis of compound 12 with the desired alkyl modification at position 8 . reagents and conditions : ( a ) k2co3 , dmf , rt , 84% ; ( b ) nh4oac , toluene , reflux , 88% ( c ) ethyl 2-bromoacetate , cs2co3 , dmf , rt , 83% ; ( d ) pd / c , h2 ( balloon ) , meoh , rt , 91% ; ( e ) bh3thf , thf , reflux , 95% ; ( f ) hatu , diea , ch2cl2 , rt , 70% ; ( g ) br2 , acoh , ch2cl2 , rt , 100% ; ( h ) arnh2 , pd2(dba)3 , xantphos , cs2co3 , dioxane , 150 c , 89% ( for example 22 , ar = 4f - ph and r1 = r2 = me ) ; ( i ) tfa , ch2cl2 , rt , 52% ( for example 22 , ar = 4f - ph and r1 = r2 = me ) . scheme 3 describes the synthesis of 6,6-dimethyl analogues . analogous chemistry to compound 12 was used for the first two steps to provide imidazole 5a . the compound was regioselectively n - alkylated with methallyl chloride to obtain the key intermediate for the piperazine cyclization step . the intramolecular hydroamination reaction proved to be difficult , and after extensive screening , treating the substrate 5a with a 5:1 mixture of acetic acid and methanesulfonic acid at 210 c produced the cyclized core with simultaneous loss of the carbamate protecting group . despite the harsh reaction condition , an acceptable yield of the product ( 39% ) was obtained and the regiochemistry was confirmed by 2d nmr of the intermediate . the cyclized core was subjected to amide coupling with n - boc glycine to yield intermediate 13 . the final steps in the sequence are analogous to scheme 2 to furnish analogue 14 . the sequence involved six steps from intermediate 5a with an overall yield of 3.7% . the synthesis of the 5,5-dimethyl modified analogues diverged from their 8,8-dimethyl modified counterparts at intermediate 7a . n - pmb protection was necessary for lactam 7a before the nah - mediated bis - methylation at the 5-position ( scheme 4 ) . the bicyclic intermediate was then deprotected and amine was subjected to a hatu mediated amidation to obtain compound 7c . the final sequence involved bromination followed by palladium - catalyzed aniline amination and boc - deprotection to furnish 5,5-dimethyl modified compound 26 . the sequence involved eight steps from intermediate 7a with an overall yield of 10.9% . reagents and conditions : ( a ) methallyl chloride , k2co3 , ki , dmf , rt , 55% ; ( b ) acoh / msoh ( 6:1 ) , 210 c , 39% ; ( c ) n - boc - glycine , hatu , diea , dmf , rt ; 57% ; ( d ) br2 , acoh , ch2cl2 , rt ; 55% ; ( e ) arnh2 , pd2(dba)3 , xantphos , cs2co3 , dioxane , 150 c ; ( f ) tfa , ch2cl2 , rt , 55% over 2 steps ( for example 25 , ar = 4f - ph ) . reagents and conditions : ( a ) pmbcl , koh , dmf , 0 c to rt , 63% ; ( b ) mei , nah , dmf , rt , 86% ; ( c ) bh3thf , thf , reflux , 100% ; ( d ) tfa , 70 c , quantitative ; ( e ) n - boc - glycine , hatu , tea , dmf , rt , 49% ; ( f ) br2 , acoh , ch2cl2 , 90% ; ( g ) arnh2 , pd2(dba)3 , xantphos , cs2co3 , dioxane , 150 c ; ( h ) tfa , ch2cl2 , rt , 46% over 2 steps for example 26 where ar = 4f - ph . we began exploring the sar on the piperazine ring by substituting at the 8-position first , and we decided to use 4-f - aniline and 3-f-4-cl aniline based on our previous work at the r6 position ( table 1 ) . adding a methyl group to the 8-position led to loss of potency by 10-fold ( cmpd 17 ) , but the compound was still equipotent to compound 15 ( scheme 5 ) . moreover , there was no profound effect on the potency by changing the absolute configuration of the methyl group . increasing the size of substituent to isopropyl group led to dramatic loss in potency ( examples 19 and 20 ) . a dimethyl group on the 8-postion not only led to more favorable compound in terms of completely blocking the benzylic carbon as indicated by our metabolite studies but also removing the stereogenic carbon . surprisingly , substitution of 3cl-4f aniline in the 8-disubstitution case provided a less potent compound when compared to other anilines ( 21 vs 22 , 23 , or 24 ) . emboldened by the increase in the potency based on the substitution on the 8-position , we decided to study the effect of dime group on the ethylene carbons of the piperazine ring . on the basis of examples 25 and 26 , it was abundantly clear that the dimethyl group on the 8-position had the most significant positive effect on potency . the substitution on the other positions of the piperazine ring only provided modest potencies when compared to 8-substitution , with activities in the double - digit nanomolar range . as the 5-substituted compounds and the 6-substituted compounds were less potent , we decided to focus our attention on the effect of the amide substituent of the piperazine nitrogen with the 8,8-dimethyl group constant . our previous efforts demonstrated that dimethyl glycine had slightly inferior potency but offered superior oral exposure . in the case of the substituted piperazines , installation of methylalanine amide provided equipotent compounds ( 15 vs 27 , 22 vs 28 , and 23 vs 29 ) . we proceeded to further understand the overall effect of the dimethylpiperazine on the minimal pharmacophore . after studying the effects of various substitutions on the piperazine ring and obtaining a significant improvement in activity , we decided to reconfirm the minimal pharmacophore needed for antiplasmodial activity . on the basis of the earlier results , we were able to achieve antiplasmodial activity with ec50 in 160400 nm range for the core devoid of the amino acid substituent on the piperazine nitrogen ( compound 31 , table 2 ) . modification of the piperazine ring into the corresponding lactam led to a 7-fold decrease in activity in the micromolar range ( 32 ) . however , the antiplasmodial activity is regained by introduction of dimethyl substituent on the 8-position with a 7-fold improvement ( 33 ) . the most optimal compound from this lactam subseries was a compound with 3cl-4f aniline substitution with an activity around 50100 nm ( 34 ) . the dimethyl substitution on the reduced piperazine core led to 10-fold improvement in potency ( 31 vs 35 ) with compounds to 10 nm range . these results led us to conclude that the amino acid substitution had minimal to no effect on the potency of the compound with substitution on the 8-position of the imidazolopiperazine core and that the basicity of the piperazine nitrogen could be modulated without complete loss in antimalarial activity . in parallel to the sar studies to optimize the potency of the compounds series , we evaluated the in vitro adme and in vivo pharmacokinetic properties of key analogues . in general , this series of compounds had very good lipinski 's rule - of - five compliance and most of the analogues were highly soluble ( > 175 m at ph 6.8 ) , with the neutral lactams being least soluble among all the compounds . table 3 summarizes the in vitro adme profiles of the important compounds . typically , a range of values in the permeability assays was observed . in the caco-2 assays , the core piperazine compounds ( both lactams as well as nonamide piperazine compounds ) exhibited high apical to basal values ( 31 , 32 , 34 , and 35 ) . the permeability of the compounds was reduced once the side chain amino acid was installed , but metabolic stability is improved ( 28 vs 35 ) . however , the % absorbed was lower in the pampa assay for compounds with poor permeability in the caco-2 assay . upon comparing the calculated log p values with pampa as well as caco-2 values unsurprisingly , log p of 4.5 or higher was needed for having excellent percentage absorbed values in pampa assay ( see supporting information ) . removing the basicity of the piperazine ring by introduction of carbonyl group improved the mouse and human microsome stability of the compound , presumably by blocking the ethylene bridge ( 31 vs 32 ) but is not evident in rat microsomes . introduction of the dimethyl group on the 8-position of the piperazine ring does not improve overall metabolic stability of the compound ( 15 vs 28 or 31 vs 35 ) despite the metabolite identification results presented earlier . this may be due to the ethylene bridge in the piperazine ring representing a metabolic soft spot and enabling metabolic switching . in general , this scaffold exhibited poor rat metabolic stability when compared to other species . one interesting finding with the substituted piperazine analogues was the improvement of activity against the herg channel as determined in the automated patch clamp assay . compared to the unsubstituted piperazine analogue 27 , we were able to decrease inhibition of the herg channel by at least 23-fold , with most analogues ( such as 22 ) suggesting a lower potential for qt interval prolongation in compounds with a substituted piperazine . compounds series found to have favorable potency and in vitro adme properties were tested in snapshot pk , an abbreviated pk study shown to accurately bin compounds into low , moderate , or high oral exposure . compounds predicted to have reasonable oral exposure would be tested in mice using both po and iv routes of administration ( table 4 ) . the in vivo clearance generally was low to moderate and significantly improved when compared to the corresponding unsubstituted analogues ( 15 , 22 ) . this resulted in higher maximum concentration as well as improvement in auc values in the oral arms as well . lactam compound 34 exhibited highest oral auc than all other analogues , and it also had lower intrinsic clearance with moderate volume of distribution . to test whether red cell partitioning was occurring to explain the high volume of distribution for the basic compounds , we carried out an in vitro experiment in which compound 22 was incubated in nave rat whole blood as well as plasma for 30 min . after centrifuging the blood and quantifying the amount of compound in the red blood cells , we determined that the concentration of the compound was 3.47 times higher in whole blood than in plasma alone , indicating that the compound is either adhering to the rbc s or getting distributed inside the cells . po dosing ; dose ( mg / kg ) , 20 ; species / strain , mouse , male balb / c ; formulation , 2.5 mg / ml in peg300/d5w , 3:1 , solution ; vd = volume of distribution ; cl = clearance ; cmax = maximal concentration ; tmax = time at maximal concentration ; auc = area under the curve ; f = oral bioavailability . because we had examples of compounds with different modifications with acceptable bioavailability , we decided to carry on the efficacy experiment in mice . the in vivo antimalarial activities of the optimized compounds were evaluated using a plasmodium berghei mouse survival model . in this test , groups of three p. berghei infected mice were treated orally with compound one day after infection . the key readouts were the percentage of parasitemia reduction on day 3 postinfection compared to the untreated mice and the prolongation of survival of the infected mice . table 5 summarizes the in vivo efficacy test results of compound in comparison with standard antimalarials chloroquine ( cq ) and artesunate ( as ) . it was observed that compounds 22 , 24 , and 29 exhibited > 99% parasitemia reduction at a low dose of 30 mg / kg with a modest survival of 15 days or higher . increasing the dose to 100 mg / kg or dosing the compound 3 30 mg / kg did not lead to increase in survival . currently , we are investigating the possibility of recrudescence of parasites or development of resistance for the compound in vivo to better explain the lack of curative activity in this model . we decided to determine the ed99 representative examples for the substituted and nonsubstituted piperazine cores and found the substituted piperazine core was 10-fold more efficacious ( compound 22 ed99 = 2.2 mg / kg ) than the unsubstituted core ( compound 1 ed99 = 26.5 mg / kg ) . compounds 34 and 36 clearly proved inferior both in terms of parasitemia reduction as well as survival in mice , indicating the need for the amino acid side chain in providing better mouse efficacy . activity = average parasitemia reduction ; survival = average lifespan after infection ( 67 days for untreated control mice ) ; compounds were formulated in 75% peg300/25% d5w ( 5% dextrose in water ) ; cq , chloroquine ; as , artesunate . 10% ethanol , 30% peg400 , 6% vitamin etpgs . on the basis of the efficacy results rat was chosen as the second animal species for oral exposure evaluation as it may serve as the in vivo preclinical toxicity species . taking into account the nonoptimal metabolic stability data for rats for this scaffold , we decided to study the oral exposure of the compound in rat for a period of 5 h in the oral arm only to get an estimation of the exposure ( table 6 ) . -methylalanine subclass of compounds like 29 did not exhibit any in vivo exposure and were not further pursued . in line with the better rat metabolic stability ( er = 0.68 ) , nonbasic lactam compound 34 demonstrated the best rat exposure despite lower mouse efficacy . po dosing ; dose ( mg / kg ) , 20 ; species / strain , rat , wistar ; formulation , 2.5 mg / ml in peg300/d5w , 3:1 , solution ; blq , below level of quantification ; na = could not be determined . as the oral exposure of compounds 22 was low in rats but demonstrated superior mouse efficacy , we decided to dose compound 22 at higher doses to make sure that sufficient multiples of exposure of the mouse efficacious dose could be achieved ( table 7 ) . we observe that when the dose was increased to 30 mg / kg from 10 mg / kg , there was overproportional 7-fold increase in auc as well as 2-fold increase in bioavailability . increasing the dose further to 100 mg / kg dose with suspension formulation led to 55-fold increase in auc with a 3-fold increase in oral bioavailability . the auc at the mouse efficacious dose ( 2 mg / kg ) was 780 hnm , therefore a 45 exposure multiple is achieved at 100 mg / kg in the rat . the multiple of exposure provides a sufficient window to use the rat as a rodent toxicology species . it is unclear at this time if this overproportional exposure at higher doses is due to saturating metabolism or an efflux transporter , but given the partial efflux observed in caco-2 , this may explain this multiple dose exposure results . compound 22 was subjected to a series of assays to evaluate the toxicity profile . this drug candidate was inactive on a panel of human cytochromes p450 ( ic50s are greater than 6 m for cyp1a2 , cyp2c19 , cyp2c9 , cyp2d6 , and cyp3a4 ) , and the observed cytotoxicity cc50 were all greater than 12 m on a panel of mammalian cell lines ( 293 t , ba / f3 , cho , hep2 , hela , huh7 ) , which translates to an adequate selectivity index ( si > 100 ) . compound 20 was found negative in the mini - ames and micronucleus tests , which indicated a low mutagenic potential . finally , considering the efficacious plasma cmax in the mouse at 2 mg / kg is low ( 75 nm ) , the risk of cardiotoxicity is considered to be minimal where the herg channel inhibitory activity ic50 = 13.4 m , providing a greater than 170-fold window . imidazolopiperazine compounds had been proven to be effective against multidrug resistant parasite strains with minimum toxicity to host - cell lines . however , early lead compound such as compound 15 and 27 still suffer from moderate potencies , relatively poor rat pk , and potent herg inhibitory activity . directed by the identification of in vitro metabolites formed by compound 1 , the piperazine core of 15 was systematically modified by a gem - dimethyl functionality at all three possible places . the 8,8-dimethyl analogues demonstrated excellent potency as well as improved oral exposure in the mouse . this also allowed us to use the glycine amide that reduced our herg risk when compared to 27 . the first generation of compounds exhibited efficacy that was comparable to the known antimalarials in the p. berghei mouse model , however , compound 22 was more efficacious than chloroquine and artesunate especially when parasitemia reduction is compared in terms of ed99 values . although compound 22 demonstrated poor rat pk at low doses , with higher doses it was able to achieve the necessary exposure multiples calculated based on the plasma concentration found to be efficacious in the mouse model . compound 22 is thus currently under preclinical safety assessment in rats as a potential candidate for human trials . plasmodium falciparum cultures were grown in o rbcs using culturing media : rpmi 1640 media ( no phenol red ) with l - glutamine and containing 0.05 mg / ml gentamicin , 0.014 mg / ml hypoxanthine , 38.4 mm hepes , 0.20% sodium bicarbonate , 0.20% glucose , 3.4 nm naoh , 5% human serum , and 1.25% albumax . using traditional p. falciparum protocols published previously , 25 ml cultures were maintained in 75 cm flasks ( fisher cat . 10 - 126 - 41 ) at 5% hematocrit and parasitemia ranging between 1% and 10% . once parasitemia reached 810% , cultures are diluted down to 1% or higher depending on needs . cultures were gassed for approximately 30 s to 1 min using a blood gas mixture to maintain a gas composition of 96% nitrogen , 3% carbon dioxide , and 1% oxygen and incubated at 37 c . on day 1 of the assay , the smear was fixed onto the slide by placing in methanol for 30 s , stained in 10% geimsa stain , and the percent of parasitized erythrocytes vs uninfected erythrocytes was determined by microscopy with a light microscope . p. falciparum strains to be used for screening were prepared with screening media ( culturing media without human serum but supplemented with 0.5% albumax ii ) and fresh erythrocytes . twenty l of screening media were dispensed via the microflo ( biotek ) into 384-well , black clear - bottom assay plates ( clear gnf custom plates by griener bio - one ) . then 50 nl of compounds were transferred using a platemate plus ( matrix ) or the gnf systems pintool into the assay plates containing screening media to a final maximum concentration of 10 m in a 12 point dose response ( 1/2 log serial dilution ) . on the basis of the measured parasitemia levels , each parasite strain was diluted to yield 0.5% parasitemia , and 50% hematocrit blood ( uninfected erythrocytes ) was added to a concentration of 4.17% . this diluted culture was used in dispensing the remaining 60% of the assay volume ( 30 l ) into the prewarmed assay plates via the microflo liquid dispenser . the final parasitemia was 0.3% and final hematocrit was 2.5% in a total volume of 50 l . assay plates were incubated at 37 c for 72 h in a gas chamber filled with low oxygen blood gas . after the 72 h incubation ( equivalent to 12 cycles during the blood stage ) , a prepared mixture of the lysis buffer ( 5 mm edta , 1.6% triton x-100 , 20 mm tris - hcl , 0.16% saponin ) in water and sybr green detection ( 0.1% ) reagent was dispensed at 10 l per well using the microflo . cultures were incubated for an additional 24 h at 25 c before measuring fluorescence intensity using the envision plate reader ( perkin - elmer ) with a 505 dichroic mirror . data were normalized based on maximum fluorescence signal values for dmso treated wells ( no inhibition by compound ) and the minimum fluorescence signal values for wells containing the highest concentration of inhibitor control compounds , for example , pyrimethamine at a final concentration of 10 m . data were analyzed on a plate - by - plate basis and compared to reference compounds that were always included on every plate , typically artemisinin , mefloquine , and pyrimethamine . ec50 values were obtained using a custom curve fitting model , and a standard logistic regression model was applied for curve fitting . the quality of the assay run was assessed by the performance of the reference compounds where the ec50 must be within 3-fold of the standard reference values for the assay plate to pass requisite data quality control needs . additionally , all compounds were typically assayed at a minimum in duplicate ( independent assay plates ) and ec50 values ideally must not vary by more than 2-fold between plates . the metabolic stability of drug candidates was determined in human , mouse , and rat liver microsomes using the compound depletion approach , quantified by lc / ms / ms . the assay measured the rate and extent of metabolism of chemical compounds by measuring the disappearance of the parent compound . the assay determined the compound s in vitro half - life ( t1/2 ) and hepatic extraction ratios ( er ) and predicted metabolic clearance in human , rat , and mouse species . the compound concentration was determined by uv absorption measured with a spectramax190 microplate spectrophotometer ( molecular devices corporation , sunnyvale , ca , usa ) at absorption wavelengths between 260 and 290 nm . the caco-2 assay was carried out in a 96-well format , and compound concentrations in each chamber were measured by lc / ms . the assay offers apparent permeability data of test compounds from the apical to the basolateral chambers [ papp ( a b ) ] and from the basolateral to the apical chambers [ papp ( b a ) ] . the results can be used to predict the in vivo oral absorption ( using papp ( a b ) ) and the transport mechanism of the test compounds across gi membrane ( ratio of papp ( b a):papp ( a b ) and log p lipophilicity data ) . the assay utilizes electrophysiology measurement on the electric current passing through herg channel that is heterologously expressed in a stable cho cell line . channels were open by a herg - specific voltage protocol , and the compound effects were directly characterized by their block of the herg current . all compounds were tested by 4-point dose response curves , and ic50 values ( between 0.2 and 30 m ) were measured . in - life studies were carried out under protocols approved by the animal care and use committee ( iacuc ) of gnf . all compounds were formulated at a concentration of 2.5 mg / ml in 75% peg300 and 25% d5w ( 5% dextrose in distilled water ) solution and were filtered using a 0.45 m syringe filter . the filtered solutions were dosed intravenously via the lateral tail vein at 5 mg / kg with a dosing volume of 2 ml / kg to male balb / c mice ( n = 3 per group ) . the filtered solutions were also administered orally at 20 mg / kg ( with a dosing volume of 8 ml / kg ) to another group of mice ( n = 3 per group ) . six blood samples of 50 l each were collected serially from each animal via retro orbital sinus up to 24 h after dosing . blood samples were collected into heparin microtainer and centrifuged and plasma separated and frozen until analysis . compounds were formulated at a concentration of 2.5 mg / ml in 75% peg300 and 25% d5w solution . in rat snapshot pk , two rats ( wistar rats ) were administrated orally at 10 mg / kg dose and four blood samples ( 100 l ) were taken at 0.5 , 1 , 3 , and 5 h post dosing . before the sample analysis , the plasma samples were pooled based on the sampling time . in the rat full pk studies , the formulations were filtered before the administration to rats . three rats were dosed intravenously ( 3 mg / kg ) , and three rats were administrated orally ( 10 mg / kg ) . six blood samples ( 100 l per sample ) were collected serially from each rat within 24 h post dosing . plasma samples ( 20 l ) were extracted with solution of acetonitrile : methanol ( 3:1 ) containing internal standard . the samples were vortexed and then centrifuged with an eppendorf centrifuge 5810r ( eppendorf , hamburg , germany ) at a setting of 4000 rpm for 5 min at 4 c . the supernatant was transferred to a clean 96-well analysis plate for lc / ms / ms analysis . an aliquot ( 10 l ) was injected onto a phenomenex c18 guard column ( 4 mm 2 mm ) followed by a zorbax sb - c8 analytical column ( 2.1 mm 30 mm , 3.5 m , agilent technologies inc . , separation was carried out using a gradient elution method with a mobile phase consisting of 0.05% formic acid in water ( solvent a ) and 0.05% formic acid in acetonitrile ( solvent b ) . the hplc system , consisting of agilent 1100 series binary pump ( agilent technologies inc . ) , agilent 1100 series micro vacuum degasser ( agilent technologies inc . ) , htc pal ctc analytics autosampler ( leap technologies , carborro , nc ) , and vici two - position actuator ( valco int . , houston , tx , usa ) was interfaced to a mds sciex api-4000 triple quadruple mass spectrometer ( mds inc . , mass spectral analyses were carried out using electrospray source in the positive ion mode and using multiple reaction monitoring ( mrm ) for quantification . mrm transition of compound 15 383.9/101.2 , compound 22 412.2/312.1 , compound 24 430.1/330.1 , compound 27 412.2/327.2 , compound 29 436.1/308.1 , compound 34 430.0/346.1 , and compound 36 355.0/272.3 was used with its optimized ms parameters , respectively . the mean value from the three animals at each time point was plotted against time to give plasma concentration time profile . pharmacokinetic parameters were determined using watson lims , version 7.2 ( thermo electron corporation , pa , usa ) , by noncompartmental analysis . pharmacokinetic parameters for compounds were calculated by noncompartmental regression analysis using an in - house fitting program developed at gnf . the oral bioavailability ( f ) was calculated as the ratio between the area under the curve ( auc ) following oral administration and the auc following intravenous administration corrected for dose ( f = aucpo doseiv / auciv dosepo ) . all in vivo efficacy studies were approved by the veterinary authorities of the canton basel - stadt . in vivo antimalarial activity was usually assessed for groups of three female nmri mice ( 2022 g ) intravenously infected on day zero with 2 10 erythrocytes parasitized with p. berghei gfp anka malaria strain ( pbgfpcon donation from a. p. waters and c. j. janse , leiden university ) . chloroquine and artesunate were formulated in 10% ethanol , 30% peg400 , and 6% vitamin e tpgs . compounds were administered orally in a volume of 10 ml / kg either as a single dose ( 24 h post infection ) or as three consecutive daily doses ( 24 , 48 , and 72 h post infection ) . with the single dose regimen , parasitemia was determined ( 72 h post infection ) and for the triple dose regimen ( 96 h post infection ) using standard flow cytometry techniques . activity was calculated as the difference between the mean percent parasitemia for the control and treated groups expressed as a percentage of the control group . the survival time in days was also recorded up to 30 days after infection . a compound was considered curative if the animal survived to day 30 after infection with no detectable parasites . unless otherwise noted , materials were obtained from commercial suppliers and were used without purification . removal of solvent under reduced pressure refers to distillation using bchi rotary evaporator attached to a vacuum pump ( 3 mmhg ) . products obtained as solids or high boiling oils were dried under vacuum ( 1 mmhg ) . purification of compounds by high pressure liquid chromatography was achieved using a waters 2487 series with ultra 120 5 m c18q column with a linear gradient from 10% solvent a ( acetonitrile with 0.035% trifluoroacetic acid ) in solvent b ( water with 0.05% trifluoroacetic acid ) to 90% a in 7.5 min , followed by 2.5 min elution with 90% a. h nmr spectra were recorded on bruker xwin - nmr ( 400 or 600 mhz ) . proton resonances are reported in parts per million ( ppm ) downfield from tetramethylsilane ( tms ) . h nmr data are reported as multiplicity ( s singlet , d doublet , t triplet , q quartet , quint quinted , sept septed , dd doublet of doublets , dt doublet of triplet , bs broad singlet ) , number of protons , and coupling constant in hertz . for spectra obtained in cdcl3 , dmso - d6 , and cd3od , the residual protons ( 7.27 , 2.50 , and 3.31 ppm , respectively ) were used as the reference . analytical thin - layer chromatography ( tlc ) was performed on commercial silica plates ( merck 60-f 254 , 0.25 mm thickness ) ; compounds were visualized by uv light ( 254 nm ) . 100c , isco ) or using silica gel ( merck kieselgel 60 , 230400 mesh ) . the purity and quantitative analysis were determined with a waters zq 2000 lc / ms system , which employed an acquity uplc system ( binary pump , column compartment , autosampler , and diode array uv detector ) . the eluent was split between the mass spectrometer and an antek chemiluminescent nitrogen detector ( clnd ) . the mobile phases used were ( a ) 95% h2o/5% meoh / ipa ( 75/25 , v / v ) + 0.05% formic acid and ( b ) meoh / ipa ( 75/25 , v / v ) + 0.035% formic acid . a gradient hplc method with flow of 0.4 ml / min started at 2% b , with a hold of 1.0 min before a linear increase to 95% b at 3.50 min , with a 30 s hold . from 4.04.25 min , the column used was a thermo syncronis c18 2.1 mm 30 mm , 3 m . the mass spectrometer was operated in positive mode , with a spray voltage of 3.2 kv and cone voltage of 30 v. the source and desolvation temperatures were 130 and 400 c , respectively , with 600 l / h of nitrogen desolvation flow . all the final compounds reported in this communication had the purity of at least 95% . elemental analyses were carried out by midwest microlabs llc , indianapolis , in , usa . compound 1a was prepared from 1 by the following method : to a stirred solution of 1 ( 12 mg , 0.03 mmol ) was added mno2 ( 52 mg , 0.60 mmol ) . the reaction mixture was stirred at room temperature ( rt ) for 2 h. lc / ms test showed that 1 was almost consumed and the desired product ( [ m + 1 ] = 428 ) was detected as one of the major peaks . the residue was subjected to a mass - triggered hplc purification to give 1a as well as 1b . h nmr for 1a ( 400 mhz , cd3od ) 7.597.63 ( m , 2h ) , 7.09 ( t , j = 6.4 hz , 2h ) , 6.957.02 ( m , 1h ) , 6.466.52 ( m , 1h ) , 6.346.38 ( m , 1h ) , 5.92 ( s , 1h ) , 4.33 ( dd , j = 4.4 , 14 hz , 1h ) , 3.96 ( dd , j = 4.8 , 13.2 hz , 1h ) , 3.703.78 ( m , 1h ) , 3.463.54 ( m , 1h ) , 1.73 ( s , 3h ) , 1.36 ( s , 3h ) . cd3od ) 7.757.78 ( m , 2h ) , 6.957.01 ( m , 3h ) , 6.446.50 ( m , 1h ) , 6.326.36 ( m , 1h ) , 4.19 ( dd , j = 1.6 , 6.8 hz , 2h ) , 4.00 ( dd , j = 1.6 , 6.8 hz , 2h ) , 1.47 ( s , 6h ) . to a solution of 2-bromo-1-(4-fluorophenyl)ethanone ( 46.5 g , 214.29 mmol ) in dmf ( 400 ml ) was added 2-(benzyloxycarbonyl)-2-methylpropanoic acid ( 55.8 g , 236.4 mmol ) and potassium carbonate ( 35.4 g , 256.5 mmol ) . the resulting solution was extracted with ethyl acetate ( 2 800 ml ) , and the combined organic layer was washed with water ( 2 800 ml ) and then brine ( 1 800 ml ) . this resulted in 67 g ( 84% ) of compound 4 as a white solid . to a solution of compound 4 ( 70 g , 187.7 mmol ) in toluene ( 700 ml ) was added nh4oac ( 144.5 g , 1.88 mol ) . the resulting solution was heated to reflux for 3 h in an oil bath . the resulting solution was extracted with ethyl acetate ( 2 500 ml ) , and the combined organic layer was washed with water ( 2 800 ml ) and brine ( 1 800 ml ) . this resulted in 58 g ( 88% ) of compound 5 as a white solid . h nmr ( 300 hz , dmso - d6 ) 11.91 ( s , nh ) , 7.807.75 ( m , 2h ) , 7.547.12 ( m , 8h ) , 4.97 ( m , 2h ) , 1.6 ( s , 6h ) . to a solution of compound 5 ( 58 g , 164.3 mmol ) in dmf ( 400 ml ) was added cesium carbonate ( 134 g , 411.0 mmol ) . this was followed by the addition of ethyl 2-bromoacetate ( 33 g , 197.6 mmol ) dropwise with stirring at rt in 30 min . the resulting solution was extracted with ethyl acetate ( 2 700 ml ) , and the combined organic layer was washed with water ( 2 800 ml ) and brine ( 1 800 ml ) . the resulting mixture was dried concentrated under vacuum . this resulted in 60 g ( 83% ) of compound 6 as a yellow solid . h nmr ( 300 hz , dmso - d6 ) 7.767.72 ( m , 2h ) , 7.327.28 ( m , 5h ) , 7.087.03 ( m , 3h ) , 5.08 ( m , 2h ) , 4.87 ( m , 2h ) , 4.23 ( q , j = 5.4 hz , 2h ) , 2.0 ( s , 6h ) , 1.28 ( t , j = 5.4 hz , 3h ) . to a solution of compound 6 ( 70 g , 159.45 mmol , 1.00 equiv ) in methanol ( 800 ml ) was added palladium on carbon ( 10 g ) . this resulted in 7 ( 38 g , 145.56 mmol , 91% ) as a white solid . lc - ms : ( es , m / z ) [ m + h ] calcd for c14h14fn3o , 260 ; found , 260 . h nmr ( 300 hz , dmso - d6 ) 7.797.74 ( m , 2h ) , 7.137.07 ( m , 3h ) , 6.35 ( s , 1h ) , 4.73 ( s , 2h ) , 1.79 ( s , 6h ) . to a stirred solution of compound 7 ( 5 g , 19.2 mmol , 1 equiv ) was dissolved in 50 ml of thf , 1 m borane / thf complex ( 57 ml , 57 mmol , 3 equiv ) was added slowly and reaction was refluxed overnight . the crude product of 8 ( 4.5 g , 18.3 mmol , 95% ) was used in the next step . lc - ms : ( es , m / z ) [ m + h ] calcd for c14h17fn3 246 , found 246 . h nmr ( dmso , 300 hz ) 7.757.70 ( m , 2h ) , 7.4 ( s , 1h ) , 7.14 ( t , j = 9.0 hz , 2h ) , 3.9 ( t , j = 5.4 hz , 2h ) , 2.51 ( t , j = 5.4 hz , 2h ) , 1.41(s , 6h ) ; nh proton not observed . to a stirred solution of compound 8 ( 2.9 g , 11.82 mmol , 1.1 equiv ) and 2-(tert - butoxycarbonylamino)acetic acid ( 2.27 g , 13 mmol , 1.1 equiv ) in 15 ml of dichloromethane were added diea ( 2.47 ml , 14.18 mmol , 1.2 equiv ) and hatu ( 5.39 g , 14.18 mmol , 1.2 equiv ) . the reaction mixture was stirred at rt for 8 h. hplc / ms analysis showed that desired product compound 9 was the major product . the organic layer was washed with washed with water ( 1 30 ml ) , followed by saturated nahco3 ( 1 30 ml ) and finally with brine ( 1 30 ml ) . the resulting oil was purified using column chromatography with hexanes / ethyl acetate ( 0100% linear gradient ) used as eluant . the desired product compound 9 was obtained as oil ( 3.3 g , 8.27 mmol , 70% ) . h nmr ( 300 hz , dmso - d6 , ) 7.777.72 ( m , 2h ) , 7.54 ( s , 1h ) , 7.207.14 ( m , 2h ) , 6.846.80 ( m , 1h ) , 4.07 ( s , 2h ) , 3.90 ( d , j = 3.0 hz , 2h ) , 3.70 ( s , 2h ) , 1.80 ( s , 6h ) , 1.40 ( s , 9h ) . lc - ms : ( es , m / z ) [ m + h ] calcd for c21h28fn4o3 , 403 ; found , 403 . to a stirred solution of compound 9 ( 3.02 g , 7.51 mmol ) in dichloromethane ( 30 ml ) was added br2 ( 0.43 ml , 8.26 mmol ) in acetic acid ( 3 ml ) . hplc / ms test showed that desired product ( ii ) was the only peak . the product was confirmed by 400 mhz proton nmr to be the title compound 10 . the product was used in the next step without further purification and exhibited quantitative mass recovery . lc - ms : ( es , m / z ) [ m + h ] calcd for c21h27brfn4o3 , 482 ; found , 482 . h nmr ( meoh - d4 , 400 hz ) 7.847.81 ( m , 2h ) , 7.14 ( t , j = 8.8 hz , 2h ) , 4.094.01 ( m , 4h ) , 3.81 ( t , j = 4.8 hz , 2h ) , 1.89 ( s , 6h ) , 1.46 ( s , 9h ) . ( s)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-methyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 17 was prepared from compound 10 ( r1 = me , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . h nmr ( meoh - d4 , 400 mhz ) 7.617.58 ( m , 2h ) , 7.167.12 ( m , 1h ) , 7.077.02 ( m , 2h ) , 6.476.42 ( m , 2h ) , 5.74 ( m , 1h ) , 4.073.68 ( m , 6h ) , 1.56 ( d , j = 6.8 hz , 3h ) . ( r)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-methyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 18 was prepared from compound 10 ( r1 = me , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . ( s)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 19 was prepared from compound 10 ( r1 = isopropyl , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . h nmr ( meoh - d4 , 400 mhz ) 7.617.58 ( m , 2h ) , 7.267.20 ( m , 3h ) , 6.456.38 ( m , 2h ) , 5.5 ( d , j = 7.9 hz , 1h ) , 4.113.75 ( m , 6h ) , 2.362.31 ( m , 1h ) , 1.15 ( d , j = 6.7 hz , 3h ) , 0.95 ( d , j = 6.7 hz , 3h ) . ( r)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 20 was prepared from compound 10 ( r1 = isopropyl , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . h nmr ( meoh - d4 , 400 mhz ) 7.697.67 ( m , 2h ) , 7.277.20 ( m , 3h ) , 6.686.65 ( dd , j = 2.5 hz , j = 11.0 hz , 1h ) , 6.606.55 ( dd , j = 2.25 hz , j = 8.7 hz , 1h ) , 5.78 ( d , j = 7.7 hz , 1h ) , 4.424.02 ( m , 4h ) , 3.943.87 ( m , 2h ) , 2.55 ( m,1h ) , 1.24(d , j = 6.8 hz , 3h ) , 1.05 ( d , j = 6.8 hz , 3h ) . m / z = 460.2 ( m + 1 ) . 2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 21 was prepared from compound 10 ( r1 = me , r2 = me ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . h nmr ( meoh - d4 , 400 mhz ) 7.597.55 ( m , 2h ) , 7.207.13 ( m , 3h ) , 6.60 ( dd , j = 11.2 , 10.8 hz ) , 1h ) , 6.53 ( dd , j = 2.4 , 0.8 hz , 1h ) , 4.0 ( m , 4h ) , 3.78 ( m , 2h ) , 2.00 ( s , 6h ) . elemental analysis ( compound + 2.0 hcl + 2.0 h2o ) : % c , 47.62 ; % h , 5.09 ; % n , 12.62 ; ( calc ) . % c = 47.54/47.38 ; % n = 12.44/12.46 ; % h = 4.62/4.57 ( experimental ) . 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : in a glass vial , cs2co3 , 4f - aniline ( 0.462 g , 4.1 mmol , 2.0 equiv ) , pd2(dba)3 ( 0.095 g , 0.104 mmol , 0.05 equiv ) , xantphos ( 0.120 g , 0.208 mmol , 0.1 equiv ) , and dioxane were stirred for 5 min at rt . compound 10 ( 1 g , 2.08 mmol , 1.0 equiv ) was added to the reaction mixture , after which the reaction mixture was degassed for 15 min and then stirred at 120 c under n2 for 8 h. hplc / ms test showed that the starting material compound 10 was consumed and desired product was formed predominantly along with some 9 . the reaction mixture was concentrated and then purified by normal phase column chromatography ( silica gel 80 g ) using a gradient of 1000% to 0100% hexane : etoac . the organic layer was concentrated at reduced pressure to yield the boc derivative ( 950 mg , 89% ) yield . lc - ms : ( es , m / z ) [ m + h ] calcd for c27h32f2n5o3 , 512 ; found , 512 . the boc compound was treated with 20% tfa in ch2cl2 ( 50 ml ) and was added to the mixture . after the completion of this reaction ( monitored by lcms ) , the resulting mixture was concentrated under reduced pressure . the resulting residue was purified by reverse phase hplc to yield product as a tfa salt . lc - ms : ( es , m / z ) [ m + h ] calcd for c22h23f2n5o , 412.2 ; found , 412.1 . h nmr ( meoh - d4 , 400 hz ) 7.617.57 ( m , 2h ) , 6.94 ( t , j = 8.8 hz , 2h ) , 6.81 ( t , j = 8.8 hz , 2h ) , 6.47 ( m , 2h ) , 3.72 ( m , 2h ) , 3.58 ( m , 2h ) , 3.42 ( m , 2h ) , 1.85 ( s , 6h ) . elemental analysis compound 22 with 0.65 equiv h2o : c , 62.44 ; n , 16.55 ; h , 5.79 ( calculated ) . c = 62.54/62.44 ; n = 16.35/16.29 ; h = 5.52/5.61 ( experimental ) . the regiochemistry of the dime groups was unambiguously assigned using 2-d nmr techniques ( table 8) . 2-amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 23 was prepared from compound 10 ( r1 = me , r2 = me ) by a pd2(dba)3 mediated amination reaction with 4-methylaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . h nmr ( meoh - d4 , 400 mhz ) 7.797.69 ( m , 2h ) , 7.21 ( t , j = 8.4 hz , 2h ) , 7.04 ( d , j = 8.0 hz , 2h ) , 6.76 ( d , j = 8.2 hz , 2h ) , 4.11 ( s , 2h ) , 3.88 ( m , 4h ) , 2.22 ( s , 3h ) , 2.12 ( s , 6h ) . lc / ms major mass : 408.1 ( m + h ) . 2-amino-1-(3-((3,4-difluorophenyl)amino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 24 was prepared from compound 10 ( r1= me , r2 = me ) by a pd2(dba)3 mediated amination reaction with 3,4-difluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . h nmr ( meoh - d4 , 400 mhz ) 7.737.69 ( m , 2h ) , 7.21 ( t , j = 8.8 hz , 2h ) , 7.067.03 ( m , 1h ) , 6.766.65 ( m , 1h ) , 6.536.51 ( m , 1h ) , 4.084.06 ( m , 4h ) , 3.863.83 ( m , 2h ) , 2.07 ( s , 6h ) . elemental analysis ( compound + 2.9 hcl ) : % c , 49.37 ; % h , 4.69 ; % n , 13.09 ; ( calc ) . % c = 49.43/49.06 ; % n = 12.89/12.77 ; % h = 4.7/4.81 ( experimental ) . 13 was prepared from compound 5a by the following way : to a stirred solution of compound 5a ( 1.1 g , 3.38 mmol , 1.00 equiv ) in dmf ( 30 ml ) was added 3-chloro-2-methylprop-1-ene ( 500 mg , 5.49 mmol , 1.50 equiv ) , potassium carbonate ( 560 mg , 4.06 mmol , 1.10 equiv ) , and potassium iodide ( 1.12 g , 6.75 mmol , 2.00 equiv ) at rt . the mixture was washed with brine ( 3 10 ml ) , dried over sodium sulfate , and concentrated under vacuum . the residue was applied onto a silica gel column with petroleum ether / etoac ( 5:1 ) to give 0.7 g ( 55% ) of compound 5a-1 as a light - yellow solid . m / z = 380 ( m + 1 ) . into a 30 ml sealed tube , was placed compound 5a-1 ( 2.0 g , 5.28 mmol , 1.00 equiv ) , acetic acid ( 12 ml ) , and methanesulfonic acid ( 2 ml ) . the reaction mixture was stirred for 12 h at 260 c ( the temperature of the sand bath ) . the aqueous layer was washed with ethyl acetate ( 3 10 ml ) . the aqueous layer was extracted with ethyl acetate ( 3 10 ml ) . the combined organic layers were washed with brine ( 3 10 ml ) , dried over sodium sulfate , and concentrated under vacuum . the solid was collected by filtration and washed with 5 ml of n - hexane to give 500 mg ( 39% ) of compound 5a-2 as a white solid . h nmr ( 400 mhz , cdcl3 ) 7.707.65 ( m , 2h ) , 7.046.9 ( m , 2h ) , 6.96 ( s , 1h ) , 4.12 ( s , 2h ) , 3.69 ( s , 2h ) , 1.23 ( s , 6h ) . to a stirred solution of compound 5a-2 ( 280 mg , 1.14 mmol , 1.0 equiv ) in dmf ( 10 ml ) was added 2-(tert - butoxycarbonyl)acetic acid ( 600 mg , 3.43 mmol , 3.0 equiv ) , hatu ( 1.3 g , 3.42 mmol , 3.0 equiv ) , and diea ( 880 mg , 6.82 mmol , 6.0 equiv ) at rt . the organic layer was washed with brine ( 3 10 ml ) , dried over sodium sulfate , and concentrated under vacuum . the residue was applied onto a silica gel column with ch2cl2/meoh ( 10:1 ) to give 280 mg ( 57% ) of compound 13 as a brown solid . h nmr ( 400 mhz , cdcl3 ) 7.727.68 ( m , 2h ) , 7.23 ( s , 1h ) , 7.137.07 ( m , 2h ) , 5.46 ( s , nh ) , 4.65 ( s , 2h ) , 4.054.04 ( m , 2h ) , 3.97 ( s , 2h ) , 1.551.39 ( m , 15h ) . the structure of 13 was verified by hmqc , cosy , hmbc , and roesy ( table 9 ) . 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-6,6-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : to a stirred solution of compound 13 ( 386 g , 0.96 mmol ) in 6 ml of dichloromethane was added br2 ( 55 l , 1.06 mmol ) in acetic acid ( 2 ml ) . after neutralization , the residue was subjected to flash chromatography ( 40 g , 0100% ethyl acetate in hexane , 50 min , dry loading ) purification to give 256 mg ( 55% ) of the title compound 13a as a colorless oil . h nmr ( 400 mhz , cdcl3 ) 7.877.83 ( m , 2h ) , 7.03 ( t , j = 8.8 hz , 2h ) , 5.46 ( s , 1h ) , 4.5 ( s , 2h ) , 3.97 ( d , j = 4.2 hz , 2h ) , 3.81 ( s , 2h ) , 1.45 ( s , 6h ) , 1.39 ( s , 9h ) . example 25was obtained from 13a by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a tfa mediated deprotection by analogy to compound 22 ( 55% yield over 2 steps ) . h nmr ( 400 mhz , dmso - d6 ) : 8.06 ( m , 2h ) , 7.98 ( m , 1h ) , 7.80 ( m , 2h ) , 7.16 ( m , 2h ) , 6.98 ( m , 2h ) , 6.60 ( m , 2h ) , 4.64 ( m , 2h ) , 4.02 ( m , 2h ) , 3.80 ( m , 2h ) , 1.38 ( s , 6h ) . 2-(4-fluorophenyl)-7-(4-methoxybenzyl)-5,5-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine : compound 7b was prepared from compound 7a ( r1 = r2 = h ) by the following way : to a solution of compound 7a ( 231 mg , 1.0 mmol ) in dmf ( 10 ml ) were added koh ( 168 mg , 3.0 mmol ) and pmbcl ( 405 l , 3.0 mmol ) at 0 c . the reaction mixture was stirred at the same temperature for 2 h and at rt for 2 additional hours . the hplc fraction were evaporated under reduced pressure and neutralized by satd nahco3 . the organic layer was concentrated under reduced pressure to give compound 7a-1 ( 221 mg , 63% ) as a white solid . h nmr ( 400 mhz , cdcl3 ) 7.677.63 ( m , 2h ) , 7.287.25 ( m , 2h ) , 7.077.02 ( m , 2h ) , 6.896.86 ( m , 2h ) , 4.74 ( s , 2h ) , 4.70 ( m , 2h ) , 4.53 ( s , 2h ) , 3.79 ( s , 3h ) . to a solution of compound 7a-1 ( 253 mg , 0.72 mmol ) in dmf ( 15 ml ) were added 60% sodium hydride ( 87 mg , 0.085 mmol ) and methyl iodide ( 0.45 ml , 7.2 mmol ) at rt . the reaction mixture was stirred at rt for 2 h. the reaction mixture was quenched with methanol and directly subjected to mass - triggered hplc purification to give 7a-2 as a white solid after evaporation of acetonitrile water in vacuo . the tfa salt was subsequently neutralized by saturated sodium bicarbonate solution and extracted with dichloromethane to get the compound as a free base . the organic layer was dried over sodium sulfate and concentrated under reduced pressure to yield the desired compound 7a-2 ( 234 mg , 86% ) . the structure of 7a-2 was verified by 2-d nmr techniques ( table 10 ) . to a solution of compound 7a-2 ( 170 mg , 0.45 mmol ) in thf ( 9 ml ) was added 1.0 n bh3thf ( 2.70 ml , 2.70 mmol ) at rt . the reaction mixture was stirred at reflux for 2 h. pd / c was added ( gas generated ) . the reaction mixture was stirred for 1 h. solid was filtered off , and solvent was removed . the product was assumed to be of 100% yield and used in the next step without further purification . h nmr ( 400 mhz , cd3od ) 8.1 ( s , 1h ) , 7.747.70 ( m , 2h ) , 7.34 ( d , j = 8.6 hz , 2h ) , 7.27 ( m , 2h ) , 6.94 ( d , j = 8.6 hz , 2h ) , 3.91 ( s , 2h ) , 3.80 ( s , 3h ) , 3.79 ( s , 2h ) , 2.87 ( s , 2h ) , 1.61 ( s , 6h ) . 7-(4-methoxybenzyl)-2-(4-fluorophenyl)-5,5-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine ( 1.07 g , 2.93 mmol , 1.00 equiv ) ( 7b ) was added to trifluoroacetic acid ( 15 ml ) . the resulting solution was stirred for 2 h at 70 c in an oil bath . the ph value of the solution was adjusted to 8 with aqueous sodium bicarbonate . 7b-1 was extracted from the aqueous layer using 3 100 ml of ethyl acetate . this resulted in 7b-1 ( 718 mg , quantitative ) as a yellow solid . to a solution of 7b-1 ( 750 mg , 3.06 mmol , 1.00 equiv ) in n , n - dimethylformamide ( 30 ml ) were added 2-(tert - butoxycarbonyl)acetic acid ( 850 mg , 4.86 mmol , 1.50 equiv ) , hatu ( 1.3 g , 3.42 mmol , 1.10 equiv ) , and triethylamine ( 650 mg , 6.44 mmol , 2.00 equiv ) . the resulting solution was stirred overnight at rt . the resulting mixture was washed with brine ( 3 100 ml ) , dried over anhydrous sodium sulfate , and concentrated under vacuum . the residue was applied onto a silica gel column with petroleum ether / ethyl acetate ( 4:1 ) . this resulted in 600 mg ( 49% ) of 7c as a pale - yellow solid . h nmr ( 300 mhz , cdcl3 ) : 7.75 ( m , 2h ) , 7.22 ( s , 1h ) , 7.137.07 ( m , 2h ) , 4.994.87 ( m , 2h ) , 4.12 ( s , 2h ) , 3.91 3.65 ( m , 2h ) , 1.57 ( m , 6h ) , 1.43 ( s , 9h ) . to a stirred solution of compound 7b-2 ( 583 g , 1.45 mmol ) in dichloromethane ( 6 ml ) was added br2 ( 82 l , 1.60 mmol ) in acetic acid ( 2 ml ) . after neutralization , the residue was subjected to flash chromatography ( 40 g , 0100% ethyl acetate in hexane , 50 min , dry loading ) purification to give 628 mg ( 90% ) of the title compound 7c as a yellow solid . h nmr ( 400 mhz , cdcl3 ) 7817.78 ( m , 2h ) , 7.07 ( t , j = 8.6 hz , 2h ) , 5.28 ( s , nh ) , 4.844.7 ( m , 2h ) , 4.073.58 ( m , 4h ) , 1.491.44 ( m , 6h ) , 1.28 ( s , 9h ) . 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-5,5-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 26 was prepared from compound 7c by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a tfa mediated deprotection in 46% yield over 2 steps . h nmr ( 400 mhz , cd3od ) : 7.57 ( m , 2h ) , 7.02 ( m , 2h ) , 6.77 ( m , 2h ) , 6.52 ( m , 2h ) , 4.974.91 ( m , 2h ) , 4.093.74 ( m , 4h ) , 1.56 ( s , 3h ) , 1.51 ( s , 3h ) . 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 28 was prepared from compound 8 ( r1 = r2 = me ) in the following fashion using a slightly modified route to the one described in scheme 2 . to a stirred solution of compound 8 ( 1.48 g , 6.04 mmol ) and net3 ( 6.0 g , 59.4 mmol ) in dichloromethane ( 20 ml ) was added 2-bromo-2-methylpropanoyl bromide ( 14 g , 60.9 mmol ) dropwise at rt . after being stirred for 3 h at rt , the reaction was then quenched by the addition of water ( 30 ml ) . the combined organic layers were washed with brine , dried over anhydrous sodium sulfate , and concentrated under vacuum . this resulted in crude product as a dark solid , which was washed with etoac : petroleum ether ( 1:10 ) to remove the impurities to produce compound 8 - 1 as a gray solid . ( 2.0 g , 5.08 mmol , 1.00 equiv ) in dmf ( 10 ml ) was nan3 ( 1.0 g , 15.38 mmol , 3.00 equiv ) at rt . the resulting mixture was washed with 3 20 ml of brine , dried over anhydrous sodium sulfate , and concentrated under vacuum . the residue was applied onto a silica gel column with petroleum ether / etoac ( 5:1 ) to give compound 8 - 2 as a white solid . m / z 357 ( m + h ) . to a stirred solution of compound 8 - 2 ( 1.2 g , 3.37 mmol , 1.00 equiv ) in methanol ( 20 ml ) was added pd / c ( 80 mg , 0.75 mmol , 0.20 equiv ) at rt . m / z 331 ( m + h ) . to a stirred solution of compound 8 - 3 ( 910 mg , 2.76 mmol , 1.00 equiv ) in thf ( 50 ml ) was added boc anhydride ( 3.2 g , 14.68 mmol , 5.00 equiv ) , followed by aqueous naoh ( 1n , 6 ml , 2.0 equiv ) at rt . the organic layer was washed with 3 10 ml of brine , dried over na2so4 , and concentrated under vacuum . the solid was collected by filtration and washed with n - hexane ( 5 ml ) to give compound 8 - 4 as a white solid . h nmr ( 300 mhz , cdcl3 ) 7.757.71 ( m , 2h ) , 7.097.02 ( m , 3h ) , 4.84(s , nh ) , 4.094.0 ( m , 4h ) , 1.98 ( s , 6h ) , 1.55 ( s , 6h ) , 1.45 ( s , 9h ) . to a stirred solution of compound 8 - 4 ( 32 mg , 0.074 mmol ) in dichloromethane ( 3 ml ) was added br2 ( 4.2 l , 0.082 mmol ) in acetic acid ( 1 ml ) . after neutralization , the residue was subjected to flash chromatography ( 4 g , 060% ethyl acetate in hexane , 16 min ) purification to give the compound 8 - 5 as colorless oil , was collected by filtration , and washed with n - hexane ( 5 ml ) to give compound 8 - 4 as a white solid . h nmr ( 400 mhz , cdcl3 ) 7.917.88 ( m , 2h ) , 7.107.04 ( m , 2h ) , 5.11(s , nh ) , 4.01 ( m , 2h ) , 3.96 ( s , 2h ) , 1.94 ( s , 6h ) , 1.51 ( s , 6h ) , 1.41 ( s , 9h ) . compound 28 was prepared from compound 8 - 5 by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a tfa mediated deprotection . h nmr ( 400 mhz , meoh - d4 ) 7.66 ( dd , j = 9.2 , 5.2 hz , 2h ) , 7.11 ( t , j = 8.8 hz , 2h ) , 6.81 ( t , j = 8.8 hz , 2h ) , 6.726.68 ( m , 2h ) , 4.043.99 ( m , 4h ) , 2.01 ( s , 6h ) , 1.66 ( s , 6h ) . 2-amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 29 was prepared from 35 by the following way : to a stirred solution of 35 ( 21 mg , 0.06 mmol ) and et3n ( 83 l , 0.60 mmol ) in dry dichloromethane ( 6 ml ) was added 2-bromo-2-methylpropanoyl bromide ( 71 l , 0.60 mmol ) . the reaction mixture was stirred at rt for 5 h. the reaction mixture was concentrated and subjected to mass - triggered lc / ms purification directly . the obtained solution was concentrated to give 22 mg ( 73% ) of compound 35 - 1as yellow oil after neutralization . h nmr ( 400 mhz , meoh - d4 ) 7.717.66 ( m , 2h ) , 7.237.19 ( m , 2h ) , 7.02 ( d , j = 8.4 hz , 2h ) , 6.66 ( d , j = 8.4 hz , 2h ) , 4.31 ( m , 2h ) , 4.09 ( m , 2h ) , 2.21 ( s , 3h ) , 2.06 ( s , 6h ) , 2.00 ( s , 6h ) . to a solution of compound 35 - 1 ( 22 mg , 0.044 mmol ) in dmf ( 3 ml ) was added nan3 ( 8.6 mg , 0.132 mmol ) at rt . the reaction mixture was stirred at rt for 2 h. the reaction mixture was directly subjected to mass - triggered hplc purification directly . the obtained mecn / aqueous solution was combined and concentrated to give 15 mg ( 75% ) of compound 35 - 2 as yellow oil after neutralization . h nmr ( 400 mhz , meoh - d4 ) 7.70 7.66 ( m , 2h ) , 7.237.19 ( m , 2h ) , 7.04 ( d , j = 8.3 hz , 2h ) , 6.66 ( d , j = 8.4 hz , 2h ) , 4.26 ( m , 2h ) , 4.074.05 ( m , 2h ) , 2.24 ( s , 3h ) , 2.03 ( s , 6h ) , 1.58 ( s , 6h ) . to a solution of compound 35 - 2 ( 15 mg , 0.033 mmol ) in meoh ( 3 ml ) was added 10% pd / c ( 4 mg , 0.003 mmol ) at rt . the reaction mixture was stirred at rt for 2 h. solid was filtered off , and solvent was removed . the reaction mixture was directly subjected to mass - triggered hplc purification to give 15 mg ( 100% ) of the title compound yellow oil . h nmr ( meoh - d4 , 400 hz ) 7.787.75 ( m , 2h ) , 7.066.97 ( m , 4h ) , 6.56 ( d , j = 8.4 hz , 2h ) , 5.16 ( s , nh ) , 4.33 ( t , j = 4.8 hz , 2h ) , 3.74 ( t , j = 4.8 hz , 2h ) , 2.29 ( s , 3h ) , 1.95 ( s , 6h ) , 1.45 ( s , 6h ) . 2-amino-1-(3-(3,4-difluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 30 was prepared from compound 8 - 5 by a pd2(dba)3 mediated amination reaction with 3,4-difluoroaniline followed by a tfa mediated deprotection.h nmr ( 400 mhz , meoh - d4 ) 7.637.60 ( m , 2h ) , 7.14 ( t , j = 8.4 hz , 2h ) , 7.01 ( dd , j = 18.8 , 8.8 hz , 1h ) , 6.686.63 ( m , 1h ) , 6.5 ( m , 1h ) , 4.03 ( m , 2h ) , 4.01 ( m , 2h ) , 2.01 ( s , 6h ) , 1.66 ( s , 6h ) . 2-(4-fluorophenyl)-n-(p - tolyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : the details of the synthesis involving an ugi reaction between 4-fluorobenzaledehyde , 2-aminopyrazine , and 1-isocyano-4-methylbenzene has been reported in the previous communication.h nmr ( 400 mhz , meoh - d4 ) 7.73 ( d , j = 6.8 hz , 2h ) , 7.006.99 ( m , 4h ) , 6.49 ( d , j = 8.0 hz , 2h ) , 4.01 ( s , 2h ) , 3.72 ( m , 2h ) , 3.15 ( m , 2h ) , 2.19 ( s , 3h ) . 2-(4-fluorophenyl)-3-(p - tolylamino)-7,8-dihydroimidazo[1,2-a]pyrazin-6(5h)-one : compound 32 was synthesized from compound 7 ( r1 = r2 = h ) pd2(dba)3 mediated coupling reaction with p - toluidine . h nmr ( 400 mhz , dmso - d6 ) 8.55 ( s , 1h ) , 7.777.75 ( m , 2h ) , 7.267.14 ( m , 4h ) , 7.016.95 ( m , 2h ) , 6.53 ( m , 1h ) , 4.53 ( m , 2h ) , 4.22 ( m , 2h ) , 2.17 ( s , 3h ) . 2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-7,8-dihydroimidazo[1,2-a]pyrazin-6(5h)-one : compound 33a was prepared from 7 by the following way : to a stirred solution of compound 7 ( 390 mg , 1.51 mmol , 1.00 equiv ) in dichloromethane ( 20 ml ) was added nbs ( 0.28 g , 1.00 equiv ) . the solid was filtered out , and the mixture was washed with a saturated solution of na2s2o3 and dried over na2so4 . the solids was purified by silica gel chromatography ( petroleum ether / etoac = 1:2 ) to result in 432 mg ( 85% ) of the title compound as a white solid . h nmr ( 300 mhz , dmso - d6 ) 8.76 ( s , 1h ) , 7.947.89 ( m , 2h ) , 7.317.25 ( m , 2h ) , 4.57 ( s , 2h ) , 1.56 ( s , 6h ) . compound 33 was prepared from compound 33a by a pd2(dba)3 mediated amination reaction with p - toluidine . h nmr ( meoh - d4 , 400 hz ) 7.627.58 ( m , 2h ) , 7.05 ( t , j = 8.4 hz , 2h ) , 6.92 ( d , j = 8.4 hz , 2h ) , 6.52 ( d , j = 8.4 hz , 2h ) , 4.42 ( s , 2h ) , 2.12 ( s , 3h ) , 1.71 ( s , 6h ) . 3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-7,8-dihydro imidazo[1,2-a]pyrazin-6(5h)-one : compound 34 was prepared from compound 7b by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline . h nmr ( meoh - d4 , 400 hz ) 7.747.70 ( m , 2h ) , 7.21 ( t , j = 8.4 hz , 1h ) , 7.08 ( m , 2h ) , 6.496.40 ( m , 2h ) , 4.43 ( s , 2h ) , 1.72 ( s , 6h ) . 2-(4-fluorophenyl)-8,8-dimethyl - n - p - tolyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : compound 35 was prepared from compound 35 - 1 ( shown below and prepared by analogy to compound 9 and using cbz - glycine ) by a pd2(dba)3 mediated amination reaction with p - toluidine followed by a 6n hcl mediated hydrolysis . h nmr ( meoh - d4 , 400 hz ) 7.787.75 ( m , 2h ) , 7.066.97 ( m , 4h ) , 6.56 ( d , j = 8.4 hz , 2h ) , 5.16 ( s , nh ) , 4.33 ( t , j = 4.8 hz , 2h ) , 3.74 ( t , j = 4.8 hz , 2h ) , 2.29 ( s , 3h ) , 1.95 ( s , 6h ) . n,2-bis(4-fluorophenyl)-8,8-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : compound 36 was prepared from compound 35 - 1 by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a 6n hcl mediated hydrolysis . h nmr ( 400 mhz , meoh - d4 ) 7.627.58 ( m , 2h ) , 7.127.08 ( m , 2h ) , 6.686.63 ( m , 2h ) , 6.67 ( m , 2h ) , 4.16 ( m , 2h ) , 3.78 ( m , 2h ) , 1.92 ( s , 6h ) . unless otherwise noted , materials were obtained from commercial suppliers and were used without purification . removal of solvent under reduced pressure refers to distillation using bchi rotary evaporator attached to a vacuum pump ( 3 mmhg ) . products obtained as solids or high boiling oils were dried under vacuum ( 1 mmhg ) . purification of compounds by high pressure liquid chromatography was achieved using a waters 2487 series with ultra 120 5 m c18q column with a linear gradient from 10% solvent a ( acetonitrile with 0.035% trifluoroacetic acid ) in solvent b ( water with 0.05% trifluoroacetic acid ) to 90% a in 7.5 min , followed by 2.5 min elution with 90% a. h nmr spectra were recorded on bruker xwin - nmr ( 400 or 600 mhz ) . proton resonances are reported in parts per million ( ppm ) downfield from tetramethylsilane ( tms ) . h nmr data are reported as multiplicity ( s singlet , d doublet , t triplet , q quartet , quint quinted , sept septed , dd doublet of doublets , dt doublet of triplet , bs broad singlet ) , number of protons , and coupling constant in hertz . for spectra obtained in cdcl3 , dmso - d6 , and cd3od , the residual protons ( 7.27 , 2.50 , and 3.31 ppm , respectively ) analytical thin - layer chromatography ( tlc ) was performed on commercial silica plates ( merck 60-f 254 , 0.25 mm thickness ) ; compounds were visualized by uv light ( 254 nm ) . 100c , isco ) or using silica gel ( merck kieselgel 60 , 230400 mesh ) . the purity and quantitative analysis were determined with a waters zq 2000 lc / ms system , which employed an acquity uplc system ( binary pump , column compartment , autosampler , and diode array uv detector ) . the eluent was split between the mass spectrometer and an antek chemiluminescent nitrogen detector ( clnd ) . the mobile phases used were ( a ) 95% h2o/5% meoh / ipa ( 75/25 , v / v ) + 0.05% formic acid and ( b ) meoh / ipa ( 75/25 , v / v ) + 0.035% formic acid . a gradient hplc method with flow of 0.4 ml / min started at 2% b , with a hold of 1.0 min before a linear increase to 95% b at 3.50 min , with a 30 s hold . from 4.04.25 min , the column used was a thermo syncronis c18 2.1 mm 30 mm , 3 m . the mass spectrometer was operated in positive mode , with a spray voltage of 3.2 kv and cone voltage of 30 v. the source and desolvation temperatures were 130 and 400 c , respectively , with 600 l / h of nitrogen desolvation flow . all the final compounds reported in this communication had the purity of at least 95% . elemental analyses were carried out by midwest microlabs llc , indianapolis , in , usa . compound 1a was prepared from 1 by the following method : to a stirred solution of 1 ( 12 mg , 0.03 mmol ) was added mno2 ( 52 mg , 0.60 mmol ) . the reaction mixture was stirred at room temperature ( rt ) for 2 h. lc / ms test showed that 1 was almost consumed and the desired product ( [ m + 1 ] = 428 ) was detected as one of the major peaks . the residue was subjected to a mass - triggered hplc purification to give 1a as well as 1b . h nmr for 1a ( 400 mhz , cd3od ) 7.597.63 ( m , 2h ) , 7.09 ( t , j = 6.4 hz , 2h ) , 6.957.02 ( m , 1h ) , 6.466.52 ( m , 1h ) , 6.346.38 ( m , 1h ) , 5.92 ( s , 1h ) , 4.33 ( dd , j = 4.4 , 14 hz , 1h ) , 3.96 ( dd , j = 4.8 , 13.2 hz , 1h ) , 3.703.78 ( m , 1h ) , 3.463.54 ( m , 1h ) , 1.73 ( s , 3h ) , 1.36 ( s , 3h ) . h nmr for 1b ( 400 mhz , cd3od ) 7.757.78 ( m , 2h ) , 6.957.01 ( m , 3h ) , 6.446.50 ( m , 1h ) , 6.326.36 ( m , 1h ) , 4.19 ( dd , j = 1.6 , 6.8 hz , 2h ) , 4.00 ( dd , j = 1.6 , 6.8 hz , 2h ) , 1.47 ( s , 6h ) . to a solution of 2-bromo-1-(4-fluorophenyl)ethanone ( 46.5 g , 214.29 mmol ) in dmf ( 400 ml ) was added 2-(benzyloxycarbonyl)-2-methylpropanoic acid ( 55.8 g , 236.4 mmol ) and potassium carbonate ( 35.4 g , 256.5 mmol ) . the resulting solution was extracted with ethyl acetate ( 2 800 ml ) , and the combined organic layer was washed with water ( 2 800 ml ) and then brine ( 1 800 ml ) . this resulted in 67 g ( 84% ) of compound 4 as a white solid . to a solution of compound 4 ( 70 g , 187.7 mmol ) in toluene ( 700 ml ) was added nh4oac ( 144.5 g , 1.88 mol ) . the resulting solution was heated to reflux for 3 h in an oil bath . the resulting solution was extracted with ethyl acetate ( 2 500 ml ) , and the combined organic layer was washed with water ( 2 800 ml ) and brine ( 1 800 ml ) . this resulted in 58 g ( 88% ) of compound 5 as a white solid . h nmr ( 300 hz , dmso - d6 ) 11.91 ( s , nh ) , 7.807.75 ( m , 2h ) , 7.547.12 ( m , 8h ) , 4.97 ( m , 2h ) , 1.6 ( s , 6h ) . to a solution of compound 5 ( 58 g , 164.3 mmol ) in dmf ( 400 ml ) was added cesium carbonate ( 134 g , 411.0 mmol ) . this was followed by the addition of ethyl 2-bromoacetate ( 33 g , 197.6 mmol ) dropwise with stirring at rt in 30 min . the resulting solution was extracted with ethyl acetate ( 2 700 ml ) , and the combined organic layer was washed with water ( 2 800 ml ) and brine ( 1 800 ml ) . the resulting mixture was dried concentrated under vacuum . this resulted in 60 g ( 83% ) of compound 6 as a yellow solid . h nmr ( 300 hz , dmso - d6 ) 7.767.72 ( m , 2h ) , 7.327.28 ( m , 5h ) , 7.087.03 ( m , 3h ) , 5.08 ( m , 2h ) , 4.87 ( m , 2h ) , 4.23 ( q , j = 5.4 hz , 2h ) , 2.0 ( s , 6h ) , 1.28 ( t , j = 5.4 hz , 3h ) . to a solution of compound 6 ( 70 g , 159.45 mmol , 1.00 equiv ) in methanol ( 800 ml ) was added palladium on carbon ( 10 g ) . this resulted in 7 ( 38 g , 145.56 mmol , 91% ) as a white solid . lc - ms : ( es , m / z ) [ m + h ] calcd for c14h14fn3o , 260 ; found , 260 . h nmr ( 300 hz , dmso - d6 ) 7.797.74 ( m , 2h ) , 7.137.07 ( m , 3h ) , 6.35 ( s , 1h ) , 4.73 ( s , 2h ) , 1.79 ( s , 6h ) . to a stirred solution of compound 7 ( 5 g , 19.2 mmol , 1 equiv ) was dissolved in 50 ml of thf , 1 m borane / thf complex ( 57 ml , 57 mmol , 3 equiv ) was added slowly and reaction was refluxed overnight . the crude product of 8 ( 4.5 g , 18.3 mmol , 95% ) was used in the next step . lc - ms : ( es , m / z ) [ m + h ] calcd for c14h17fn3 246 , found 246 . h nmr ( dmso , 300 hz ) 7.757.70 ( m , 2h ) , 7.4 ( s , 1h ) , 7.14 ( t , j = 9.0 hz , 2h ) , 3.9 ( t , j = 5.4 hz , 2h ) , 2.51 ( t , j = 5.4 hz , 2h ) , 1.41(s , 6h ) ; nh proton not observed . to a stirred solution of compound 8 ( 2.9 g , 11.82 mmol , 1.1 equiv ) and 2-(tert - butoxycarbonylamino)acetic acid ( 2.27 g , 13 mmol , 1.1 equiv ) in 15 ml of dichloromethane were added diea ( 2.47 ml , 14.18 mmol , 1.2 equiv ) and hatu ( 5.39 g , 14.18 mmol , 1.2 equiv ) . the reaction mixture was stirred at rt for 8 h. hplc / ms analysis showed that desired product compound 9 was the major product . the organic layer was washed with washed with water ( 1 30 ml ) , followed by saturated nahco3 ( 1 30 ml ) and finally with brine ( 1 30 ml ) . the resulting oil was purified using column chromatography with hexanes / ethyl acetate ( 0100% linear gradient ) used as eluant . the desired product compound 9 was obtained as oil ( 3.3 g , 8.27 mmol , 70% ) . h nmr ( 300 hz , dmso - d6 , ) 7.777.72 ( m , 2h ) , 7.54 ( s , 1h ) , 7.207.14 ( m , 2h ) , 6.846.80 ( m , 1h ) , 4.07 ( s , 2h ) , 3.90 ( d , j = 3.0 hz , 2h ) , 3.70 ( s , 2h ) , 1.80 ( s , 6h ) , 1.40 ( s , 9h ) . lc - ms : ( es , m / z ) [ m + h ] calcd for c21h28fn4o3 , 403 ; found , 403 . to a stirred solution of compound 9 ( 3.02 g , 7.51 mmol ) in dichloromethane ( 30 ml ) was added br2 ( 0.43 ml , 8.26 mmol ) in acetic acid ( 3 ml ) . hplc / ms test showed that desired product ( ii ) was the only peak . the product was confirmed by 400 mhz proton nmr to be the title compound 10 . the product was used in the next step without further purification and exhibited quantitative mass recovery . lc - ms : ( es , m / z ) [ m + h ] calcd for c21h27brfn4o3 , 482 ; found , 482 . h nmr ( meoh - d4 , 400 hz ) 7.847.81 ( m , 2h ) , 7.14 ( t , j = 8.8 hz , 2h ) , 4.094.01 ( m , 4h ) , 3.81 ( t , j = 4.8 hz , 2h ) , 1.89 ( s , 6h ) , 1.46 ( s , 9h ) . ( s)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-methyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 17 was prepared from compound 10 ( r1 = me , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . h nmr ( meoh - d4 , 400 mhz ) 7.617.58 ( m , 2h ) , 7.167.12 ( m , 1h ) , 7.077.02 ( m , 2h ) , 6.476.42 ( m , 2h ) , 5.74 ( m , 1h ) , 4.073.68 ( m , 6h ) , 1.56 ( d , j = 6.8 hz , 3h ) . r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . ( s)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 19 was prepared from compound 10 ( r1 = isopropyl , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . h nmr ( meoh - d4 , 400 mhz ) 7.617.58 ( m , 2h ) , 7.267.20 ( m , 3h ) , 6.456.38 ( m , 2h ) , 5.5 ( d , j = 7.9 hz , 1h ) , 4.113.75 ( m , 6h ) , 2.362.31 ( m , 1h ) , 1.15 ( d , j = 6.7 hz , 3h ) , 0.95 ( d , j = 6.7 hz , 3h ) . ( r)-2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8-isopropyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 20 was prepared from compound 10 ( r1 = isopropyl , r2 = h ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . h nmr ( meoh - d4 , 400 mhz ) 7.697.67 ( m , 2h ) , 7.277.20 ( m , 3h ) , 6.686.65 ( dd , j = 2.5 hz , j = 11.0 hz , 1h ) , 6.606.55 ( dd , j = 2.25 hz , j = 8.7 hz , 1h ) , 5.78 ( d , j = 7.7 hz , 1h ) , 4.424.02 ( m , 4h ) , 3.943.87 ( m , 2h ) , 2.55 ( m,1h ) , 1.24(d , j = 6.8 hz , 3h ) , 1.05 ( d , j = 6.8 hz , 3h ) . 2-amino-1-(3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 21 was prepared from compound 10 ( r1 = me , r2 = me ) by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . h nmr ( meoh - d4 , 400 mhz ) 7.597.55 ( m , 2h ) , 7.207.13 ( m , 3h ) , 6.60 ( dd , j = 11.2 , 10.8 hz ) , 1h ) , 6.53 ( dd , j = 2.4 , 0.8 hz , 1h ) , 4.0 ( m , 4h ) , 3.78 ( m , 2h ) , 2.00 ( s , 6h ) . elemental analysis ( compound + 2.0 hcl + 2.0 h2o ) : % c , 47.62 ; % h , 5.09 ; % n , 12.62 ; ( calc ) . % c = 47.54/47.38 ; % n = 12.44/12.46 ; % h = 4.62/4.57 ( experimental ) . 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : in a glass vial , cs2co3 , 4f - aniline ( 0.462 g , 4.1 mmol , 2.0 equiv ) , pd2(dba)3 ( 0.095 g , 0.104 mmol , 0.05 equiv ) , xantphos ( 0.120 g , 0.208 mmol , 0.1 equiv ) , and dioxane were stirred for 5 min at rt . compound 10 ( 1 g , 2.08 mmol , 1.0 equiv ) was added to the reaction mixture , after which the reaction mixture was degassed for 15 min and then stirred at 120 c under n2 for 8 h. hplc / ms test showed that the starting material compound 10 was consumed and desired product was formed predominantly along with some 9 . the reaction mixture was concentrated and then purified by normal phase column chromatography ( silica gel 80 g ) using a gradient of 1000% to 0100% hexane : etoac . the organic layer was concentrated at reduced pressure to yield the boc derivative ( 950 mg , 89% ) yield . lc - ms : ( es , m / z ) [ m + h ] calcd for c27h32f2n5o3 , 512 ; found , 512 . the boc compound was treated with 20% tfa in ch2cl2 ( 50 ml ) and was added to the mixture . after the completion of this reaction ( monitored by lcms ) , the resulting mixture was concentrated under reduced pressure . the resulting residue was purified by reverse phase hplc to yield product as a tfa salt . lc - ms : ( es , m / z ) [ m + h ] calcd for c22h23f2n5o , 412.2 ; found , 412.1 . h nmr ( meoh - d4 , 400 hz ) 7.617.57 ( m , 2h ) , 6.94 ( t , j = 8.8 hz , 2h ) , 6.81 ( t , j = 8.8 hz , 2h ) , 6.47 ( m , 2h ) , 3.72 ( m , 2h ) , 3.58 ( m , 2h ) , 3.42 ( m , 2h ) , 1.85 ( s , 6h ) . elemental analysis compound 22 with 0.65 equiv h2o : c , 62.44 ; n , 16.55 ; h , 5.79 ( calculated ) . c = 62.54/62.44 ; n = 16.35/16.29 ; h = 5.52/5.61 ( experimental ) . the regiochemistry of the dime groups was unambiguously assigned using 2-d nmr techniques ( table 8) . 2-amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 23 was prepared from compound 10 ( r1 = me , r2 = me ) by a pd2(dba)3 mediated amination reaction with 4-methylaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . h nmr ( meoh - d4 , 400 mhz ) 7.797.69 ( m , 2h ) , 7.21 ( t , j = 8.4 hz , 2h ) , 7.04 ( d , j = 8.0 hz , 2h ) , 6.76 ( d , j = 8.2 hz , 2h ) , 4.11 ( s , 2h ) , 3.88 ( m , 4h ) , 2.22 ( s , 3h ) , 2.12 ( s , 6h ) . lc / ms major mass : 408.1 ( m + h ) . 2-amino-1-(3-((3,4-difluorophenyl)amino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 24 was prepared from compound 10 ( r1= me , r2 = me ) by a pd2(dba)3 mediated amination reaction with 3,4-difluoroaniline followed by a tfa mediated deprotection in a protocol similar to compound 22 . h nmr ( meoh - d4 , 400 mhz ) 7.737.69 ( m , 2h ) , 7.21 ( t , j = 8.8 hz , 2h ) , 7.067.03 ( m , 1h ) , 6.766.65 ( m , 1h ) , 6.536.51 ( m , 1h ) , 4.084.06 ( m , 4h ) , 3.863.83 ( m , 2h ) , 2.07 ( s , 6h ) . elemental analysis ( compound + 2.9 hcl ) : % c , 49.37 ; % h , 4.69 ; % n , 13.09 ; ( calc ) . % c = 49.43/49.06 ; % n = 12.89/12.77 ; % h = 4.7/4.81 ( experimental ) . 13 was prepared from compound 5a by the following way : to a stirred solution of compound 5a ( 1.1 g , 3.38 mmol , 1.00 equiv ) in dmf ( 30 ml ) was added 3-chloro-2-methylprop-1-ene ( 500 mg , 5.49 mmol , 1.50 equiv ) , potassium carbonate ( 560 mg , 4.06 mmol , 1.10 equiv ) , and potassium iodide ( 1.12 g , 6.75 mmol , 2.00 equiv ) at rt . the mixture was washed with brine ( 3 10 ml ) , dried over sodium sulfate , and concentrated under vacuum . the residue was applied onto a silica gel column with petroleum ether / etoac ( 5:1 ) to give 0.7 g ( 55% ) of compound 5a-1 as a light - yellow solid . m / z = 380 ( m + 1 ) . into a 30 ml sealed tube , was placed compound 5a-1 ( 2.0 g , 5.28 mmol , 1.00 equiv ) , acetic acid ( 12 ml ) , and methanesulfonic acid ( 2 ml ) . the reaction mixture was stirred for 12 h at 260 c ( the temperature of the sand bath ) . the aqueous layer was washed with ethyl acetate ( 3 10 ml ) . the aqueous layer was extracted with ethyl acetate ( 3 10 ml ) . the combined organic layers were washed with brine ( 3 10 ml ) , dried over sodium sulfate , and concentrated under vacuum . the solid was collected by filtration and washed with 5 ml of n - hexane to give 500 mg ( 39% ) of compound 5a-2 as a white solid . h nmr ( 400 mhz , cdcl3 ) 7.707.65 ( m , 2h ) , 7.046.9 ( m , 2h ) , 6.96 ( s , 1h ) , 4.12 ( s , 2h ) , 3.69 ( s , 2h ) , 1.23 ( s , 6h ) . to a stirred solution of compound 5a-2 ( 280 mg , 1.14 mmol , 1.0 equiv ) in dmf ( 10 ml ) was added 2-(tert - butoxycarbonyl)acetic acid ( 600 mg , 3.43 mmol , 3.0 equiv ) , hatu ( 1.3 g , 3.42 mmol , 3.0 equiv ) , and diea ( 880 mg , 6.82 mmol , 6.0 equiv ) at rt . the organic layer was washed with brine ( 3 10 ml ) , dried over sodium sulfate , and concentrated under vacuum . the residue was applied onto a silica gel column with ch2cl2/meoh ( 10:1 ) to give 280 mg ( 57% ) of compound 13 as a brown solid . h nmr ( 400 mhz , cdcl3 ) 7.727.68 ( m , 2h ) , 7.23 ( s , 1h ) , 7.137.07 ( m , 2h ) , 5.46 ( s , nh ) , 4.65 ( s , 2h ) , 4.054.04 ( m , 2h ) , 3.97 ( s , 2h ) , 1.551.39 ( m , 15h ) . the structure of 13 was verified by hmqc , cosy , hmbc , and roesy ( table 9 ) . 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-6,6-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : to a stirred solution of compound 13 ( 386 g , 0.96 mmol ) in 6 ml of dichloromethane was added br2 ( 55 l , 1.06 mmol ) in acetic acid ( 2 ml ) . after neutralization , the residue was subjected to flash chromatography ( 40 g , 0100% ethyl acetate in hexane , 50 min , dry loading ) purification to give 256 mg ( 55% ) of the title compound 13a as a colorless oil . h nmr ( 400 mhz , cdcl3 ) 7.877.83 ( m , 2h ) , 7.03 ( t , j = 8.8 hz , 2h ) , 5.46 ( s , 1h ) , 4.5 ( s , 2h ) , 3.97 ( d , j = 4.2 hz , 2h ) , 3.81 ( s , 2h ) , 1.45 ( s , 6h ) , 1.39 ( s , 9h ) . example 25was obtained from 13a by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a tfa mediated deprotection by analogy to compound 22 ( 55% yield over 2 steps ) . h nmr ( 400 mhz , dmso - d6 ) : 8.06 ( m , 2h ) , 7.98 ( m , 1h ) , 7.80 ( m , 2h ) , 7.16 ( m , 2h ) , 6.98 ( m , 2h ) , 6.60 ( m , 2h ) , 4.64 ( m , 2h ) , 4.02 ( m , 2h ) , 3.80 ( m , 2h ) , 1.38 ( s , 6h ) . 2-(4-fluorophenyl)-7-(4-methoxybenzyl)-5,5-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine : compound 7b was prepared from compound 7a ( r1 = r2 = h ) by the following way : to a solution of compound 7a ( 231 mg , 1.0 mmol ) in dmf ( 10 ml ) were added koh ( 168 mg , 3.0 mmol ) and pmbcl ( 405 l , 3.0 mmol ) at 0 c . the reaction mixture was stirred at the same temperature for 2 h and at rt for 2 additional hours . the hplc fraction were evaporated under reduced pressure and neutralized by satd nahco3 . the organic layer was concentrated under reduced pressure to give compound 7a-1 ( 221 mg , 63% ) as a white solid . h nmr ( 400 mhz , cdcl3 ) 7.677.63 ( m , 2h ) , 7.287.25 ( m , 2h ) , 7.077.02 ( m , 2h ) , 6.896.86 ( m , 2h ) , 4.74 ( s , 2h ) , 4.70 ( m , 2h ) , 4.53 ( s , 2h ) , 3.79 ( s , 3h ) . to a solution of compound 7a-1 ( 253 mg , 0.72 mmol ) in dmf ( 15 ml ) were added 60% sodium hydride ( 87 mg , 0.085 mmol ) and methyl iodide ( 0.45 ml , 7.2 mmol ) at rt . the reaction mixture was stirred at rt for 2 h. the reaction mixture was quenched with methanol and directly subjected to mass - triggered hplc purification to give 7a-2 as a white solid after evaporation of acetonitrile water in vacuo . the tfa salt was subsequently neutralized by saturated sodium bicarbonate solution and extracted with dichloromethane to get the compound as a free base . the organic layer was dried over sodium sulfate and concentrated under reduced pressure to yield the desired compound 7a-2 ( 234 mg , 86% ) . the structure of 7a-2 was verified by 2-d nmr techniques ( table 10 ) . to a solution of compound 7a-2 ( 170 mg , 0.45 mmol ) in thf ( 9 ml ) was added 1.0 n bh3thf ( 2.70 ml , 2.70 mmol ) at rt . the reaction mixture was stirred at reflux for 2 h. pd / c was added ( gas generated ) . the reaction mixture was stirred for 1 h. solid was filtered off , and solvent was removed . the product was assumed to be of 100% yield and used in the next step without further purification . h nmr ( 400 mhz , cd3od ) 8.1 ( s , 1h ) , 7.747.70 ( m , 2h ) , 7.34 ( d , j = 8.6 hz , 2h ) , 7.27 ( m , 2h ) , 6.94 ( d , j = 8.6 hz , 2h ) , 3.91 ( s , 2h ) , 3.80 ( s , 3h ) , 3.79 ( s , 2h ) , 2.87 ( s , 2h ) , 1.61 ( s , 6h ) . 7-(4-methoxybenzyl)-2-(4-fluorophenyl)-5,5-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine ( 1.07 g , 2.93 mmol , 1.00 equiv ) ( 7b ) was added to trifluoroacetic acid ( 15 ml ) . the resulting solution was stirred for 2 h at 70 c in an oil bath . the ph value of the solution was adjusted to 8 with aqueous sodium bicarbonate . 7b-1 was extracted from the aqueous layer using 3 100 ml of ethyl acetate . this resulted in 7b-1 ( 718 mg , quantitative ) as a yellow solid . to a solution of 7b-1 ( 750 mg , 3.06 mmol , 1.00 equiv ) in n , n - dimethylformamide ( 30 ml ) were added 2-(tert - butoxycarbonyl)acetic acid ( 850 mg , 4.86 mmol , 1.50 equiv ) , hatu ( 1.3 g , 3.42 mmol , 1.10 equiv ) , and triethylamine ( 650 mg , 6.44 mmol , 2.00 equiv ) . the resulting solution was stirred overnight at rt . the resulting mixture was washed with brine ( 3 100 ml ) , dried over anhydrous sodium sulfate , and concentrated under vacuum . the residue was applied onto a silica gel column with petroleum ether / ethyl acetate ( 4:1 ) . this resulted in 600 mg ( 49% ) of 7c as a pale - yellow solid . h nmr ( 300 mhz , cdcl3 ) : 7.75 ( m , 2h ) , 7.22 ( s , 1h ) , 7.137.07 ( m , 2h ) , 4.994.87 ( m , 2h ) , 4.12 ( s , 2h ) , 3.91 3.65 ( m , 2h ) , 1.57 ( m , 6h ) , 1.43 ( s , 9h ) . to a stirred solution of compound 7b-2 ( 583 g , 1.45 mmol ) in dichloromethane ( 6 ml ) was added br2 ( 82 l , 1.60 mmol ) in acetic acid ( 2 ml ) . after neutralization , the residue was subjected to flash chromatography ( 40 g , 0100% ethyl acetate in hexane , 50 min , dry loading ) purification to give 628 mg ( 90% ) of the title compound 7c as a yellow solid . h nmr ( 400 mhz , cdcl3 ) 7817.78 ( m , 2h ) , 7.07 ( t , j = 8.6 hz , 2h ) , 5.28 ( s , nh ) , 4.844.7 ( m , 2h ) , 4.073.58 ( m , 4h ) , 1.491.44 ( m , 6h ) , 1.28 ( s , 9h ) . 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-5,5-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)ethanone : compound 26 was prepared from compound 7c by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a tfa mediated deprotection in 46% yield over 2 steps . h nmr ( 400 mhz , cd3od ) : 7.57 ( m , 2h ) , 7.02 ( m , 2h ) , 6.77 ( m , 2h ) , 6.52 ( m , 2h ) , 4.974.91 ( m , 2h ) , 4.093.74 ( m , 4h ) , 1.56 ( s , 3h ) , 1.51 ( s , 3h ) . 2-amino-1-(2-(4-fluorophenyl)-3-(4-fluorophenylamino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 28 was prepared from compound 8 ( r1 = r2 = me ) in the following fashion using a slightly modified route to the one described in scheme 2 . to a stirred solution of compound 8 ( 1.48 g , 6.04 mmol ) and net3 ( 6.0 g , 59.4 mmol ) in dichloromethane ( 20 ml ) was added 2-bromo-2-methylpropanoyl bromide ( 14 g , 60.9 mmol ) dropwise at rt . after being stirred for 3 h at rt , the reaction was then quenched by the addition of water ( 30 ml ) . the combined organic layers were washed with brine , dried over anhydrous sodium sulfate , and concentrated under vacuum . this resulted in crude product as a dark solid , which was washed with etoac : petroleum ether ( 1:10 ) to remove the impurities to produce compound 8 - 1 as a gray solid . ( 2.0 g , 5.08 mmol , 1.00 equiv ) in dmf ( 10 ml ) was nan3 ( 1.0 g , 15.38 mmol , 3.00 equiv ) at rt . the resulting mixture was washed with 3 20 ml of brine , dried over anhydrous sodium sulfate , and concentrated under vacuum . the residue was applied onto a silica gel column with petroleum ether / etoac ( 5:1 ) to give compound 8 - 2 as a white solid . m / z 357 ( m + h ) . to a stirred solution of compound 8 - 2 ( 1.2 g , 3.37 mmol , 1.00 equiv ) in methanol ( 20 ml ) was added pd / c ( 80 mg , 0.75 mmol , 0.20 equiv ) at rt . m / z 331 ( m + h ) . to a stirred solution of compound 8 - 3 ( 910 mg , 2.76 mmol , 1.00 equiv ) in thf ( 50 ml ) was added boc anhydride ( 3.2 g , 14.68 mmol , 5.00 equiv ) , followed by aqueous naoh ( 1n , 6 ml , 2.0 equiv ) at rt . the organic layer was washed with 3 10 ml of brine , dried over na2so4 , and concentrated under vacuum . the solid was collected by filtration and washed with n - hexane ( 5 ml ) to give compound 8 - 4 as a white solid . h nmr ( 300 mhz , cdcl3 ) 7.757.71 ( m , 2h ) , 7.097.02 ( m , 3h ) , 4.84(s , nh ) , 4.094.0 ( m , 4h ) , 1.98 ( s , 6h ) , 1.55 ( s , 6h ) , 1.45 ( s , 9h ) . to a stirred solution of compound 8 - 4 ( 32 mg , 0.074 mmol ) in dichloromethane ( 3 ml ) was added br2 ( 4.2 l , 0.082 mmol ) in acetic acid ( 1 ml ) . after neutralization , the residue was subjected to flash chromatography ( 4 g , 060% ethyl acetate in hexane , 16 min ) purification to give the compound 8 - 5 as colorless oil , was collected by filtration , and washed with n - hexane ( 5 ml ) to give compound 8 - 4 as a white solid . h nmr ( 400 mhz , cdcl3 ) 7.917.88 ( m , 2h ) , 7.107.04 ( m , 2h ) , 5.11(s , nh ) , 4.01 ( m , 2h ) , 3.96 ( s , 2h ) , 1.94 ( s , 6h ) , 1.51 ( s , 6h ) , 1.41 ( s , 9h ) . compound 28 was prepared from compound 8 - 5 by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a tfa mediated deprotection . h nmr ( 400 mhz , meoh - d4 ) 7.66 ( dd , j = 9.2 , 5.2 hz , 2h ) , 7.11 ( t , j = 8.8 hz , 2h ) , 6.81 ( t , j = 8.8 hz , 2h ) , 6.726.68 ( m , 2h ) , 4.043.99 ( m , 4h ) , 2.01 ( s , 6h ) , 1.66 ( s , 6h ) . 2-amino-1-(2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 29 was prepared from 35 by the following way : to a stirred solution of 35 ( 21 mg , 0.06 mmol ) and et3n ( 83 l , 0.60 mmol ) in dry dichloromethane ( 6 ml ) was added 2-bromo-2-methylpropanoyl bromide ( 71 l , 0.60 mmol ) . the reaction mixture was stirred at rt for 5 h. the reaction mixture was concentrated and subjected to mass - triggered lc / ms purification directly . the obtained solution was concentrated to give 22 mg ( 73% ) of compound 35 - 1as yellow oil after neutralization . h nmr ( 400 mhz , meoh - d4 ) 7.717.66 ( m , 2h ) , 7.237.19 ( m , 2h ) , 7.02 ( d , j = 8.4 hz , 2h ) , 6.66 ( d , j = 8.4 hz , 2h ) , 4.31 ( m , 2h ) , 4.09 ( m , 2h ) , 2.21 ( s , 3h ) , 2.06 ( s , 6h ) , 2.00 ( s , 6h ) . to a solution of compound 35 - 1 ( 22 mg , 0.044 mmol ) in dmf ( 3 ml ) was added nan3 ( 8.6 mg , 0.132 mmol ) at rt . the reaction mixture was stirred at rt for 2 h. the reaction mixture was directly subjected to mass - triggered hplc purification directly . the obtained mecn / aqueous solution was combined and concentrated to give 15 mg ( 75% ) of compound 35 - 2 as yellow oil after neutralization . h nmr ( 400 mhz , meoh - d4 ) 7.70 7.66 ( m , 2h ) , 7.237.19 ( m , 2h ) , 7.04 ( d , j = 8.3 hz , 2h ) , 6.66 ( d , j = 8.4 hz , 2h ) , 4.26 ( m , 2h ) , 4.074.05 ( m , 2h ) , 2.24 ( s , 3h ) , 2.03 ( s , 6h ) , 1.58 ( s , 6h ) . to a solution of compound 35 - 2 ( 15 mg , 0.033 mmol ) in meoh ( 3 ml ) was added 10% pd / c ( 4 mg , 0.003 mmol ) at rt . the reaction mixture was stirred at rt for 2 h. solid was filtered off , and solvent was removed . the reaction mixture was directly subjected to mass - triggered hplc purification to give 15 mg ( 100% ) of the title compound yellow oil . h nmr ( meoh - d4 , 400 hz ) 7.787.75 ( m , 2h ) , 7.066.97 ( m , 4h ) , 6.56 ( d , j = 8.4 hz , 2h ) , 5.16 ( s , nh ) , 4.33 ( t , j = 4.8 hz , 2h ) , 3.74 ( t , j = 4.8 hz , 2h ) , 2.29 ( s , 3h ) , 1.95 ( s , 6h ) , 1.45 ( s , 6h ) . 2-amino-1-(3-(3,4-difluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8h)-yl)-2-methylpropan-1-one : compound 30 was prepared from compound 8 - 5 by a pd2(dba)3 mediated amination reaction with 3,4-difluoroaniline followed by a tfa mediated deprotection.h nmr ( 400 mhz , meoh - d4 ) 7.637.60 ( m , 2h ) , 7.14 ( t , j = 8.4 hz , 2h ) , 7.01 ( dd , j = 18.8 , 8.8 hz , 1h ) , 6.686.63 ( m , 1h ) , 6.5 ( m , 1h ) , 4.03 ( m , 2h ) , 4.01 ( m , 2h ) , 2.01 ( s , 6h ) , 1.66 ( s , 6h ) . 2-(4-fluorophenyl)-n-(p - tolyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : the details of the synthesis involving an ugi reaction between 4-fluorobenzaledehyde , 2-aminopyrazine , and 1-isocyano-4-methylbenzene has been reported in the previous communication.h nmr ( 400 mhz , meoh - d4 ) 7.73 ( d , j = 6.8 hz , 2h ) , 7.006.99 ( m , 4h ) , 6.49 ( d , j = 8.0 hz , 2h ) , 4.01 ( s , 2h ) , 3.72 ( m , 2h ) , 3.15 ( m , 2h ) , 2.19 ( s , 3h ) . 2-(4-fluorophenyl)-3-(p - tolylamino)-7,8-dihydroimidazo[1,2-a]pyrazin-6(5h)-one : compound 32 was synthesized from compound 7 ( r1 = r2 = h ) pd2(dba)3 mediated coupling reaction with p - toluidine . h nmr ( 400 mhz , dmso - d6 ) 8.55 ( s , 1h ) , 7.777.75 ( m , 2h ) , 7.267.14 ( m , 4h ) , 7.016.95 ( m , 2h ) , 6.53 ( m , 1h ) , 4.53 ( m , 2h ) , 4.22 ( m , 2h ) , 2.17 ( s , 3h ) . 2-(4-fluorophenyl)-8,8-dimethyl-3-(p - tolylamino)-7,8-dihydroimidazo[1,2-a]pyrazin-6(5h)-one : compound 33a was prepared from 7 by the following way : to a stirred solution of compound 7 ( 390 mg , 1.51 mmol , 1.00 equiv ) in dichloromethane ( 20 ml ) was added nbs ( 0.28 g , 1.00 equiv ) . the solid was filtered out , and the mixture was washed with a saturated solution of na2s2o3 and dried over na2so4 . the solids was purified by silica gel chromatography ( petroleum ether / etoac = 1:2 ) to result in 432 mg ( 85% ) of the title compound as a white solid . h nmr ( 300 mhz , dmso - d6 ) 8.76 ( s , 1h ) , 7.947.89 ( m , 2h ) , 7.317.25 ( m , 2h ) , 4.57 ( s , 2h ) , 1.56 ( s , 6h ) . compound 33 was prepared from compound 33a by a pd2(dba)3 mediated amination reaction with p - toluidine . h nmr ( meoh - d4 , 400 hz ) 7.627.58 ( m , 2h ) , 7.05 ( t , j = 8.4 hz , 2h ) , 6.92 ( d , j = 8.4 hz , 2h ) , 6.52 ( d , j = 8.4 hz , 2h ) , 4.42 ( s , 2h ) , 2.12 ( s , 3h ) , 1.71 ( s , 6h ) . 3-(4-chloro-3-fluorophenylamino)-2-(4-fluorophenyl)-8,8-dimethyl-7,8-dihydro imidazo[1,2-a]pyrazin-6(5h)-one : compound 34 was prepared from compound 7b by a pd2(dba)3 mediated amination reaction with 4-chloro-3-fluoroaniline . h nmr ( meoh - d4 , 400 hz ) 7.747.70 ( m , 2h ) , 7.21 ( t , j = 8.4 hz , 1h ) , 7.08 ( m , 2h ) , 6.496.40 ( m , 2h ) , 4.43 ( s , 2h ) , 1.72 ( s , 6h ) . 2-(4-fluorophenyl)-8,8-dimethyl - n - p - tolyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : compound 35 was prepared from compound 35 - 1 ( shown below and prepared by analogy to compound 9 and using cbz - glycine ) by a pd2(dba)3 mediated amination reaction with p - toluidine followed by a 6n hcl mediated hydrolysis . h nmr ( meoh - d4 , 400 hz ) 7.787.75 ( m , 2h ) , 7.066.97 ( m , 4h ) , 6.56 ( d , j = 8.4 hz , 2h ) , 5.16 ( s , nh ) , 4.33 ( t , j = 4.8 hz , 2h ) , 3.74 ( t , j = 4.8 hz , 2h ) , 2.29 ( s , 3h ) , 1.95 ( s , 6h ) . n,2-bis(4-fluorophenyl)-8,8-dimethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : compound 36 was prepared from compound 35 - 1 by a pd2(dba)3 mediated amination reaction with 4-fluoroaniline followed by a 6n hcl mediated hydrolysis . h nmr ( 400 mhz , meoh - d4 ) 7.627.58 ( m , 2h ) , 7.127.08 ( m , 2h ) , 6.686.63 ( m , 2h ) , 6.67 ( m , 2h ) , 4.16 ( m , 2h ) , 3.78 ( m , 2h ) , 1.92 ( s , 6h ) .

on the basis of the initial success of optimization of a novel series of imidazolopiperazines , a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties . these changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies . the optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency , in vitro metabolic stability profile and , as a result , enhanced oral exposure in vivo in mice . the optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model . they exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies .

Introduction Chemistry Results and Discussion Summary Experimental Section Experimental Protocols (Chemistry)

with support from the wellcome trust and medicines for malaria venture ( mmv ) , the ngbs consortium ( the novartis institute for tropical diseases ( nitd ) , the genomics institute of the novartis research foundation ( gnf ) , the biomedical primate research centre ( bprc ) , and the swiss tropical and public health institute ( stphi ) ) was formed and set up to discover new antimalarials which are effective against multidrug resistant parasite strains here we report our continued effort to optimize a series of imidazolopiperazine compounds in search of a preclinical candidate . as described previously , an extensive sar study on the three peripheral parts had yielded decent in vitro and in vivo antimalarial activities as well as favorable physiochemical and pharmacokinetic properties . our efforts toward the first - generation compounds led to candidates ( like compound 1 ) with double - digit nanomolar potency and moderate oral exposure in mice . however , most potent compounds with an unsubstituted piperazine had poor oral exposure which resulted in inferior in vivo efficacy when administered orally , for example , compounds 28 and 31 ( scheme 1 ) in the previous communication ( figure 1 ) . we were intrigued by the possibility of further refining the core structure of the molecule to further improve the properties . our hypothesis was further reinforced by chemical stability and mouse in vitro metabolic stability results which led to the identification of two biologically inactive metabolites ( 1a and 1b ) arising from the oxidation of piperazine ring . our plan was to substitute three possible positions ( positions 5 , 6 , and 8) to evaluate the corresponding effects on potency as well as a means to improve oral exposure . on the basis of the designed molecules , it was clear that previous synthetic routes were not amenable for the desired compounds because we could not utilize the 3 + 2 cyclization . distinct syntheses were designed and executed to obtain substitution at different positions of a piperazine ring . the intramolecular hydroamination reaction proved to be difficult , and after extensive screening , treating the substrate 5a with a 5:1 mixture of acetic acid and methanesulfonic acid at 210 c produced the cyclized core with simultaneous loss of the carbamate protecting group . we began exploring the sar on the piperazine ring by substituting at the 8-position first , and we decided to use 4-f - aniline and 3-f-4-cl aniline based on our previous work at the r6 position ( table 1 ) . moreover , there was no profound effect on the potency by changing the absolute configuration of the methyl group . emboldened by the increase in the potency based on the substitution on the 8-position , we decided to study the effect of dime group on the ethylene carbons of the piperazine ring . on the basis of examples 25 and 26 , it was abundantly clear that the dimethyl group on the 8-position had the most significant positive effect on potency . the substitution on the other positions of the piperazine ring only provided modest potencies when compared to 8-substitution , with activities in the double - digit nanomolar range . as the 5-substituted compounds and the 6-substituted compounds were less potent , we decided to focus our attention on the effect of the amide substituent of the piperazine nitrogen with the 8,8-dimethyl group constant . in the case of the substituted piperazines , installation of methylalanine amide provided equipotent compounds ( 15 vs 27 , 22 vs 28 , and 23 vs 29 ) . we proceeded to further understand the overall effect of the dimethylpiperazine on the minimal pharmacophore . after studying the effects of various substitutions on the piperazine ring and obtaining a significant improvement in activity , we decided to reconfirm the minimal pharmacophore needed for antiplasmodial activity . on the basis of the earlier results , we were able to achieve antiplasmodial activity with ec50 in 160400 nm range for the core devoid of the amino acid substituent on the piperazine nitrogen ( compound 31 , table 2 ) . modification of the piperazine ring into the corresponding lactam led to a 7-fold decrease in activity in the micromolar range ( 32 ) . these results led us to conclude that the amino acid substitution had minimal to no effect on the potency of the compound with substitution on the 8-position of the imidazolopiperazine core and that the basicity of the piperazine nitrogen could be modulated without complete loss in antimalarial activity . in parallel to the sar studies to optimize the potency of the compounds series , we evaluated the in vitro adme and in vivo pharmacokinetic properties of key analogues . in general , this series of compounds had very good lipinski 's rule - of - five compliance and most of the analogues were highly soluble ( > 175 m at ph 6.8 ) , with the neutral lactams being least soluble among all the compounds . table 3 summarizes the in vitro adme profiles of the important compounds . typically , a range of values in the permeability assays was observed . in the caco-2 assays , the core piperazine compounds ( both lactams as well as nonamide piperazine compounds ) exhibited high apical to basal values ( 31 , 32 , 34 , and 35 ) . the permeability of the compounds was reduced once the side chain amino acid was installed , but metabolic stability is improved ( 28 vs 35 ) . removing the basicity of the piperazine ring by introduction of carbonyl group improved the mouse and human microsome stability of the compound , presumably by blocking the ethylene bridge ( 31 vs 32 ) but is not evident in rat microsomes . introduction of the dimethyl group on the 8-position of the piperazine ring does not improve overall metabolic stability of the compound ( 15 vs 28 or 31 vs 35 ) despite the metabolite identification results presented earlier . this may be due to the ethylene bridge in the piperazine ring representing a metabolic soft spot and enabling metabolic switching . compounds series found to have favorable potency and in vitro adme properties were tested in snapshot pk , an abbreviated pk study shown to accurately bin compounds into low , moderate , or high oral exposure . compounds predicted to have reasonable oral exposure would be tested in mice using both po and iv routes of administration ( table 4 ) . the in vivo clearance generally was low to moderate and significantly improved when compared to the corresponding unsubstituted analogues ( 15 , 22 ) . this resulted in higher maximum concentration as well as improvement in auc values in the oral arms as well . to test whether red cell partitioning was occurring to explain the high volume of distribution for the basic compounds , we carried out an in vitro experiment in which compound 22 was incubated in nave rat whole blood as well as plasma for 30 min . after centrifuging the blood and quantifying the amount of compound in the red blood cells , we determined that the concentration of the compound was 3.47 times higher in whole blood than in plasma alone , indicating that the compound is either adhering to the rbc s or getting distributed inside the cells . because we had examples of compounds with different modifications with acceptable bioavailability , we decided to carry on the efficacy experiment in mice . the in vivo antimalarial activities of the optimized compounds were evaluated using a plasmodium berghei mouse survival model . we decided to determine the ed99 representative examples for the substituted and nonsubstituted piperazine cores and found the substituted piperazine core was 10-fold more efficacious ( compound 22 ed99 = 2.2 mg / kg ) than the unsubstituted core ( compound 1 ed99 = 26.5 mg / kg ) . on the basis of the efficacy results rat was chosen as the second animal species for oral exposure evaluation as it may serve as the in vivo preclinical toxicity species . taking into account the nonoptimal metabolic stability data for rats for this scaffold , we decided to study the oral exposure of the compound in rat for a period of 5 h in the oral arm only to get an estimation of the exposure ( table 6 ) . -methylalanine subclass of compounds like 29 did not exhibit any in vivo exposure and were not further pursued . as the oral exposure of compounds 22 was low in rats but demonstrated superior mouse efficacy , we decided to dose compound 22 at higher doses to make sure that sufficient multiples of exposure of the mouse efficacious dose could be achieved ( table 7 ) . the auc at the mouse efficacious dose ( 2 mg / kg ) was 780 hnm , therefore a 45 exposure multiple is achieved at 100 mg / kg in the rat . finally , considering the efficacious plasma cmax in the mouse at 2 mg / kg is low ( 75 nm ) , the risk of cardiotoxicity is considered to be minimal where the herg channel inhibitory activity ic50 = 13.4 m , providing a greater than 170-fold window . directed by the identification of in vitro metabolites formed by compound 1 , the piperazine core of 15 was systematically modified by a gem - dimethyl functionality at all three possible places . the 8,8-dimethyl analogues demonstrated excellent potency as well as improved oral exposure in the mouse . the first generation of compounds exhibited efficacy that was comparable to the known antimalarials in the p. berghei mouse model , however , compound 22 was more efficacious than chloroquine and artesunate especially when parasitemia reduction is compared in terms of ed99 values . although compound 22 demonstrated poor rat pk at low doses , with higher doses it was able to achieve the necessary exposure multiples calculated based on the plasma concentration found to be efficacious in the mouse model . then 50 nl of compounds were transferred using a platemate plus ( matrix ) or the gnf systems pintool into the assay plates containing screening media to a final maximum concentration of 10 m in a 12 point dose response ( 1/2 log serial dilution ) . on the basis of the measured parasitemia levels , each parasite strain was diluted to yield 0.5% parasitemia , and 50% hematocrit blood ( uninfected erythrocytes ) was added to a concentration of 4.17% . after the 72 h incubation ( equivalent to 12 cycles during the blood stage ) , a prepared mixture of the lysis buffer ( 5 mm edta , 1.6% triton x-100 , 20 mm tris - hcl , 0.16% saponin ) in water and sybr green detection ( 0.1% ) reagent was dispensed at 10 l per well using the microflo . the metabolic stability of drug candidates was determined in human , mouse , and rat liver microsomes using the compound depletion approach , quantified by lc / ms / ms . the assay measured the rate and extent of metabolism of chemical compounds by measuring the disappearance of the parent compound . the caco-2 assay was carried out in a 96-well format , and compound concentrations in each chamber were measured by lc / ms . the results can be used to predict the in vivo oral absorption ( using papp ( a b ) ) and the transport mechanism of the test compounds across gi membrane ( ratio of papp ( b a):papp ( a b ) and log p lipophilicity data ) . the assay utilizes electrophysiology measurement on the electric current passing through herg channel that is heterologously expressed in a stable cho cell line . all compounds were tested by 4-point dose response curves , and ic50 values ( between 0.2 and 30 m ) were measured . in - life studies were carried out under protocols approved by the animal care and use committee ( iacuc ) of gnf . all compounds were formulated at a concentration of 2.5 mg / ml in 75% peg300 and 25% d5w ( 5% dextrose in distilled water ) solution and were filtered using a 0.45 m syringe filter . , mass spectral analyses were carried out using electrospray source in the positive ion mode and using multiple reaction monitoring ( mrm ) for quantification . all in vivo efficacy studies were approved by the veterinary authorities of the canton basel - stadt . compounds were administered orally in a volume of 10 ml / kg either as a single dose ( 24 h post infection ) or as three consecutive daily doses ( 24 , 48 , and 72 h post infection ) . activity was calculated as the difference between the mean percent parasitemia for the control and treated groups expressed as a percentage of the control group . purification of compounds by high pressure liquid chromatography was achieved using a waters 2487 series with ultra 120 5 m c18q column with a linear gradient from 10% solvent a ( acetonitrile with 0.035% trifluoroacetic acid ) in solvent b ( water with 0.05% trifluoroacetic acid ) to 90% a in 7.5 min , followed by 2.5 min elution with 90% a. h nmr spectra were recorded on bruker xwin - nmr ( 400 or 600 mhz ) . elemental analyses were carried out by midwest microlabs llc , indianapolis , in , usa . after neutralization , the residue was subjected to flash chromatography ( 40 g , 0100% ethyl acetate in hexane , 50 min , dry loading ) purification to give 256 mg ( 55% ) of the title compound 13a as a colorless oil . the product was assumed to be of 100% yield and used in the next step without further purification . after neutralization , the residue was subjected to flash chromatography ( 40 g , 0100% ethyl acetate in hexane , 50 min , dry loading ) purification to give 628 mg ( 90% ) of the title compound 7c as a yellow solid . the residue was applied onto a silica gel column with petroleum ether / etoac ( 5:1 ) to give compound 8 - 2 as a white solid . 2-(4-fluorophenyl)-n-(p - tolyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : the details of the synthesis involving an ugi reaction between 4-fluorobenzaledehyde , 2-aminopyrazine , and 1-isocyano-4-methylbenzene has been reported in the previous communication.h nmr ( 400 mhz , meoh - d4 ) 7.73 ( d , j = 6.8 hz , 2h ) , 7.006.99 ( m , 4h ) , 6.49 ( d , j = 8.0 hz , 2h ) , 4.01 ( s , 2h ) , 3.72 ( m , 2h ) , 3.15 ( m , 2h ) , 2.19 ( s , 3h ) . the solids was purified by silica gel chromatography ( petroleum ether / etoac = 1:2 ) to result in 432 mg ( 85% ) of the title compound as a white solid . purification of compounds by high pressure liquid chromatography was achieved using a waters 2487 series with ultra 120 5 m c18q column with a linear gradient from 10% solvent a ( acetonitrile with 0.035% trifluoroacetic acid ) in solvent b ( water with 0.05% trifluoroacetic acid ) to 90% a in 7.5 min , followed by 2.5 min elution with 90% a. h nmr spectra were recorded on bruker xwin - nmr ( 400 or 600 mhz ) . elemental analyses were carried out by midwest microlabs llc , indianapolis , in , usa . the crude product of 8 ( 4.5 g , 18.3 mmol , 95% ) was used in the next step . after neutralization , the residue was subjected to flash chromatography ( 40 g , 0100% ethyl acetate in hexane , 50 min , dry loading ) purification to give 256 mg ( 55% ) of the title compound 13a as a colorless oil . the product was assumed to be of 100% yield and used in the next step without further purification . after neutralization , the residue was subjected to flash chromatography ( 40 g , 0100% ethyl acetate in hexane , 50 min , dry loading ) purification to give 628 mg ( 90% ) of the title compound 7c as a yellow solid . 2-(4-fluorophenyl)-n-(p - tolyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-amine : the details of the synthesis involving an ugi reaction between 4-fluorobenzaledehyde , 2-aminopyrazine , and 1-isocyano-4-methylbenzene has been reported in the previous communication.h nmr ( 400 mhz , meoh - d4 ) 7.73 ( d , j = 6.8 hz , 2h ) , 7.006.99 ( m , 4h ) , 6.49 ( d , j = 8.0 hz , 2h ) , 4.01 ( s , 2h ) , 3.72 ( m , 2h ) , 3.15 ( m , 2h ) , 2.19 ( s , 3h ) . the solids was purified by silica gel chromatography ( petroleum ether / etoac = 1:2 ) to result in 432 mg ( 85% ) of the title compound as a white solid .

active ageing ( aa ) is the global goal of present ageing world for meeting the challenges of older persons and for improving their quality of life . active ageing can be explained as a concept and walker ( 2002 ) has defined aa as profound strategy to maximize participation and wellbeing as people grow older . world health organization ( who ) defined aa as the way of thinking and working on the process of optimizing opportunities for health , participation and security in order to enhance quality of life as people age . the concept of aa can be encapsulated as engaged in life and it has been recognized as a latent construct which has no specific clear dependent variable to measure it and it may be determined by various latent ( unobserved ) factors . the aa construct is influenced by several groups of determinants or determinant factors including the cross - cutting determinants ( gender and culture ) . these determinant factors are unobserved or latent and each of them can be presented by some indicators [ 2 , 3 ] . the pace of recent population ageing in thailand is faster than other asian countries and even far more faster than developed countries in the west [ 4 , 5 ] , and proportion of elderly ( aged 60 and over ) is projected to reach more than 30% within the next three decades in thailand . thailand has become an ageing society and is going to increase its older population . a rapid proceeding pace of population ageing , along with demographic changes in thailand , is posing critical challenges for thai government to maintain economic growth , social stability , and living standard of people in the nation . all determinant factors of aa with their indicators ( hence active ageing level , aal ) need to be better understood for developing policies and programs focused on active ageing in an ageing society . moreover , comparison of aal regarding cross - cutting determinants of aa , gender and culture , should be unveiled for better policy implications for the older persons and for the nation as a whole . using exploratory factor analysis , one study by haque et al . found active ageing determinant factors models with six determinant factors for female and male older persons in thailand . but the study did not validate the models ( i.e. , how does the model fit with the sample data ? ) . ( or any other study so far in knowledge ) did not compare aal for culture of different regions in thailand . culture , defined as people 's behavior , attitude , or way of life , varies depending on region of residence ( e.g. , urban or rural , different region of a country ) of individuals . therefore , people 's behavior , attitude , or way of life depends on the place or region of society where the society is situated . though thailand is perceived as southeast asia 's most ethnically homogeneous nation state and strong unity of thai culture , thailand government 's use of regional terms ( central , north , northeast , and south ) indicates some regional cultural diversity in thailand . despite the important strength and unity of thai culture , so , doing research or formulating policies on active ageing in thailand , the variations of active ageing depending on region of residence ( central , north , northeast , and south ) should be considered or should be borne in mind . the objective of this study was to gain empirical knowledge on active ageing determinant factors model for thai older persons including their active ageing level . the main specific objectives of this study are ( i ) to test the validity of active ageing determinant factors model for thai older persons and ( ii ) to test the similarity of active ageing level for various regions ( bangkok , central ( excluding bangkok ) , north , northeast , and south ) in thailand . the data for this study come from a nationally representative sample from the 2011 survey of older persons in thailand ( sopt ) . the sample of sopt consists of 62,840 persons aged 50 years and over , 54% of whom were 60 years and older . the data have been collected in sopt by using stratified two - stage sampling method . the secondary sampling units were private dwellings selected by random sampling from the list of all enumerated households in each village or block of the first sampling units . the 2011 sopt is a nationally representative survey which covers data for population subgroups or geographic subareas ( e.g. , regions and urban - rural areas ) . further details of the methodology , including sampling strategy , for sopt are available at nso 's report on the 2011 survey of older persons in thailand . study ) aged 60 years and over ( n = 23,801 ; female = 14,369 and male = 9,432 ) . for validating the active ageing determinant factors structure in thailand ( suggested by haque et al . ) , the variables used for this study in confirmatory factor analysis ( cfa ) are provided in table 1 . the study by haque et al . , a pioneer study for finding a model for active ageing determinants , suggested two separate determinant factor structures for each gender . using the indicator variables , listed in table 1 , the suggested active ageing determinant factors models of female and male older persons are provided in figures 1 and 2 , respectively . in the hypothesized models in figures 1 and 2 , the error terms of indicator variables are indicated by . the asterisks indicate parameters ( regression coefficient ( standardized ) , covariances of independent variables in the model ) to be estimated . meanwhile , error terms in cfa are considered as independent variables and their variances should be estimated . unidirectional arrows in the hypothesized models indicate directional linear influences ( i.e. , linear effect of one variable on another ) , and both - way directional arrows indicate correlation between connected variables but no directional influence of one on others is hypothesized . parameters of each of the hypothesized models can be estimated in cfa framework which was done using stata 12 ( model fit statistics , in cfa , are available after estimation of parameters ) . maximum likelihood ( ml ) estimation method is currently one of the most used methods for parameter estimation . details of parameter estimation procedure in cfa are available elsewhere [ 1012 ] . as this paper has no aim to estimate the parameters of those models ( but has aim to evaluate those models ) , any estimated model should be tested for how well the model fits to the sample data . this study used chi - square ( ) , root mean squared error of approximation ( rmsea ) , comparative fit index ( cfi ) , and standardized root mean squared residual ( srmr ) . the is a traditional measure of overall model fit ( it assesses the magnitude of differences between sample and estimated covariance matrices ) . that is , the null hypothesis for model evaluation is as h0 : there is no difference between sample covariance and estimated covariance ; that is , the model fits well to the data . active ageing level can be exhibited by computing the active ageing index ( aai ) of older persons . the aai ranges from 0 to 1 and a higher value of aai indicates higher active ageing level . ( 2016 ) has suggested that aai should be calculated separately for female and male older persons in thailand . using nationally representative sample and exploratory factor analysis ( efa ) , haque et al . the weakness of results from a model constructed from efa is that there is no scope to test the model fit . haque and colleagues study used only efa and the model ( from which aai has been obtained ) was not validated . for overcoming the weakness in efa , the study by haque and his colleagues used efa in thai national data for establishing the preliminary active ageing model of older persons and the study has identified six determinant factors for each gender . using the same factors ( with their corresponding indicator variables ) as in haque et al . 's study , this study runs the model in cfa framework ( in stata 12 ) which provides the predicted factor scores of each factor . from the predicted factor score , the factor index for each factor has been estimated using the following formula : index of fi:(1)fi = score of fiminscore of fimaxscore of fiminscore of fi , where fi is the ith factor . calculation of the aai , from cfa , used the following formula:(2)aai=i=1n=6fi6,where fi is the index of factor i ( n = 6 , because there were 6 factors ) . then an overall average aai has been calculated using all values of aai for all individuals . for simplicity , the aai is a composite index which combines a number of factor indices . because of social change over time and of cultural diversities in different places , possible changes may occur in individual aal over time , as has occurred in other aspects of individual or population in thailand ; for example , between 1994 and 2007 thailand experienced rapid social changes ( population ageing , socioeconomic development , health policies , etc . ) and these changes affect health across groups ( rural - urban ) and overall . as has been stated in introduction , there exists cultural variability in thailand depending on region of residence ( bangkok , central , north , northeast , and south ) . it also may be hypothesized that under the circumstances of cultural diversities in different cultural settings , there would be variability of aa level in various cultural contexts , that is , in various regions within a country . aa level quartiles would be the one method of comparing changes in the aal at a community over time or for comparing it in different places or for groups . aa level quartiles can be calculated by ranking individuals from lowest aai to the highest value and then dividing list into four groups or quartiles . each quartile for individuals , in whole thailand , for instance , contains 25% of individuals ( along with specific mean level of aai ) . then we may compare those distributions of quartiles ( percentage distribution ) for individuals overall ( national ) with different places ( e.g. , bangkok , central , north , northeast , and south ) . this quartile method of comparison will provide only the overview of difference(s ) but is unable to test the significance of the differences . similarity with significance of aa levels in different regions could be tested by using one - way analysis of variance ( anova ) . one - way anova is appropriate for one categorical variable with k levels ( e.g. , region of residence with 5 levels in this study ) or k groups and there should be a population of interest for which there is a quantitative variable for each of the k levels of the categorical variable ( e.g. , aai in this study ) . the aais for each group have mean parameters ( population means ) that we may label as 1 through k . the number of individuals ( older persons ) in group i ( 1 i k ) is defined as ni and n = i=1ni is the total sample size . the null hypothesis to be tested in one - way anova states that all the population means are equal ; that is , h0 : 1 = 2 = 3 = 4 = 5 ( for 5 regions of this study ) . the anova technique can identify significant differences ( if there were any ) but is unable to specify where the significant differences exist . so , post hoc comparisons using a built - in test in stata ( e.g. , bonferroni test ) were conducted . it has been stated , in materials and methods , that the model fit statistics can be obtained after the model estimation in cfa . the hypothesized models have been run in cfa in stata 12 and the obtained model fit indices are summarized in table 3 . the chi - square ( ) statistics for two models are very high and indicated that none of the models fits well to the sample data . but the statistic is very sensitive to sample size ; usually for large sample ( which is the case for two models ) it shows significant result which rejects the null hypothesis ( h0 : there is no difference between sample covariance and estimated covariance ; i.e. , the model fits well to the data ) . for correcting the sensitivity of statistic to large sample ( i.e. , tendency to reject the null hypothesis ) , there are other test statistics which are most widely used ( e.g. , root mean squared error of approximation ( rmsea ) , comparative fit index ( cfi ) , and standardized root mean squared residual ( srmr ) ) . however , comparing the estimated rmsea , cfi , and srmr fit indices presented in table 3 with the threshold values of those indices presented in table 2 suggests that both the estimated models seemed to fit well to the data . that is , active ageing determinant factors model for each gender can be adequately described by those six respective ( to each model ) correlated factors . study , relationships of indicator variables with determinant factors of active ageing ( factor structure of active ageing , i.e. , the active ageing determinant factors model ) are different for female and male older persons , separate active ageing index has been estimated for female and male older persons . the calculated mean aais for female and male older persons are 0.64 and 0.61 , respectively . comparing these mean aais with undp 's human development index ( hdi ) , female and male older persons in thailand had moderate level mean aai . female and male older persons have been divided into four groups according to their mean aai ( i.e. , they are divided according to quartiles of mean aai ) ; mean aai for each group has been presented in table 4 . an independent - samples t - test was run to determine if there were differences in mean aai of female and male older persons . this study found that active ageing level of female is bit higher than active ageing level of male older persons . the calculated statistic , t(23799 ) = 21.554 with p < 0.001 , indicates a statistical significant difference of two populations ' mean level of active ageing ( may reject the null hypothesis that there is no difference between the mean of aai of female and male older population ) . it implies that mean active ageing levels of female older population and male older population , in thailand , are significantly different . the calculated average active ageing levels of female and male older persons for every five regions ( bangkok , central , north , northeast , and south ) have been portrayed in figure 3 . region - wise mean active ageing level of older persons represented in figure 3 shows the glimpse of situation only . but distribution of older persons according to aai quartiles and region of residence enables showing the apparent disparities of aal in different regions . distribution of older persons according to aai quartiles and region of residence is provided in tables 5 and 7 . the results in table 5 exhibited that highest proportion of female older persons in bangkok was the lowest group of mean aai whereas in north and northeast region highest proportion was in medium highest group of mean aai . the highest percentage of female older persons in south region was in highest group of mean aai . female older persons from the north and northeast region had equal mean aai and higher mean aai than other regions . comparing with undp 's hdi , overall mean aais of female elderly in all five regions were in moderate level . a one - way anova between female elderly persons was conducted to compare the mean aai for regions ( bangkok , central , north , northeast , and south ) . there was a significant difference of mean aai at the p < 0.001 level for the five regions [ f(4,14364 ) = 56.79 ; p = 0.00 ] . the results of anova found significant differences but could not specify where the significant differences exist . so , post hoc comparisons using bonferroni test were conducted and the results are presented in table 6 . post hoc comparisons by bonferroni test indicated that the mean aai of female older persons is significantly lower in bangkok than all other regions . mean aai of female older persons is significantly higher in central region than bangkok but the first two regions are lower than the other three regions ( north , northeast , and south ) . there is no significant difference in female elderly 's mean aai in north , northeast , and south of thailand . the results in table 7 revealed that highest proportion of male older persons in bangkok and in central region were in the lowest group of mean aai whereas in north region highest proportion were in medium highest group of mean aai . the highest percentage of male older persons in northeast and in south region was in highest group of mean aai . overall , it can be mentioned from the sample results that male older persons from northeast and south region had equal mean aai and higher mean aai than that of other regions . comparing with undp 's hdi , overall mean aai male older persons in all five regions were in moderate level . a one - way anova between male elderly persons was conducted to compare the mean aai for regions ( bangkok , central , north , northeast , and south ) . there was a significant difference of mean aai at the p < 0.001 level for the five regions [ f(4,9427 ) = 28.58 ; p = 0.00 ] . the results of anova found significant differences but could not specify where the significant differences exist . so , post hoc comparisons using bonferroni test were conducted and the results are presented in table 8 . post hoc comparisons by bonferroni test indicated that the mean aai of male older persons is significantly lower in bangkok than all other regions . mean aai of male older persons is significantly higher in central region than bangkok but the first two regions ( bangkok and central ) are lower than the other three regions ( north , northeast , and south ) . male older persons ' mean aai is significantly similar in north , northeast , and south region . as it has been stated and also proved that active ageing determinant factors model varies for female and male older persons , their active ageing level also varies ; for instance , results revealed that the overall mean aai for female and male older persons was revealed as 0.64 and 0.61 , respectively . these estimates proved the hypothesis regarding the one cross - cutting determinant , gender , ( i.e. , active ageing determinant factors model and active ageing level vary depending on gender in thailand ) as true . the numerical values of aai for both female and male older persons must aim for further improvement . because active ageing index shows that even top performing female older persons still fall short by almost 36% to the highest desired status possible ( i.e. if we look at region - wise active ageing level , then it is evident that active ageing level of older persons in central region is comparatively lower than other regions . results for comparing active ageing level in different regions revealed that there were disparities in mean aai for female older population . female older persons from bangkok region had the lowest level of mean aai among the five regions . even though mean aai of female older persons in the three regions ( north , northeast , and south ) seemed significantly the same , female older persons in central region had significantly lower mean aai than the north , northeast , and south region . on the other hand , as stated earlier that overall estimated mean aai of male older persons is lower than female older persons , it also revealed from the results that the above is also true for every five regions . also , the test results for regional differences for mean aai of male older persons follow the same pattern as for female older persons . as active ageing level in thailand is not high ( evidenced as far behind the goal ) , so , for promoting active ageing , focus should be given to the identified determinant factors of active ageing . as determinant factors are latent , hence focus should be given to their corresponding indicator variables ( i.e. , measured variables ) aiming to promote active ageing . to make results more understandable , relation of directly measured variables with active ageing level should be interpreted which may be helpful for other researchers and policy makers . using measured variables for each model , correlations between aai and measured variables could be estimated . correlation coefficient between measured variables and aai for older persons has been calculated in the study by haque et al . 's study , so recommendations depending on the results regarding correlation coefficients ( as in haque et al 's study urged that good health condition , opportunities of longer working lives , continuous income , participation in social group(s ) , and lifelong learning influence active ageing among the thai older persons . so , an integrated active ageing package ( iaap ) would be helpful for increasing older persons ' active ageing level . the iaap should include policies for older persons to improve their health and economic security , to promote participation in social groups and longer working lives , and to arrange learning programs . as active ageing level of older persons in thailand is not high , so , some policy recommendations should be considered to increase the active ageing level of future older persons . the thai government 's national policy on the elderly ( npe ) should include a new strategy on increasing active ageing level of elderly . special emphasis should be given to central region , as older persons in central region are lower in respect to active ageing level compared to other regions . policy should be focused for older persons on improving health and economic security , on promoting participation in social groups and longer working lives , and on arranging education ( e.g. , lifelong learning ) programs for increasing their active ageing level . all the above policies should be integrated into one package as integrated active ageing package ( iaap ) . along with the policy for extension of compulsory retirement age , incentives for elderly workers , and incentives for employers ( who employed older persons ) , the iaap should include some programs . the iaap programs may include health programs curative care for illness and continuous mental health care , easy access for assistive devices ( e.g. , walker / mover , eye glasses ) , and preventive and promotive health measures such as exercise and annual health checkup ; community and elderly group participation programs ; tobacco cessation counseling program ; special knowledge and information for quality of life development program for elderly ; and vocational training ( including occupational retraining ) program . the proposed strategy , for npe , on increasing active ageing level of older persons can be fulfilled by implementing the iaap . the ministry of social development and human security ( mosdhs ) , thailand , may lead the implementation of iaap . the mosdhs may collaborate with the ministry of health and the department of non - formal education for full implementation of those programs . implementation of iaap would be helpful for increasing active ageing level of older persons which , in turn , is helpful for prolonging good health and independent living of older persons , that is , for increasing the quality of older persons ' lives .

active ageing level and its discrepancy in different regions ( bangkok , central , north , northeast , and south ) of thailand have been examined for prioritizing the policy agenda to be implemented . attempt has been made to test preliminary active ageing models for thai older persons and hence active ageing index ( aai , ranges from 0 to 1 ) has been estimated . using nationally representative data and confirmatory factor analysis approach , this study justified active ageing models for female and male older persons in thailand . results revealed that active ageing level of thai older persons is not high ( mean aais for female and male older persons are 0.64 and 0.61 , resp . , and those are significantly different ( p < 0.001 ) ) . mean aai in central region is lower than north , northeast , and south regions but there is no significant difference in the latter three regions of thailand . special emphasis should be given to central region and policy should be undertaken for increasing active ageing level . implementation of an integrated active ageing package ( iaap ) , containing policies for older persons to improve their health and economic security , to promote participation in social groups and longer working lives , and to arrange learning programs , would be helpful for increasing older persons ' active ageing level in thailand .

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

active ageing ( aa ) is the global goal of present ageing world for meeting the challenges of older persons and for improving their quality of life . active ageing can be explained as a concept and walker ( 2002 ) has defined aa as profound strategy to maximize participation and wellbeing as people grow older . the concept of aa can be encapsulated as engaged in life and it has been recognized as a latent construct which has no specific clear dependent variable to measure it and it may be determined by various latent ( unobserved ) factors . the pace of recent population ageing in thailand is faster than other asian countries and even far more faster than developed countries in the west [ 4 , 5 ] , and proportion of elderly ( aged 60 and over ) is projected to reach more than 30% within the next three decades in thailand . a rapid proceeding pace of population ageing , along with demographic changes in thailand , is posing critical challenges for thai government to maintain economic growth , social stability , and living standard of people in the nation . all determinant factors of aa with their indicators ( hence active ageing level , aal ) need to be better understood for developing policies and programs focused on active ageing in an ageing society . moreover , comparison of aal regarding cross - cutting determinants of aa , gender and culture , should be unveiled for better policy implications for the older persons and for the nation as a whole . using exploratory factor analysis , one study by haque et al . found active ageing determinant factors models with six determinant factors for female and male older persons in thailand . but the study did not validate the models ( i.e. ( or any other study so far in knowledge ) did not compare aal for culture of different regions in thailand . , urban or rural , different region of a country ) of individuals . though thailand is perceived as southeast asia 's most ethnically homogeneous nation state and strong unity of thai culture , thailand government 's use of regional terms ( central , north , northeast , and south ) indicates some regional cultural diversity in thailand . despite the important strength and unity of thai culture , so , doing research or formulating policies on active ageing in thailand , the variations of active ageing depending on region of residence ( central , north , northeast , and south ) should be considered or should be borne in mind . the objective of this study was to gain empirical knowledge on active ageing determinant factors model for thai older persons including their active ageing level . the main specific objectives of this study are ( i ) to test the validity of active ageing determinant factors model for thai older persons and ( ii ) to test the similarity of active ageing level for various regions ( bangkok , central ( excluding bangkok ) , north , northeast , and south ) in thailand . the data for this study come from a nationally representative sample from the 2011 survey of older persons in thailand ( sopt ) . the data have been collected in sopt by using stratified two - stage sampling method . the 2011 sopt is a nationally representative survey which covers data for population subgroups or geographic subareas ( e.g. further details of the methodology , including sampling strategy , for sopt are available at nso 's report on the 2011 survey of older persons in thailand . study ) aged 60 years and over ( n = 23,801 ; female = 14,369 and male = 9,432 ) . for validating the active ageing determinant factors structure in thailand ( suggested by haque et al . ) , the variables used for this study in confirmatory factor analysis ( cfa ) are provided in table 1 . , a pioneer study for finding a model for active ageing determinants , suggested two separate determinant factor structures for each gender . using the indicator variables , listed in table 1 , the suggested active ageing determinant factors models of female and male older persons are provided in figures 1 and 2 , respectively . in the hypothesized models in figures 1 and 2 , the error terms of indicator variables are indicated by . the asterisks indicate parameters ( regression coefficient ( standardized ) , covariances of independent variables in the model ) to be estimated . meanwhile , error terms in cfa are considered as independent variables and their variances should be estimated . unidirectional arrows in the hypothesized models indicate directional linear influences ( i.e. , linear effect of one variable on another ) , and both - way directional arrows indicate correlation between connected variables but no directional influence of one on others is hypothesized . maximum likelihood ( ml ) estimation method is currently one of the most used methods for parameter estimation . as this paper has no aim to estimate the parameters of those models ( but has aim to evaluate those models ) , any estimated model should be tested for how well the model fits to the sample data . this study used chi - square ( ) , root mean squared error of approximation ( rmsea ) , comparative fit index ( cfi ) , and standardized root mean squared residual ( srmr ) . the is a traditional measure of overall model fit ( it assesses the magnitude of differences between sample and estimated covariance matrices ) . that is , the null hypothesis for model evaluation is as h0 : there is no difference between sample covariance and estimated covariance ; that is , the model fits well to the data . active ageing level can be exhibited by computing the active ageing index ( aai ) of older persons . the aai ranges from 0 to 1 and a higher value of aai indicates higher active ageing level . ( 2016 ) has suggested that aai should be calculated separately for female and male older persons in thailand . using nationally representative sample and exploratory factor analysis ( efa ) , haque et al . the weakness of results from a model constructed from efa is that there is no scope to test the model fit . haque and colleagues study used only efa and the model ( from which aai has been obtained ) was not validated . for overcoming the weakness in efa , the study by haque and his colleagues used efa in thai national data for establishing the preliminary active ageing model of older persons and the study has identified six determinant factors for each gender . using the same factors ( with their corresponding indicator variables ) as in haque et al . 's study , this study runs the model in cfa framework ( in stata 12 ) which provides the predicted factor scores of each factor . from the predicted factor score , the factor index for each factor has been estimated using the following formula : index of fi:(1)fi = score of fiminscore of fimaxscore of fiminscore of fi , where fi is the ith factor . calculation of the aai , from cfa , used the following formula:(2)aai=i=1n=6fi6,where fi is the index of factor i ( n = 6 , because there were 6 factors ) . then an overall average aai has been calculated using all values of aai for all individuals . because of social change over time and of cultural diversities in different places , possible changes may occur in individual aal over time , as has occurred in other aspects of individual or population in thailand ; for example , between 1994 and 2007 thailand experienced rapid social changes ( population ageing , socioeconomic development , health policies , etc . ) as has been stated in introduction , there exists cultural variability in thailand depending on region of residence ( bangkok , central , north , northeast , and south ) . it also may be hypothesized that under the circumstances of cultural diversities in different cultural settings , there would be variability of aa level in various cultural contexts , that is , in various regions within a country . aa level quartiles would be the one method of comparing changes in the aal at a community over time or for comparing it in different places or for groups . each quartile for individuals , in whole thailand , for instance , contains 25% of individuals ( along with specific mean level of aai ) . , bangkok , central , north , northeast , and south ) . this quartile method of comparison will provide only the overview of difference(s ) but is unable to test the significance of the differences . similarity with significance of aa levels in different regions could be tested by using one - way analysis of variance ( anova ) . one - way anova is appropriate for one categorical variable with k levels ( e.g. , region of residence with 5 levels in this study ) or k groups and there should be a population of interest for which there is a quantitative variable for each of the k levels of the categorical variable ( e.g. , aai in this study ) . the aais for each group have mean parameters ( population means ) that we may label as 1 through k . the number of individuals ( older persons ) in group i ( 1 i k ) is defined as ni and n = i=1ni is the total sample size . the null hypothesis to be tested in one - way anova states that all the population means are equal ; that is , h0 : 1 = 2 = 3 = 4 = 5 ( for 5 regions of this study ) . so , post hoc comparisons using a built - in test in stata ( e.g. it has been stated , in materials and methods , that the model fit statistics can be obtained after the model estimation in cfa . the hypothesized models have been run in cfa in stata 12 and the obtained model fit indices are summarized in table 3 . but the statistic is very sensitive to sample size ; usually for large sample ( which is the case for two models ) it shows significant result which rejects the null hypothesis ( h0 : there is no difference between sample covariance and estimated covariance ; i.e. , tendency to reject the null hypothesis ) , there are other test statistics which are most widely used ( e.g. , root mean squared error of approximation ( rmsea ) , comparative fit index ( cfi ) , and standardized root mean squared residual ( srmr ) ) . however , comparing the estimated rmsea , cfi , and srmr fit indices presented in table 3 with the threshold values of those indices presented in table 2 suggests that both the estimated models seemed to fit well to the data . that is , active ageing determinant factors model for each gender can be adequately described by those six respective ( to each model ) correlated factors . study , relationships of indicator variables with determinant factors of active ageing ( factor structure of active ageing , i.e. , the active ageing determinant factors model ) are different for female and male older persons , separate active ageing index has been estimated for female and male older persons . the calculated mean aais for female and male older persons are 0.64 and 0.61 , respectively . comparing these mean aais with undp 's human development index ( hdi ) , female and male older persons in thailand had moderate level mean aai . female and male older persons have been divided into four groups according to their mean aai ( i.e. , they are divided according to quartiles of mean aai ) ; mean aai for each group has been presented in table 4 . an independent - samples t - test was run to determine if there were differences in mean aai of female and male older persons . this study found that active ageing level of female is bit higher than active ageing level of male older persons . the calculated statistic , t(23799 ) = 21.554 with p < 0.001 , indicates a statistical significant difference of two populations ' mean level of active ageing ( may reject the null hypothesis that there is no difference between the mean of aai of female and male older population ) . it implies that mean active ageing levels of female older population and male older population , in thailand , are significantly different . the calculated average active ageing levels of female and male older persons for every five regions ( bangkok , central , north , northeast , and south ) have been portrayed in figure 3 . region - wise mean active ageing level of older persons represented in figure 3 shows the glimpse of situation only . but distribution of older persons according to aai quartiles and region of residence enables showing the apparent disparities of aal in different regions . distribution of older persons according to aai quartiles and region of residence is provided in tables 5 and 7 . the results in table 5 exhibited that highest proportion of female older persons in bangkok was the lowest group of mean aai whereas in north and northeast region highest proportion was in medium highest group of mean aai . the highest percentage of female older persons in south region was in highest group of mean aai . female older persons from the north and northeast region had equal mean aai and higher mean aai than other regions . comparing with undp 's hdi , overall mean aais of female elderly in all five regions were in moderate level . a one - way anova between female elderly persons was conducted to compare the mean aai for regions ( bangkok , central , north , northeast , and south ) . there was a significant difference of mean aai at the p < 0.001 level for the five regions [ f(4,14364 ) = 56.79 ; p = 0.00 ] . the results of anova found significant differences but could not specify where the significant differences exist . post hoc comparisons by bonferroni test indicated that the mean aai of female older persons is significantly lower in bangkok than all other regions . mean aai of female older persons is significantly higher in central region than bangkok but the first two regions are lower than the other three regions ( north , northeast , and south ) . there is no significant difference in female elderly 's mean aai in north , northeast , and south of thailand . the results in table 7 revealed that highest proportion of male older persons in bangkok and in central region were in the lowest group of mean aai whereas in north region highest proportion were in medium highest group of mean aai . the highest percentage of male older persons in northeast and in south region was in highest group of mean aai . overall , it can be mentioned from the sample results that male older persons from northeast and south region had equal mean aai and higher mean aai than that of other regions . comparing with undp 's hdi , overall mean aai male older persons in all five regions were in moderate level . a one - way anova between male elderly persons was conducted to compare the mean aai for regions ( bangkok , central , north , northeast , and south ) . there was a significant difference of mean aai at the p < 0.001 level for the five regions [ f(4,9427 ) = 28.58 ; p = 0.00 ] . post hoc comparisons by bonferroni test indicated that the mean aai of male older persons is significantly lower in bangkok than all other regions . mean aai of male older persons is significantly higher in central region than bangkok but the first two regions ( bangkok and central ) are lower than the other three regions ( north , northeast , and south ) . male older persons ' mean aai is significantly similar in north , northeast , and south region . as it has been stated and also proved that active ageing determinant factors model varies for female and male older persons , their active ageing level also varies ; for instance , results revealed that the overall mean aai for female and male older persons was revealed as 0.64 and 0.61 , respectively . these estimates proved the hypothesis regarding the one cross - cutting determinant , gender , ( i.e. , active ageing determinant factors model and active ageing level vary depending on gender in thailand ) as true . the numerical values of aai for both female and male older persons must aim for further improvement . because active ageing index shows that even top performing female older persons still fall short by almost 36% to the highest desired status possible ( i.e. if we look at region - wise active ageing level , then it is evident that active ageing level of older persons in central region is comparatively lower than other regions . results for comparing active ageing level in different regions revealed that there were disparities in mean aai for female older population . female older persons from bangkok region had the lowest level of mean aai among the five regions . even though mean aai of female older persons in the three regions ( north , northeast , and south ) seemed significantly the same , female older persons in central region had significantly lower mean aai than the north , northeast , and south region . on the other hand , as stated earlier that overall estimated mean aai of male older persons is lower than female older persons , it also revealed from the results that the above is also true for every five regions . also , the test results for regional differences for mean aai of male older persons follow the same pattern as for female older persons . as active ageing level in thailand is not high ( evidenced as far behind the goal ) , so , for promoting active ageing , focus should be given to the identified determinant factors of active ageing . as determinant factors are latent , hence focus should be given to their corresponding indicator variables ( i.e. , measured variables ) aiming to promote active ageing . to make results more understandable , relation of directly measured variables with active ageing level should be interpreted which may be helpful for other researchers and policy makers . correlation coefficient between measured variables and aai for older persons has been calculated in the study by haque et al . 's study , so recommendations depending on the results regarding correlation coefficients ( as in haque et al 's study urged that good health condition , opportunities of longer working lives , continuous income , participation in social group(s ) , and lifelong learning influence active ageing among the thai older persons . so , an integrated active ageing package ( iaap ) would be helpful for increasing older persons ' active ageing level . the iaap should include policies for older persons to improve their health and economic security , to promote participation in social groups and longer working lives , and to arrange learning programs . as active ageing level of older persons in thailand is not high , so , some policy recommendations should be considered to increase the active ageing level of future older persons . the thai government 's national policy on the elderly ( npe ) should include a new strategy on increasing active ageing level of elderly . special emphasis should be given to central region , as older persons in central region are lower in respect to active ageing level compared to other regions . policy should be focused for older persons on improving health and economic security , on promoting participation in social groups and longer working lives , and on arranging education ( e.g. , lifelong learning ) programs for increasing their active ageing level . all the above policies should be integrated into one package as integrated active ageing package ( iaap ) . along with the policy for extension of compulsory retirement age , incentives for elderly workers , and incentives for employers ( who employed older persons ) , the iaap should include some programs . , walker / mover , eye glasses ) , and preventive and promotive health measures such as exercise and annual health checkup ; community and elderly group participation programs ; tobacco cessation counseling program ; special knowledge and information for quality of life development program for elderly ; and vocational training ( including occupational retraining ) program . the proposed strategy , for npe , on increasing active ageing level of older persons can be fulfilled by implementing the iaap . the ministry of social development and human security ( mosdhs ) , thailand , may lead the implementation of iaap . the mosdhs may collaborate with the ministry of health and the department of non - formal education for full implementation of those programs . implementation of iaap would be helpful for increasing active ageing level of older persons which , in turn , is helpful for prolonging good health and independent living of older persons , that is , for increasing the quality of older persons ' lives .

clathrin - coated pits represent the major port of entry into higher eukaryotic cells ( conner and schmid , 2003a ) . within the clathrin lattice , the ap2 adaptor complex interprets signals in the cytoplasmic domains of transmembrane cargo , thus allowing its inclusion into coated pits . the ap2 complex is composed of two 100-kd subunits ( - and 2-adaptin ) , a 50-kd subunit ( 2 ) , and a 17-kd subunit ( 2 ; kirchhausen , 1999 ) . while clathrin binds to ap2 via the 2 subunit ( shih et al . , 1995 ) , the 2 subunit interacts with cargo , specifically recognizing tyrosine - based internalization motifs ( yxx ; y , tyrosine ; , bulky hydrophobic ; x , polar ) in the cytoplasmic tails of receptors ( ohno et al . , ap2 recruitment to the plasma membrane appears to be mediated at least partly because of interactions with phosphoinositides ( gaidarov and keen , 1999 ) . recent reports have demonstrated that 2 phosphorylation regulates the interaction of ap2 with cargo . in permeabilized cells , it was shown that 2 phosphorylation was required for the incorporation of transferrin bound to its receptor into newly formed coated pits ( olusanya et al . , 2001 ) . furthermore , transfection of hela cells with mutant forms of 2 , which could not be phosphorylated , resulted in an inhibition of transferrin endocytosis , indicating the importance of 2 phosphorylation in vivo ( olusanya et al . , , measuring affinities of ap2 for peptides containing tyrosine - based internalization motifs , demonstrated that 2 phosphorylation significantly enhances the association of ap2 with these motifs ( ricotta et al . , 2002 ) . the x - ray structure of the ap2 complex ( lacking the hinge and ear domains of - and 2-adaptin ) has revealed that the binding site for cargo on 2 is partially blocked by 2-adaptin in the unphosphorylated complex ( collins et al . , 2002 ) . this suggests that a conformational change is required to reveal the binding site , and such a change could be effected by phosphorylation . in particular , the phosphorylation site on 2 , thr156 ( pauloin and thurieau , 1993 ) , is located on an exposed loop of 2 and is thus ideally situated to elicit such a change ( collins et al . , 2002 ) . interestingly , the paradigm of phosphorylation of subunits regulating cargo interactions has been further supported by the demonstration that phosphorylation of 1 , the medium subunit of the ap1 adaptor complex found in clathrin - coated vesicles budding from the tgn , also enhances its association with cargo ( ghosh and kornfeld , 2003 ) . these data support the premise that reversible cycles of phosphorylation are important in the control of the coated vesicle cycle . however , they raise the question of how 2 phosphorylation is itself regulated so that ap2 adaptors are activated only in the correct functional context . previous reports have shown that ap2 within clathrin coats has a higher affinity for sorting signals , suggesting that the processes of coated vesicle formation might be coupled to cargo sorting ( rapoport et al . , 1997 ) . here , we provide a mechanistic explanation for how this occurs by showing that clathrin can directly regulate the levels of phosphorylated 2 in vivo and in vitro via activation of the 2 kinase . aak1 is a likely candidate for the kinase that phosphorylates 2 in vivo and is closely associated with ap2 ( conner and schmid , 2002 ; ricotta et al . , 2002 ) . it is composed of an nh2-terminal serine / threonine kinase domain and a cooh - terminal domain containing motifs mediating both ap2 binding ( owen et al . , 1999 ) and clathrin binding ( morgan et al . , 2000 ) . addition of purified clathrin to incubations containing 2 kinase and ap2 adaptor complex resulted in a pronounced increase in 2 phosphorylation ( fig . 1 a ) . to investigate the mechanism of kinase activation , we isolated 2 kinase and ap2 under conditions that dissociate the kinase from the ap2 complex ( pauloin and thurieau , 1993 ) , and measured 2 kinase activity in the presence of increasing concentrations of clathrin and ap2 substrate ( fig . these data showed that clathrin activates the 2 kinase by reducing the apparent km for ap2 without affecting the apparent vmax ( fig . clathrin may either increase the affinity of the kinase for 2 or may remove a competitive inhibitor , e.g. , a pseudosubstrate as is common for many serine / threonine kinases ( kemp and pearson , 1991 ) . as a member of the ark / prk family of protein kinases , 2 kinase recognizes the consensus sequence ( l(i)xxqxtg ) ( pauloin and thurieau , 1993 ; smythe and ayscough , 2003 ) , and thus one potential pseudosubstrate is the sequence lvnqslg at the cooh terminus . in the aak1 primary sequence , the nh2- and cooh - terminal domains are connected via a long central region unusually rich in prolines , alanines , and glutamines ( conner and schmid , 2002 ) . sequences with this composition are generally flexible ( perham et al . , 1987 ) . hence , this region is ideally suited to allow the activated nh2-terminal kinase domain the freedom of movement needed to phosphorylate 2 on ap2 molecules within the tight confines of a coated pit , perhaps while tethered ( via its cooh - terminal domain ) to a clathrin molecule ( see fig . ( a ) autoradiography showing incorporation of p into 2 after incubation of the 2 kinase with ap2 in the presence or absence of clathrin . ( b ) the activity of the 2 kinase was assayed using ap2 as a substrate in the presence of increasing concentrations of clathrin as indicated . results are from a typical experiment where each point is the mean of two samples that differed by < 10% . the inset shows a double - reciprocal plot of 1/v versus 1/[ap2 ] for clathrin activation of 2 kinase . ( c ) kinase assay showing the effect of adding clathrin , heavy chain only , light chain , or reconstituted heavy and light chains . results are from a typical experiment where each point is the mean and range of two samples . the coomassie gels indicate the relative proportions of clathrin heavy and light chains added to each incubation . results are from a typical experiment where each point is the mean and range of two samples . however , previous reports have suggested that clathrin light chains might regulate 2 phosphorylation ( pauloin and jolles , 1984 ) . to investigate whether light chains could activate the purified kinase , we separated clathrin heavy chain and light chains as described previously ( winkler and stanley , 1983 ) , and tested each separately and together in a kinase assay . 1 c ; light chains prepared by heat denaturation were also inactive [ unpublished data ] ) . this result is expected given the spatial organization of coated pits with light chains on the outside of the lattice and is consistent with the model described above . to rule out the possibility that the enhancement of 2 phosphorylation was caused by clathrin aggregating the ap2 complexes , we tested the clathrin activation of 2 kinase using a peptide substrate corresponding to residues 148161 of 2 and found that clathrin could also enhance phosphorylation of this substrate ( fig . , we generated anti - peptide antibodies to a phosphopeptide that corresponds to the thr156 phosphorylation site on 2 . in addition , we purified ap2 from bovine brain , under conditions whereby 2 kinase remains associated with the ap2 complex ( campbell et al . untreated ap2 , or ap2 that had been incubated with mgatp , were immunoblotted either with an antibody that recognizes the brain - specific insert of -adaptin or with the anti - phospho - thr156 2 antibodies ( fig . although blotting with the -adaptin antibodies showed that equal amounts of ap2 were present in each lane , immunoreactivity with anti - phospho - thr156 2 antibodies was maximal where ap2 had been incubated with mgatp ( fig . 2 a , lane 2 ) . treatment of the phosphorylated complex with alkaline phosphatase resulted in a loss of immunoreactivity ( fig . 2 a , lane 3 ) . reactivity against phosphorylated 2 was also lost when the antibody was incubated in the presence of a 100-fold molar excess of phosphopeptide ( fig . 2 a , lane 4 ) , but not when incubated with nonphosphorylated peptide ( fig . 2 a , lane 5 ) . together , these data confirm that the antibody recognizes phospho - thr156 2 only . aps were incubated with mgatp for 15 min at 30c , and then in the presence or absence of calf alkaline phosphatase for 10 min at 30c before immunoblotting . the blots were probed with antibodies that recognize the brain - specific insert of -adaptin or with antibodies generated against phospho - thr156 2 . lane 4 , anti - phospho - thr156 2 antibodies were incubated in the presence of a 100-fold molar excess of phosphorylated peptide used to generate the antibodies . lane 5 , anti - phospho - thr156 2 antibodies were incubated with 100-fold molar excess of the nonphosphorylated peptide . ( b ) the sequestration of biotinylated transferrin into new clathrin coated pits was measured by inaccessibility to avidin with the modifications described in materials and methods . ( c ) permeabilized cell membranes incubated as indicated were reisolated and subjected to immunoblotting with antibodies against the brain - specific insert of -adaptin or with anti - phospho - thr156 2 antibodies . ( d ) a histogram showing quantitation of the levels of 2 phosphorylation in permeabilized cell membranes in the presence or absence of clathrin . permeabilized cell assay mixes were incubated aps that were phosphorylated before either with atp ( middle ) or [ p]atp ( bottom ) and in the absence or presence of microcystin as indicated . the membranes were reisolated at the indicated times , and after sds - page , the amount of phosphorylated 2 was measured using phospho - thr156 2 antibodies or by autoradiography . we used these phosphospecific antibodies to investigate whether clathrin might regulate 2 phosphorylation in the context of a coated pit using a permeabilized cell assay that reconstitutes the sequestration of cargo into newly formed coated pits ( smythe et al . briefly , this assay uses biotinylated transferrin that binds to transferrin receptors on the cell surface . the sequestration of biotinylated transferrin into newly formed coated pits is measured by inaccessibility to exogenously added avidin . in the permeabilized cell system , new coated pit formation is dependent on the addition of purified ap2 when ap2 is limiting in the cytosol . previous experiments have demonstrated that 2 phosphorylation is required for ligand sequestration into newly formed coated pits ( olusanya et al . , 2001 ) and that clathrin required for new pit formation is recruited from the permeabilized cell membranes ( mclauchlan et al . , 1998 ) . we modified the assay system to make it more dependent on the addition of exogenous clathrin by incubating membrane and soluble components with the protein phosphatase inhibitor microcystin ( see materials and methods ) . 2 b shows that when treated under these conditions , exogenous clathrin is now required for coat protein dependent stimulation of sequestration , presumably because dephosphorylation is required for clathrin recruitment from the permeabilized cell membranes . b were reisolated from the assay mixes and subjected to immunoblotting ( fig . 2 c ) . the blots were probed with an antibody that recognizes the brain - specific insert of -adaptin , and so recognized only ap2 that was added to the assay mix in purified form or in cytosol . incubations performed in the presence of aps show that added ap2 associated with the permeabilized cell membranes and this amount was independent of added clathrin . however , phospho - thr156 2 immunoreactivity was significantly increased ( greater than twofold ; fig . 2 d ) in those incubations containing clathrin and where ligand sequestration occurs . this indicates that clathrin is needed to maximize phospho - thr156 2 at the plasma membrane , and this correlates with the ability of newly formed clathrin - coated pits to sequester ligand . when permeabilized cell membranes were incubated with aps prelabeled with [ p]atp , there was a time - dependent decline in radioactivity that was prevented by microcystin ( fig . when this experiment was repeated with aps prelabeled with cold atp , there was an initial decline in phospho-2 immunoreactivity followed by an increase ( fig . however , the level of phospho-2 immunoreactivity after 30 min in the absence of microcystin was less than in its presence for the same time period ( fig . 2 e , middle ) . this strongly suggests that the level of phospho-2 is determined by the net activities of both kinase and phosphatases . to investigate the connection between clathrin and 2 phosphorylation in vivo , we examined the level of phospho - thr156 2 in the chicken dt40 b cell line dko - r . in this cell line , both endogenous alleles of clathrin heavy chain have been inactivated by homologous recombination and replaced with clathrin under the control of a tetracycline - responsive repressor ( wettey et al . , 2002 ) . from est database searches ( http://chick.umist.ac.uk ) , we confirmed that the amino acid sequence of chicken 2 is 98% identical to the mammalian protein , and shows perfect conservation in the region surrounding thr156 ( unpublished data ) . we analyzed the distribution of phosphorylated 2 in assembled and unassembled pools of clathrin prepared from dko - r cells under clathrin - expressing conditions . 3 a ) , consistent with the high endocytic capacity of the dko - r cell line ( wettey et al . , 2002 ) . immunoblotting of 2 showed that the ap2 complex distributed more equally between the assembled and unassembled fractions . however , almost all the phospho - thr156 2 was found in the assembled fraction , indicating that 2 phosphorylation occurs primarily when ap2 is associated with the assembled pool of clathrin . in dko - r cells treated with doxycycline for 96 h , 3 b ) . using a more sensitive clathrin triskelion elisa ( cheetham et al . , 1996 ) , residual expression corresponding to 0.5% of the unrepressed level was detected under these conditions ( unpublished data ) . as judged by immunoblotting , clathrin depletion did not affect the level of 2 ( fig . 3 b ) or , in separate experiments , - and -adaptin ( unpublished data ) . however , the level of 2 phosphorylation was strikingly reduced by clathrin removal ( fig . dko - r cells were homogenized , and membrane ( a ) and cytosolic ( u ) fractions were prepared to separate assembled and unassembled pools of clathrin . the fractions were subjected to sds - page , and after transfer to nitrocellulose were immunoblotted with antibodies to clathrin , 2 , and phospho - thr156 2 . ( b ) phosphorylated 2 is substantially reduced in clathrin - depleted dko - r cells . exponentially growing chicken dko - r cells treated for 96 h without ( lane 1 ) or with ( lane 2 ) 0.1 g / ml doxycycline were lysed and subjected to immunoblotting with antibodies to clathrin , 2 , and phospho - thr156 2 . by activating the 2 kinase , we propose that clathrin provides a crucial regulatory link between ap2 phosphorylation and functional coated pit assembly ( fig . 4 ) . to explain the low level of phospho - thr156 2 found in cytosolic ap2 , we suggest that the 2 kinase associated with cytosolic ap2 has a low basal activity . this implies that ap2 must first bind the plasma membrane via a cargo - independent interaction , and is consistent with the observation that , in the permeabilized cell system , ap2 bound membranes independently of clathrin , although ligand sequestration was facilitated by clathrin via its effect on 2 phosphorylation . it is also consistent with our observation that in dko - r cells , clathrin depletion did not prevent the binding of ap2 to the plasma membrane , but did inhibit endocytosis ( wettey et al . , 2002 ) . one likely possibility for this cargo - independent binding is via the phosphoinositide - binding sites of -adaptin ( gaidarov and keen , 1999 ) and 2 ( rohde et al . , 2002 ) . this could provide an initial targeting of ap2 as the plasma membrane is enriched in phosphatidylinositol 4,5-bisphosphate . once ap2 was associated with the correct membrane this would induce a conformational change in ap2 , allowing 2 to bind cargo receptors . our model explains the long - standing observation that the proteolytic sensitivity of 2 dramatically increases when ap2 is incorporated into clathrin cages ( matsui and kirchhausen , 1990 ) , and is consistent with previous experiments showing that the incorporation of ap2 into clathrin coats increases its affinity for cargo ( rapoport et al . , 1997 ) . it will be of considerable interest to investigate if clathrin also regulates 1cargo interactions via a similar mechanism . recent reports have shown that overexpression of wild - type and kinase - dead aak1 resulted in an inhibition of transferrin endocytosis . a likely interpretation , consistent with our results , is that excess aak1 , independently of its kinase activity , acts in a dominant - negative fashion by binding either ap2 and/or clathrin and preventing their interaction . ap2 recruitment to the membrane occurs independently of 2 thr156 phosphorylation , most likely via interactions with phosphatidylinositol 4,5-bisphosphate and other protein components at the membrane . at this stage , the 2 kinase has basal activity ( red circles ) , and hence , there is minimal cargo recruitment . after clathrin recruitment , the 2 kinase is activated ( green circles ) and maximal cargo recruitment occurs . the insert indicates a possible mechanism by which clathrin may activate the 2 kinase , which is most likely to be aak1 , by inducing a conformational change in aak1 so that the flexible p- , a- , g- , q - rich domain can extend allowing the nh2-terminal kinase domain free access to phosphorylate 2 within the coated pit . in summary , our results point to a novel regulatory feature for clathrin that is independent of its structural role and that provides elegant spatial control of ap2 and cargo interactions , ensuring that ap2 is only activated at the correct cellular location and in the correct functional context . antibodies to the brain - specific insert of -adaptin ( aa 706722 ) were prepared as described previously ( ball et al . , 1995 ) . antibodies to the phosphorylated form of 2 were generated by injection of the phosphopeptide ceeqsqitsqvt(p)gqigwrrr coupled to bsa into a sheep . mabs to 2 were obtained from transduction laboratories and were a gift from margaret robinson , cambridge university , uk . coat proteins were prepared by differential centrifugation followed by gel filtration as described previously ( campbell et al . , 1984 ) . further preparation of ap2 from the 2 kinase was performed on hydroxyapatite as described previously ( pauloin and thurieau , 1993 ) . some batches of ap2 and kinase were prepared by linda adams ( university of dundee , dundee , uk ) and sophia semerdjieva ( university of sheffield , sheffield , uk ) . clathrin heavy and light chains were separated as described previously ( winkler and stanley , 1983 ) . kinase assays ( 20-l final volume ) contained 2 kinase , ap2 , 1 mm mgcl2 , and 100 m [p]atp ( specific activity of 0.5 ci / nmol ) in 10 mm tris , ph 7.5 . incubations were performed for 10 min at 30c , reactions were stopped by addition of 5 laemmli sample buffer , and the samples were heated at 60c for 5 min before electrophoresis . gels were fixed , dried , and radioactivity was quantified using a phosphorimager ( fuji ) followed by analysis using fuji science lab . incubations were as described above , except that atp was used at a specific activity of 2 ci / nmol ; incubations were processed as described previously ( cross and smythe , 1998 ) . the permeabilized cell assays were performed as described previously ( smythe et al . , 1994 ; mclauchlan et al . , 1998 ) with the following modifications : assay mixes were pretreated with 1 m microcystin at 4c for 10 min before allowing the reaction to proceed . maintenance of dko - r cells and doxycycline treatment were as described previously ( wettey et al . , 2002 ) . 5 10 cells were lysed in pbs containing 0.1% triton x-100 , 0.2 mm pmsf , and 50 mm naf , and were centrifuged at 1,000 g for 5 min . to separate assembled from unassembled clathrin , cells were lysed in 40 mm hepes , ph 7.4 , 75 mm kcl , 4.5 mm mgcl2 , 1.5 mm cacl2 , 0.5% np-40 , 0.01% pmsf , and 50 mm naf , and were centrifuged at 1,000 g. after centrifugation at 100,000 g for 20 min , the supernatant ( unassembled clathrin ) was removed and the pellet ( assembled clathrin ) was resuspended in the same volume of lysis buffer as the supernatant ( cheetham et al . , 1996 ) . both fractions were subjected to immunoblotting as above . antibodies to the brain - specific insert of -adaptin ( aa 706722 ) were prepared as described previously ( ball et al . , 1995 ) . antibodies to the phosphorylated form of 2 were generated by injection of the phosphopeptide ceeqsqitsqvt(p)gqigwrrr coupled to bsa into a sheep . mabs to 2 were obtained from transduction laboratories and were a gift from margaret robinson , cambridge university , uk . coat proteins were prepared by differential centrifugation followed by gel filtration as described previously ( campbell et al . , 1984 ) . this allowed separation of clathrin from the aps . further preparation of ap2 from the 2 kinase was performed on hydroxyapatite as described previously ( pauloin and thurieau , 1993 ) . some batches of ap2 and kinase were prepared by linda adams ( university of dundee , dundee , uk ) and sophia semerdjieva ( university of sheffield , sheffield , uk ) . clathrin heavy and light chains were separated as described previously ( winkler and stanley , 1983 ) . kinase assays ( 20-l final volume ) contained 2 kinase , ap2 , 1 mm mgcl2 , and 100 m [p]atp ( specific activity of 0.5 ci / nmol ) in 10 mm tris , ph 7.5 . incubations were performed for 10 min at 30c , reactions were stopped by addition of 5 laemmli sample buffer , and the samples were heated at 60c for 5 min before electrophoresis . gels were fixed , dried , and radioactivity was quantified using a phosphorimager ( fuji ) followed by analysis using fuji science lab . incubations were as described above , except that atp was used at a specific activity of 2 ci / nmol ; incubations were processed as described previously ( cross and smythe , 1998 ) . the permeabilized cell assays were performed as described previously ( smythe et al . , 1994 ; mclauchlan et al . , 1998 ) with the following modifications : assay mixes were pretreated with 1 m microcystin at 4c for 10 min before allowing the reaction to proceed . maintenance of dko - r cells and doxycycline treatment were as described previously ( wettey et al . , 2002 ) . 5 10 cells were lysed in pbs containing 0.1% triton x-100 , 0.2 mm pmsf , and 50 mm naf , and were centrifuged at 1,000 g for 5 min . , cells were lysed in 40 mm hepes , ph 7.4 , 75 mm kcl , 4.5 mm mgcl2 , 1.5 mm cacl2 , 0.5% np-40 , 0.01% pmsf , and 50 mm naf , and were centrifuged at 1,000 g. after centrifugation at 100,000 g for 20 min , the supernatant ( unassembled clathrin ) was removed and the pellet ( assembled clathrin ) was resuspended in the same volume of lysis buffer as the supernatant ( cheetham et al . , 1996 ) . both fractions were subjected to immunoblotting as above .

endocytic cargo such as the transferrin receptor is incorporated into clathrin - coated pits by associating , via tyrosine - based motifs , with the ap2 complex . cargo ap2 interactions occur via the 2 subunit of ap2 , which needs to be phosphorylated for endocytosis to occur . the most likely role for 2 phosphorylation is in cargo recruitment because 2 phosphorylation enhances its binding to internalization motifs . here , we investigate the control of 2 phosphorylation . we identify clathrin as a specific activator of the 2 kinase and , in permeabilized cells , we show that ligand sequestration , driven by exogenous clathrin , results in elevated levels of 2 phosphorylation . furthermore , we show that ap2 containing phospho-2 is mainly associated with assembled clathrin in vivo , and that the level of phospho-2 is strongly reduced in a chicken b cell line depleted of clathrin heavy chain . our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-2 levels .

Introduction Results and discussion Materials and methods Antibodies Clathrin, AP2, and 2 kinase Kinase assays Permeabilized cell assay Growth and preparation of DKO-R cells

clathrin - coated pits represent the major port of entry into higher eukaryotic cells ( conner and schmid , 2003a ) . within the clathrin lattice , the ap2 adaptor complex interprets signals in the cytoplasmic domains of transmembrane cargo , thus allowing its inclusion into coated pits . the ap2 complex is composed of two 100-kd subunits ( - and 2-adaptin ) , a 50-kd subunit ( 2 ) , and a 17-kd subunit ( 2 ; kirchhausen , 1999 ) . while clathrin binds to ap2 via the 2 subunit ( shih et al . , 1995 ) , the 2 subunit interacts with cargo , specifically recognizing tyrosine - based internalization motifs ( yxx ; y , tyrosine ; , bulky hydrophobic ; x , polar ) in the cytoplasmic tails of receptors ( ohno et al . recent reports have demonstrated that 2 phosphorylation regulates the interaction of ap2 with cargo . in permeabilized cells , it was shown that 2 phosphorylation was required for the incorporation of transferrin bound to its receptor into newly formed coated pits ( olusanya et al . furthermore , transfection of hela cells with mutant forms of 2 , which could not be phosphorylated , resulted in an inhibition of transferrin endocytosis , indicating the importance of 2 phosphorylation in vivo ( olusanya et al . , , measuring affinities of ap2 for peptides containing tyrosine - based internalization motifs , demonstrated that 2 phosphorylation significantly enhances the association of ap2 with these motifs ( ricotta et al . the x - ray structure of the ap2 complex ( lacking the hinge and ear domains of - and 2-adaptin ) has revealed that the binding site for cargo on 2 is partially blocked by 2-adaptin in the unphosphorylated complex ( collins et al . in particular , the phosphorylation site on 2 , thr156 ( pauloin and thurieau , 1993 ) , is located on an exposed loop of 2 and is thus ideally situated to elicit such a change ( collins et al . interestingly , the paradigm of phosphorylation of subunits regulating cargo interactions has been further supported by the demonstration that phosphorylation of 1 , the medium subunit of the ap1 adaptor complex found in clathrin - coated vesicles budding from the tgn , also enhances its association with cargo ( ghosh and kornfeld , 2003 ) . these data support the premise that reversible cycles of phosphorylation are important in the control of the coated vesicle cycle . however , they raise the question of how 2 phosphorylation is itself regulated so that ap2 adaptors are activated only in the correct functional context . previous reports have shown that ap2 within clathrin coats has a higher affinity for sorting signals , suggesting that the processes of coated vesicle formation might be coupled to cargo sorting ( rapoport et al . here , we provide a mechanistic explanation for how this occurs by showing that clathrin can directly regulate the levels of phosphorylated 2 in vivo and in vitro via activation of the 2 kinase . aak1 is a likely candidate for the kinase that phosphorylates 2 in vivo and is closely associated with ap2 ( conner and schmid , 2002 ; ricotta et al . addition of purified clathrin to incubations containing 2 kinase and ap2 adaptor complex resulted in a pronounced increase in 2 phosphorylation ( fig . to investigate the mechanism of kinase activation , we isolated 2 kinase and ap2 under conditions that dissociate the kinase from the ap2 complex ( pauloin and thurieau , 1993 ) , and measured 2 kinase activity in the presence of increasing concentrations of clathrin and ap2 substrate ( fig . these data showed that clathrin activates the 2 kinase by reducing the apparent km for ap2 without affecting the apparent vmax ( fig . clathrin may either increase the affinity of the kinase for 2 or may remove a competitive inhibitor , e.g. as a member of the ark / prk family of protein kinases , 2 kinase recognizes the consensus sequence ( l(i)xxqxtg ) ( pauloin and thurieau , 1993 ; smythe and ayscough , 2003 ) , and thus one potential pseudosubstrate is the sequence lvnqslg at the cooh terminus . in the aak1 primary sequence , the nh2- and cooh - terminal domains are connected via a long central region unusually rich in prolines , alanines , and glutamines ( conner and schmid , 2002 ) . ( a ) autoradiography showing incorporation of p into 2 after incubation of the 2 kinase with ap2 in the presence or absence of clathrin . ( b ) the activity of the 2 kinase was assayed using ap2 as a substrate in the presence of increasing concentrations of clathrin as indicated . the inset shows a double - reciprocal plot of 1/v versus 1/[ap2 ] for clathrin activation of 2 kinase . ( c ) kinase assay showing the effect of adding clathrin , heavy chain only , light chain , or reconstituted heavy and light chains . the coomassie gels indicate the relative proportions of clathrin heavy and light chains added to each incubation . however , previous reports have suggested that clathrin light chains might regulate 2 phosphorylation ( pauloin and jolles , 1984 ) . to investigate whether light chains could activate the purified kinase , we separated clathrin heavy chain and light chains as described previously ( winkler and stanley , 1983 ) , and tested each separately and together in a kinase assay . this result is expected given the spatial organization of coated pits with light chains on the outside of the lattice and is consistent with the model described above . to rule out the possibility that the enhancement of 2 phosphorylation was caused by clathrin aggregating the ap2 complexes , we tested the clathrin activation of 2 kinase using a peptide substrate corresponding to residues 148161 of 2 and found that clathrin could also enhance phosphorylation of this substrate ( fig . in addition , we purified ap2 from bovine brain , under conditions whereby 2 kinase remains associated with the ap2 complex ( campbell et al . untreated ap2 , or ap2 that had been incubated with mgatp , were immunoblotted either with an antibody that recognizes the brain - specific insert of -adaptin or with the anti - phospho - thr156 2 antibodies ( fig . although blotting with the -adaptin antibodies showed that equal amounts of ap2 were present in each lane , immunoreactivity with anti - phospho - thr156 2 antibodies was maximal where ap2 had been incubated with mgatp ( fig . treatment of the phosphorylated complex with alkaline phosphatase resulted in a loss of immunoreactivity ( fig . reactivity against phosphorylated 2 was also lost when the antibody was incubated in the presence of a 100-fold molar excess of phosphopeptide ( fig . together , these data confirm that the antibody recognizes phospho - thr156 2 only . aps were incubated with mgatp for 15 min at 30c , and then in the presence or absence of calf alkaline phosphatase for 10 min at 30c before immunoblotting . lane 4 , anti - phospho - thr156 2 antibodies were incubated in the presence of a 100-fold molar excess of phosphorylated peptide used to generate the antibodies . lane 5 , anti - phospho - thr156 2 antibodies were incubated with 100-fold molar excess of the nonphosphorylated peptide . ( b ) the sequestration of biotinylated transferrin into new clathrin coated pits was measured by inaccessibility to avidin with the modifications described in materials and methods . ( d ) a histogram showing quantitation of the levels of 2 phosphorylation in permeabilized cell membranes in the presence or absence of clathrin . the membranes were reisolated at the indicated times , and after sds - page , the amount of phosphorylated 2 was measured using phospho - thr156 2 antibodies or by autoradiography . we used these phosphospecific antibodies to investigate whether clathrin might regulate 2 phosphorylation in the context of a coated pit using a permeabilized cell assay that reconstitutes the sequestration of cargo into newly formed coated pits ( smythe et al . in the permeabilized cell system , new coated pit formation is dependent on the addition of purified ap2 when ap2 is limiting in the cytosol . previous experiments have demonstrated that 2 phosphorylation is required for ligand sequestration into newly formed coated pits ( olusanya et al . we modified the assay system to make it more dependent on the addition of exogenous clathrin by incubating membrane and soluble components with the protein phosphatase inhibitor microcystin ( see materials and methods ) . 2 b shows that when treated under these conditions , exogenous clathrin is now required for coat protein dependent stimulation of sequestration , presumably because dephosphorylation is required for clathrin recruitment from the permeabilized cell membranes . the blots were probed with an antibody that recognizes the brain - specific insert of -adaptin , and so recognized only ap2 that was added to the assay mix in purified form or in cytosol . incubations performed in the presence of aps show that added ap2 associated with the permeabilized cell membranes and this amount was independent of added clathrin . this indicates that clathrin is needed to maximize phospho - thr156 2 at the plasma membrane , and this correlates with the ability of newly formed clathrin - coated pits to sequester ligand . however , the level of phospho-2 immunoreactivity after 30 min in the absence of microcystin was less than in its presence for the same time period ( fig . this strongly suggests that the level of phospho-2 is determined by the net activities of both kinase and phosphatases . to investigate the connection between clathrin and 2 phosphorylation in vivo , we examined the level of phospho - thr156 2 in the chicken dt40 b cell line dko - r . in this cell line , both endogenous alleles of clathrin heavy chain have been inactivated by homologous recombination and replaced with clathrin under the control of a tetracycline - responsive repressor ( wettey et al . from est database searches ( http://chick.umist.ac.uk ) , we confirmed that the amino acid sequence of chicken 2 is 98% identical to the mammalian protein , and shows perfect conservation in the region surrounding thr156 ( unpublished data ) . we analyzed the distribution of phosphorylated 2 in assembled and unassembled pools of clathrin prepared from dko - r cells under clathrin - expressing conditions . 3 a ) , consistent with the high endocytic capacity of the dko - r cell line ( wettey et al . immunoblotting of 2 showed that the ap2 complex distributed more equally between the assembled and unassembled fractions . however , almost all the phospho - thr156 2 was found in the assembled fraction , indicating that 2 phosphorylation occurs primarily when ap2 is associated with the assembled pool of clathrin . as judged by immunoblotting , clathrin depletion did not affect the level of 2 ( fig . however , the level of 2 phosphorylation was strikingly reduced by clathrin removal ( fig . dko - r cells were homogenized , and membrane ( a ) and cytosolic ( u ) fractions were prepared to separate assembled and unassembled pools of clathrin . the fractions were subjected to sds - page , and after transfer to nitrocellulose were immunoblotted with antibodies to clathrin , 2 , and phospho - thr156 2 . ( b ) phosphorylated 2 is substantially reduced in clathrin - depleted dko - r cells . exponentially growing chicken dko - r cells treated for 96 h without ( lane 1 ) or with ( lane 2 ) 0.1 g / ml doxycycline were lysed and subjected to immunoblotting with antibodies to clathrin , 2 , and phospho - thr156 2 . by activating the 2 kinase , we propose that clathrin provides a crucial regulatory link between ap2 phosphorylation and functional coated pit assembly ( fig . to explain the low level of phospho - thr156 2 found in cytosolic ap2 , we suggest that the 2 kinase associated with cytosolic ap2 has a low basal activity . this implies that ap2 must first bind the plasma membrane via a cargo - independent interaction , and is consistent with the observation that , in the permeabilized cell system , ap2 bound membranes independently of clathrin , although ligand sequestration was facilitated by clathrin via its effect on 2 phosphorylation . it is also consistent with our observation that in dko - r cells , clathrin depletion did not prevent the binding of ap2 to the plasma membrane , but did inhibit endocytosis ( wettey et al . this could provide an initial targeting of ap2 as the plasma membrane is enriched in phosphatidylinositol 4,5-bisphosphate . once ap2 was associated with the correct membrane this would induce a conformational change in ap2 , allowing 2 to bind cargo receptors . our model explains the long - standing observation that the proteolytic sensitivity of 2 dramatically increases when ap2 is incorporated into clathrin cages ( matsui and kirchhausen , 1990 ) , and is consistent with previous experiments showing that the incorporation of ap2 into clathrin coats increases its affinity for cargo ( rapoport et al . a likely interpretation , consistent with our results , is that excess aak1 , independently of its kinase activity , acts in a dominant - negative fashion by binding either ap2 and/or clathrin and preventing their interaction . ap2 recruitment to the membrane occurs independently of 2 thr156 phosphorylation , most likely via interactions with phosphatidylinositol 4,5-bisphosphate and other protein components at the membrane . at this stage , the 2 kinase has basal activity ( red circles ) , and hence , there is minimal cargo recruitment . after clathrin recruitment , the 2 kinase is activated ( green circles ) and maximal cargo recruitment occurs . the insert indicates a possible mechanism by which clathrin may activate the 2 kinase , which is most likely to be aak1 , by inducing a conformational change in aak1 so that the flexible p- , a- , g- , q - rich domain can extend allowing the nh2-terminal kinase domain free access to phosphorylate 2 within the coated pit . in summary , our results point to a novel regulatory feature for clathrin that is independent of its structural role and that provides elegant spatial control of ap2 and cargo interactions , ensuring that ap2 is only activated at the correct cellular location and in the correct functional context . antibodies to the phosphorylated form of 2 were generated by injection of the phosphopeptide ceeqsqitsqvt(p)gqigwrrr coupled to bsa into a sheep . further preparation of ap2 from the 2 kinase was performed on hydroxyapatite as described previously ( pauloin and thurieau , 1993 ) . kinase assays ( 20-l final volume ) contained 2 kinase , ap2 , 1 mm mgcl2 , and 100 m [p]atp ( specific activity of 0.5 ci / nmol ) in 10 mm tris , ph 7.5 . gels were fixed , dried , and radioactivity was quantified using a phosphorimager ( fuji ) followed by analysis using fuji science lab . incubations were as described above , except that atp was used at a specific activity of 2 ci / nmol ; incubations were processed as described previously ( cross and smythe , 1998 ) . to separate assembled from unassembled clathrin , cells were lysed in 40 mm hepes , ph 7.4 , 75 mm kcl , 4.5 mm mgcl2 , 1.5 mm cacl2 , 0.5% np-40 , 0.01% pmsf , and 50 mm naf , and were centrifuged at 1,000 g. after centrifugation at 100,000 g for 20 min , the supernatant ( unassembled clathrin ) was removed and the pellet ( assembled clathrin ) was resuspended in the same volume of lysis buffer as the supernatant ( cheetham et al . antibodies to the phosphorylated form of 2 were generated by injection of the phosphopeptide ceeqsqitsqvt(p)gqigwrrr coupled to bsa into a sheep . further preparation of ap2 from the 2 kinase was performed on hydroxyapatite as described previously ( pauloin and thurieau , 1993 ) . some batches of ap2 and kinase were prepared by linda adams ( university of dundee , dundee , uk ) and sophia semerdjieva ( university of sheffield , sheffield , uk ) . kinase assays ( 20-l final volume ) contained 2 kinase , ap2 , 1 mm mgcl2 , and 100 m [p]atp ( specific activity of 0.5 ci / nmol ) in 10 mm tris , ph 7.5 . incubations were performed for 10 min at 30c , reactions were stopped by addition of 5 laemmli sample buffer , and the samples were heated at 60c for 5 min before electrophoresis . gels were fixed , dried , and radioactivity was quantified using a phosphorimager ( fuji ) followed by analysis using fuji science lab . incubations were as described above , except that atp was used at a specific activity of 2 ci / nmol ; incubations were processed as described previously ( cross and smythe , 1998 ) . , 1998 ) with the following modifications : assay mixes were pretreated with 1 m microcystin at 4c for 10 min before allowing the reaction to proceed . 5 10 cells were lysed in pbs containing 0.1% triton x-100 , 0.2 mm pmsf , and 50 mm naf , and were centrifuged at 1,000 g for 5 min . , cells were lysed in 40 mm hepes , ph 7.4 , 75 mm kcl , 4.5 mm mgcl2 , 1.5 mm cacl2 , 0.5% np-40 , 0.01% pmsf , and 50 mm naf , and were centrifuged at 1,000 g. after centrifugation at 100,000 g for 20 min , the supernatant ( unassembled clathrin ) was removed and the pellet ( assembled clathrin ) was resuspended in the same volume of lysis buffer as the supernatant ( cheetham et al .

lipid rafts contain lipids , glycosylphosphatidylinositol- ( gpi- ) anchored proteins and various signal transduction proteins [ 1 , 2 ] that engage the microdomain in cell signaling and cell adhesion events . capacitation is a process whereby spermatozoa gain the ability to fertilize through a series of membrane changes including a reorganization of membrane lipids and proteins [ 4 , 5 ] . lipid rafts in head membranes of uncapacitated sperm are uniformly distributed but become restricted to the periacrosomal region during capacitation , suggesting rafts are associated with capacitation . one particular protein involved in cell signaling during capacitation , na k - atpase , is extremely sensitive to its lipid environment and its function is uniquely damaged when sperm are cryopreserved , a process that also reduces fertility . loss of na k - atpase function postthaw , whether caused by cryopreservation - induced direct damage to the protein and/or alterations in the protein 's lipid environment , could cause the loss of fertility . na k - atpase is a ubiquitous transmembrane protein consisting of alpha ( ) and beta ( ) subunits [ 10 , 11 ] , which is well known to function as an ion - transporting enzyme , but also has been recently shown to act as a transmembrane signalling molecule in somatic cells and even more recently in sperm . although na k - atpase is not associated with lipid rafts in brain synaptosomes , mdck cells , or enterocytes , it is enriched in lipid raft fractions of gastric luminal cells , hepatocytes , renal epithelial , outer medulla kidney , and cardiac cells . interestingly , na k - atpase is stimulated in muscle fibers and fish gill epithelium by a relocation which is mediated by lipid rafts [ 19 , 20 ] . the specific effects of lipid environment on na k - atpase in sperm membranes is unknown . liposomes provide a model membrane system to study protein function [ 21 , 22 ] , facilitating assessment of integral membrane proteins without interference from other cell components that may obscure results . many proteoliposome studies attempt to reproduce a typical membrane by mixing a few commercial lipids as a base composition [ 2426 ] , but , logically , using extracted endogenous membrane phospholipids would even more closely mimic the native lipid bilayer including lipid rafts . functional protein reconstitution into vesicles is dependent on method of preparation and lipid composition of the vesicles , necessitating determination of degree of incorporation of a protein into the liposomes before detailed protein function can be reliably and repeatedly evaluated . we hypothesized that lipid composition would affect na k - atpase insertion into liposomes , and therefore measured incorporation , function and orientation of na k - atpase in liposomes made from lipids extracted from the head plasma membrane ( hpm ) of bull sperm , and then altered the lipid composition of the proteoliposomes to determine the effect of lipid composition on the nature of the incorporation of na k - atpase into lipid vesicles . adenosine 5-triphosphate ( atp ) , sodium chloride ( nacl ) , hepes , ouabain octahydrate , -amylase ( type ii - a from bacillus species ) , bovine serum albumin ( bsa ) , dog kidney na k - atpase , osmium tetroxide , imidazole , ammonium molybdate l - histidine , carbonyl cyanide 3-chlorophenylhydrazone ( cccp ) , valinomycin , phosphatidylcholine ( pc ; 16 : 0 , 18 : 0 , 18 : 1 , and 18 : 2 ) , phosphatidylethanolamine ( pe ) , sphingomyelin ( sph ; 18 : 0 ) , and phosphatidylserine ( ps ; 16 : 0 ) were purchased from sigma - aldrich ( mississauga , on , canada ) . n-(dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , triethylammonium salt ( dansyl dhpe ) was from molecular probes . thermo fisher scientific was the supplier for chloroform , methanol , ascorbic acid , hydrochloric acid ( hcl ) , sodium citrate dihydrate , sodium arsenite , and glacial acetic acid . phosphatidylcholine ( 20 : 4 ) , sph ( 16 : 0 ) , ps ( 18 : 0 and 18 : 2 ) , and phosphatidylinositol ( pi ; 18 : 1 ) were purchased from avanti ( alabaster , al ) , and pc ( 22 : 6 ) and pi ( 16 : 0 ) were from matreya ( pleasant gap , pa ) . lipids were extracted from the head plasma membrane ( hpm ) of whole bull sperm ( 33 ejaculates from 14 different bulls ) using established methods [ 8 , 9 ] , pooled , dried , weighed , resolubilized in chloroform methanol ( 2 : 1 , v / v ) , divided into equal aliquots containing 1.5 mg lipids , and stored at 70c under nitrogen gas to provide identical lipid compositions for all liposomes . dog kidney na k - atpase was purified using an ouabain affinity column , freeze dried and stored at 20c . freeze - dried samples were resuspended in hepes - buffered saline ( hbs ; 150 mm nacl ; 8 mm hepes ; ph 7.2 ) , pooled and re - aliquoted to provide identical working samples . one aliquot of stored lipids was warmed to room temperature and dried ( stream of n2 gas to visible dryness then 1 hr vacuum desiccation ) . dried lipids were solubilized by incubation in hbs ( 0.75 mg lipid / ml ; 60c , 60 seconds ) , then vortexing ( 30 seconds ) with scraping and further incubation ( 60c , 30 seconds ) . vortexing and incubation were repeated thrice and the resultant lipid suspension was either used for liposome preparation or placed on ice to cool while the protein was prepared . protein ( purified na k - atpase , -amylase , bsa ) was diluted in ice - cold hbs ( final concentration , 0.075 or 0.15 mg protein / ml ) , added to the cooled lipid suspension and vortexed ( 15 seconds ) . the resultant lipid protein ratio was 20 : 1 or 10 : 1 ( w / w ) in 3.0 ml ( 0.5 mg lipid / ml and 0.025 or 0.05 mg protein / ml ) . liposomes or proteoliposomes were created using high pressure nitrogen filtration , which was the best of five methods for consistency of producing small , unilamellar liposomes using bull sperm hpm lipids . the lipid or lipid / protein suspension was forced through a 0.1 m nuclepore polycarbonate membrane ( whatman inc . clifton , new jersey , usa ) using high - pressure nitrogen gas ( 35 kg / cm ) . resulting liposomes or proteoliposomes were maintained under n2 gas until measurements were taken ( within 30 minutes ) . dynamic light scattering ( dls)proteoliposome size was assessed by dls with a malvern microfluidizer , model m-110-s ( malvern instruments , herrenberg , germany ) , which provided six independent readings that were then converted by an internal algorithm into liposome diameter . proteoliposome size was assessed by dls with a malvern microfluidizer , model m-110-s ( malvern instruments , herrenberg , germany ) , which provided six independent readings that were then converted by an internal algorithm into liposome diameter . scanning electron microscopy ( sem)proteoliposomes were settled on polished carbon planchettes , fixed ( 2 hours , 0.5% ( w / v ) oso4 in 0.05 m veronal acetate and 0.2 m imidazole ) , rinsed ( 3x , 0.2 m imidazole ) , and then dehydrated in increasing concentrations of ethanol ( 50% , 70% , 90% , then 3x in 100% ; ) . planchettes were made conductive by sputter coating with argon to 15 nm thick using emscope k550 sputter coater , ( ashford , kent , uk ) and were viewed using a hitachi s-570 scanning electron microscope ( tokyo , japan ) . proteoliposomes were settled on polished carbon planchettes , fixed ( 2 hours , 0.5% ( w / v ) oso4 in 0.05 m veronal acetate and 0.2 m imidazole ) , rinsed ( 3x , 0.2 m imidazole ) , and then dehydrated in increasing concentrations of ethanol ( 50% , 70% , 90% , then 3x in 100% ; ) . planchettes were made conductive by sputter coating with argon to 15 nm thick using emscope k550 sputter coater , ( ashford , kent , uk ) and were viewed using a hitachi s-570 scanning electron microscope ( tokyo , japan ) . vancouver , canada ) . fluorescence resonance energy transfer ( fret ) tested incorporation of protein into the bilayer , assessing the fluorescence emission of the fret probe n-(dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , triethylammonium salt ( dansyl dhpe ) with a spectrofluorometer ( photon technology international , model a-1010 , london , on , canada ) . excitation scans first established optimal excitation and emission wavelengths for tryptophan and dansyl ( excitation ranges 220315 and 215400 nm , resp . ; emission ranges 350500 nm and 425500 nm , resp . ) , selecting final conditions of excitation : 278 nm ; emission range : 300520 nm ; and scan rate : 1 nm / sec . liposomes or proteoliposomes for fret incorporated dansyl dhpe in chloroform / methanol into hpm lipids in darkness to preserve the fluorescence of the dansyl ( final concentration : 2 mol% , based on preliminary experiments comparing 1 and 2 mol% ) , and vesicles were formed as before . emission intensity was assessed with 2.0 ml liposome / proteoliposome in a quartz cuvette in a 4-place temperature controlled sample chamber filled with n2 gas to minimize any oxidation of protein , which reduces tryptophan fluorescence . each replicate ( n = 3 ) measured fluorescence emission on 4 samples : buffer , liposomes ( no protein ) , atpase proteoliposomes , and amylase proteoliposomes ; all contained 2 mol% dansyl dhpe . each sample within each replicate the function of na k - atpase was determined ( n = 4 ) as the time - dependent release of inorganic phosphate ( po4 ) from atp ( adapted from [ 31 , 32 ] ) in the presence of atpase proteoliposomes , liposomes ( no protein ) , and also na k - atpase alone ( no liposomes present ) ; all samples were prepared as per liposome / proteoliposome methods . the assay was first optimized in 96-well plates for liposome / proteoliposome testing : sample volume ( 10 , 20 , 30 , 40 , 50 , 75 , and 100 l / well ; n = 3 ) , lipid : protein ratio ( 20 : 1 or 10 : 1 ; wt : wt ; n = 4 ) , assay incubation times ( 20 , 30 , 40 , and 60 minutes ; n = 4 ) and optimal atp concentration ( 6 mg / ml or 12 mg / ml ; n = 1 ) . conditions for the final assay were as follows : proteoliposomes ( 50.0 g lipid and 2.25 g protein / well ) , lipid - only liposomes ( 50.0 g lipid / well ) , and enzyme - alone ( 2.25 g protein / well ) . the reaction was initiated by the addition of 120 g atp / well . the reaction was stopped by addition of a stop reagent ( equal parts of 1% ( w / v ) ammonium molybdate in 12% sds ( w / v in distilled h2o ) and 6% ( w / v ) ascorbic acid in 1 n hcl ) , incubated ( 58 minutes , room temperature ) , stabilized ( 2% sodium citrate/2% sodium arsenite/2% acetic acid ) , and colour allowed to develop ( 20 minutes , room temperature ) . absorbance was then read at 750 nm using a powerwave x 340 microplate scanning spectrophotometer ( bio - tek instruments inc . , winooski , vermont , canada ) . a control assay included -amylase and bsa proteoliposomes ( 0.025 mg protein / ml ; n = 1 for each protein ) . the orientation of na k - atpase within the proteoliposomes was assessed with an adapted sidedness assay . sidedness expresses whether the protein is located in the proteoliposome bilayer in one of three orientations : inside - out ( subunit facing outwards ) , right - side - out ( subunit facing outwards ) , or not incorporated ( not incorporated into the bilayer ) . proteoliposomes were made as before , but in a na base buffer ( 130 mm na , 4 mm mg and 30 mm histidine , ph 7.0 ) and identical aliquots were subjected to six treatments ( n = 3 ) , intact proteoliposomes with no k ( no k ) 3 mm ouabain ; intact proteoliposomes plus k ionophores ( carbonyl cyanide 3-chlorophenylhydrazone ( cccp ) and valinomycin at 1.0 mg / ml and 0.5 mg / ml ethanol resp . ) so that k was on the inside and outside of the proteoliposomes ( k ) 3 mm ouabain , and liposomes opened with detergent ( dodecyloctaethylene glycol monoether ( c12e8 ) , 0.2 mg / ml ) in the presence of k ( open k ) 3 mm ouabain . the six resulting treatments ( table 1 ; no k ( b ) , no k + ouabain ( d ) , k ( a ) , k + ouabain ( c ) , open k ( f ) , and open k + ouabain ( g ) ) were then incubated with atp ( 60 minutes ; 37c ) to measure enzyme activity as before . immediately after addition of 0 or 3 mm ouabain , proteoliposomes were provided atp ( final atp concentration , 3 or 6 mg / ml ) and assessed for atpase function , measured as po4 production ( detailed above ) . phosphate production was then input into calculations to determine orientation / sidedness as specified by cornelius based on the treatment groups . treatment a ( k ) measures both inside - out and unincorporated na k - atpase , while ouabain ( treatment c ; k + ouabain ) inhibits this function . treatments b ( no k ) and d ( no k + ouabain ) are a baseline measure of any background activity . treatment f ( opened ) measures total na k - atpase activity in all orientations , while treatment g ( opened + ouabain ) inhibits all activity and thus measures basal na k - atpase activity . orientation of na k - atpase is thus determined as : ( 1)inside out : ( a - b)(f - g ) or ( c - d)(f - g),right side out : ( f a)(f - g),not incorporated : ( b g)(f - g ) . to determine the impact of lipid composition on na k - atpase incorporation and orientation in proteoliposomes , atpase proteoliposomes were composed of 100% hpm lipids 50% select lipids ( , hpm : sl 1 : 1 ( w / w ) ; n = 3 ) . sl were a complex mix of various phospholipids with known fatty acid composition ( pc : pe : sph : ps : pi ratio 21 : 26 : 42 : 5 : 5 ) , with a total saturated : unsaturated fatty acid ratio of approximately 85 : 15 . all data were analyzed using sas 9.1 ( sas 9.1 for windows ts level 1m3 , the sas institute inc . equaliy of variances was accounted for in all data sets . for the function assay ( po4 production ) , general linear model ( glm ) evaluated differences in po4 production among liposomes , proteoliposomes , and na k - atpase . data were checked for normality and square root transformed if necessary to stabilize the variances . least squares means ( lsm ) analysis assessed specific differences where type by method interactions were significant , and contrast statements assessed differences among main effects . differences between these sample types for lipid : protein ratios of 10 : and 20 : 1 were carried out in the same way . t - test compared po4 production by proteoliposomes at a lipid : protein of 10 : 1 or 20 : 1 at 60 minutes of incubation . a glm analyzed the fret raw emission values , determining differences within each replicate using a tukey 's test . glm also analyzed the ratios calculated for emission from atpase proteoliposomes and amylase proteoliposomes expressed relative to the emission intensity of the pure lipid liposomes ( set to 100% ) within each replicate . calculated amounts of na k - atpase in each orientation ( inside out , right side out and not incorporated ) were analyzed using glm , and compared by lsm . overall effect of ouabain was analyzed with glm , comparing pooled ouabain treatments with lsm of log - transformed data . dynamic light scattering showed that the diameters of proteoliposomes created using high pressure nitrogen filtration ranged from 5872 nm and were not statistically different than those of the lipid - only liposomes ( 5482 nm ) . the dls results were verified with sem micrographs ( figure 1 ) . both na k - atpase and -amylase contain endogenous tryptophan [ 12 , 35 ] , which was excited during fret and emitted light at 345 nm , the wavelength of excitation of the fluorophore dansyl which was tagged on the dhpe . the dansyl absorbed this light and the tryptophan - excited dansyl emitted at the empirically measured 500 nm ( similar to the published maxima of 497 nm ; ) . bsa proteoliposomes produced negligible emission , consistent with the fact that bsa is a serum protein and should not incorporate into a membrane . emission intensity differed overall among different vesicles ( p .001 ; figure 2 ) and specifically when emission of liposomes with no protein was set to 100% ( 100 0% < 114.9 2.9% < 161.6 29.3% for liposomes , atpase , and amylase , respectively ; p = .052 ) . it is interesting to note that -amylase proteoliposomes had a greater emission at 345 nm , the wavelength of tryptophan emission , than the atpase liposomes ( figure 2 ) , in accordance with the greater tryptophan content in the amylase compared to atpase [ 12 , 35 ] . the na k - atpase incorporated into hpm lipid vesicles was functionally competent to cleave atp and release po4 . proteoliposomes and enzyme - only produced more po4 with increasing protein concentration in a linear fashion ( p < .001 ) , while liposomes did not ( figure 3 ) . in data pooled from lipid to protein ratios of 10 : 1 and 20 : 1 , proteoliposomes produced more po4 than liposomes and enzyme - only ( p < .0001 ) . increasing na k - atpase content increased po4 production overall ( 10 : 1 lipid : protein > 20 : 1 ; p < .0001 ) and at 60 minutes of incubation ( figure 4 ; p = .046 ) , but individual earlier time points did not differ significantly ( data not shown ) . therefore , all subsequent assays were conducted with a 10 : 1 lipid : protein ratio and a 60 minutes incubation time . proteoliposomes containing bsa or -amylase were much less effective at cleaving po4 than atpase - proteoliposomes ( 1.9 and 1.4 versus 7.0 nmol in a pilot trial ) . incubation of na k - atpase - proteoliposomes with the specific inhibitor ouabain lowered po4 production ( p .0001 ) . the sidedness assay determined that most na k - atpase in the proteoliposomes was not incorporated into the bilayer ( 68.3% ) , but all enzymes that were incorporated were facing right side out ( 31.7% ) and none was inside out ( figure 5 ) . the orientation of na k - atpase in proteoliposomes made from 100% hpm lipids ( as before ) differed from that of proteoliposomes made of a mixture of hpm and sl lipids ( 1 : 1 ; wt : wt ; hpm : sl proteoliposomes ) , as assessed by the sidedness assay . although , as before the na k - atpase in both hpm and hpm : sl proteoliposomes was largely not incorporated ( 84.4% ) , inclusion of sl lipids caused all atpase that was incorporated to be inside out ( 15.6% ) , and none to be right side out , differing significantly from the exclusively right side out atpase in the pure hpm liposomes ( p = .038 ; figure 6 ) . these results convincingly demonstrate that atpase enzyme can be functionally incorporated into an artificial lipid bilayer and that the lipid composition impacts the orientation and functional ability of amphipathic proteins , consistent with raft theory . the incorporation of na k - atpase into liposomes did not affect the size of small vesicles made by filtration , when compared to lipid - only liposomes . small , unilamellar proteoliposomes , such as those produced by high pressure filtration methods established here , are therefore appropriate for the study of protein function . a measurement of the proximity of lipid and protein molecules in relation to each other using fluorescence resonance energy transfer can be employed to determine if a protein is incorporated into a lipid bilayer . fret uses the energy from one molecule , the donor , here the tryptophan present in na k - atpase or -amylase ( see [ 12 , 35 ] , resp . ) , to emit light at a wavelength ( tryptophan , 345 nm ) that excites a second acceptor molecule , here dansyl . the key is that the energy transfer can only occur over very short distance , so the donor and acceptor must be in close enough physical proximity to interact in this way . if they are not , emission from the donor molecule will not be absorbed by the acceptor and thereby excite it : the unabsorbed light emitted by the donor will therefore be emitted . optimal conditions were first established to detect resonance energy transfer , using 2 mol% dansyl ( energy acceptor ) , a lipid : protein ratio of 10 : 1 ( hpm : na k - atpase ) which also was the optimum lipid : protein ratio to measure atpase function . this is similar to the lipid : protein ratios commonly used for na k - atpase function studies . our identified nonoxidizing n2 atmosphere parallels the need because oxidation of tryptophan decreases its fluorescence . emission from atpase proteoliposomes was consistently significantly higher than in liposomes alone , indicating that atpase was incorporated into the lipid bilayer and its tryptophan excited . amylase proteoliposomes elicited significantly higher dansyl emission than atpase proteoliposomes because of the approximate 2-fold greater number of tryptophan moieties in amylase compared to na k - atpase [ 12 , 35 ] . the -amylase proteoliposomes showed a large peak at the tryptophan emission wavelength , which both corresponds with the reported high level of tryptophan present and suggests that the tryptophan emission exceeded the absorbance capacity of the dansyl molecules in the membrane . functionality of atpase in liposomes was assessed by measuring the enzymatic ability to cleave phosphate from atp . proteoliposomes containing na k - atpase produced significantly more po4 than bsa or -amylase proteoliposomes , or liposomes alone , indicating that the incorporated na k - atpase enzyme is indeed functional under the optimal assay conditions of 10 : 1 lipid : protein incubated for 60 minutes incubation time , which is in the range used by others . having demonstrated a reliable method to produce proteoliposomes with functional incorporated atpase in the bilayer , it was important to know the orientation of the enzyme . certainly the lipids in sperm head plasma membranes are not randomly distributed [ 3941 ] . furthermore , since hpm lipids from boar sperm form distinct domains of differing fluidities , which change over time , or with cryopreservation and/or with the medium in which sperm are preserved , the hpm lipids in the liposomes here were expected to similarly organize in a nonrandom manner . if the nak - atpase preferentially interacted with certain lipids , its orientation should be nonrandom , which was exactly what the sidedness assay indicated . all the enzyme that was incorporated , was incorporated in a right - side - out orientation ( subunit facing outwards ) . others have found that the sidedness of na k - atpase in artificial membrane differs depending on the source of protein , the method of reconstitution , and especially on the lipid composition of the vesicles . vesicles of pc made by detergent dialysis caused 50% of atpase to orient inside out , while in liposomes made of pe , pc , pi , and cholesterol by bio bead detergent removal , the ratio for na k - atpase right side out : inside out : not oriented was 65 : 15 : 20 . the liposomes here made by nitrogen filtration with native sperm hpm lipids allowed effective incorporation of functional na k - atpase , albeit at a low level . altering the lipid composition of the vesicles did not affect the amount of protein that was not incorporated but did affect the orientation of na k - atpase in the membrane . when the bilayers contained endogenous native lipids , all the atpase was oriented with its subunit in the outer leaflet , but replacing the hpm with 50% selected mixed lipids significantly increased the amount of na k - atpase oriented with its subunit in the inner leaflet . na k - atpase orientation in reconstituted vesicles is affected by lipid composition , more specifically by the acyl chain lengths of phospholipids , the degree of saturation of the phospholipids , and the amount of cholesterol present [ 47 , 48 ] . acyl chain lengths of phospholipids are important for determining the thickness of membrane bilayers . bilayers that are too thick or too thin to accommodate the hydrophobic region of na k - atpase may result in improper folding , altered function , and differing levels of inclusion of atpase . increasing the acyl chain length of monounsaturated pc results in an increase in the amount of na k - atpase that is not incorporated into liposomes , and the optimal amount of protein incorporated into liposomes occurs at 14- and 20-carbon chain length for unsaturated and saturated pc , respectively . cholesterol inclusion works to increase the thickness and order of plasma membranes and it has been suggested that some membrane domains are formed only in the presence of certain concentrations of cholesterol . lipid rafts are enriched in cholesterol , sphingomyelin , lipids with saturated long chain acyl chains , and in sperm , the plasma membrane which lays over the acrosome displays rafts enriched in gangliosides and cholesterol [ 53 , 54 ] . cholesterol content of membranes both above and below native membrane content ( 16 mol% of total lipids ) decreases na k - atpase activity in pc liposomes using pig kidney atpase . while the addition of 40 mol% cholesterol into pc liposomes had no effect on the amount of na k - atpase that was not incorporated into liposomes , it doubled the amount of enzyme that was inside out ( right - side - out enzyme was not measured ; ) . the extracted hpm lipids were measured and consisted of 45.2% pc , 9.7% pe , 5.7% sph , 5.4% pi , 4.2% ps , and 29.8% cholesterol , with 29.3% of the total phospholipids being saturated . most saturated phospholipids were 16 carbons long , while the majority of the unsaturated phospholipids had 22 carbon acyl chains . therefore liposomes made with 50% hpm : 50% selected lipids would have increased quantities of sph ( 16 : 0 and 18 : 0 ) and pe ( 18 : 0 ) , along with moderate increases in other phospholipids . the amount of overall cholesterol would be decreased by 50% . although reducing the cholesterol did not affect the amount of atpase incorporated into the liposomes , either here or for cornelius , the lower cholesterol content could have caused the altered orientation , since removal of cholesterol from sperm membranes alters raft structure , changing the size , distribution , and content of glycoproteins [ 5456 ] . it would be interesting to test atpase orientation in liposomes with systematically altered cholesterol content . lipid rafts have been variously suggested to break down or become enriched [ 3 , 54 ] during sperm capacitation . regardless of how rafts change , it is clear that the lipid rafts in sperm membranes change during capacitation . seminolipid , a sperm - specific glycolipid concentrated in the outer leaflet of the plasma membrane on the apical ridge of the sperm head [ 56 , 58 ] , may have the ability to stabilize the plasma membrane , preventing the acrosome reaction [ 56 , 59 ] . consistent with activation of na k - atpase by raft - mediated relocation in other cells [ 19 , 20 ] , atpase in sperm may be relocated in the plasma membrane by the efflux of cholesterol during capacitation so that it is accessible to bind ouabain and initiate signalling cascade . cholesterol efflux from sperm membranes during capacitation occurs more readily in nonraft or fluid fractions of the membrane . certainly the current results demonstrate that na k - atpase is sensitive to its lipid environment , and that lipid environment alters its orientation , and therefore its accessibility to external stimuli such as ouabain . high pressure nitrogen filtration applied to native lipids extracted from sperm membranes reliably creates consistently sized liposomes and proteoliposomes . fret elegantly proves that the proteoliposomes have actually incorporated the amylase or atpase proteins into the lipid bilayer . incorporated atpase is functional and , in a bilayer of native lipids , is oriented exclusively in a right side - out orientation . alteration of the lipid composition changes the orientation of na k - atpase in the lipid bilayer , confirming that specific lipids effectively influence integral membrane proteins . these results support recent results suggesting that inclusion of raft - associated lipids and proteins may increase the quantity and stabilize the orientation of na k - atpase in model , or cellular , membranes .

sperm membranes change in structure and composition upon ejaculation to undergo capacitation , a molecular transformation which enables spermatozoa to undergo the acrosome reaction and be capable of fertilization . changes to the membrane environment including lipid composition , specifically lipid microdomains , may be responsible for enabling capacitation . to study the effect of lipid environment on proteins , liposomes were created using lipids extracted from bull sperm membranes , with or without a protein ( na+ k+-atpase or -amylase ) . protein incorporation , function , and orientation were determined . fluorescence resonance energy transfer ( fret ) confirmed protein inclusion in the lipid bilayer , and protein function was confirmed using a colourometric assay of phosphate production from atp cleavage . in the native lipid liposomes , atpase was oriented with the subunit facing the outer leaflet , while changing the lipid composition to 50% native lipids and 50% exogenous lipids significantly altered this orientation of na+ k+-atpase within the membranes .

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

lipid rafts in head membranes of uncapacitated sperm are uniformly distributed but become restricted to the periacrosomal region during capacitation , suggesting rafts are associated with capacitation . one particular protein involved in cell signaling during capacitation , na k - atpase , is extremely sensitive to its lipid environment and its function is uniquely damaged when sperm are cryopreserved , a process that also reduces fertility . loss of na k - atpase function postthaw , whether caused by cryopreservation - induced direct damage to the protein and/or alterations in the protein 's lipid environment , could cause the loss of fertility . although na k - atpase is not associated with lipid rafts in brain synaptosomes , mdck cells , or enterocytes , it is enriched in lipid raft fractions of gastric luminal cells , hepatocytes , renal epithelial , outer medulla kidney , and cardiac cells . the specific effects of lipid environment on na k - atpase in sperm membranes is unknown . liposomes provide a model membrane system to study protein function [ 21 , 22 ] , facilitating assessment of integral membrane proteins without interference from other cell components that may obscure results . many proteoliposome studies attempt to reproduce a typical membrane by mixing a few commercial lipids as a base composition [ 2426 ] , but , logically , using extracted endogenous membrane phospholipids would even more closely mimic the native lipid bilayer including lipid rafts . functional protein reconstitution into vesicles is dependent on method of preparation and lipid composition of the vesicles , necessitating determination of degree of incorporation of a protein into the liposomes before detailed protein function can be reliably and repeatedly evaluated . we hypothesized that lipid composition would affect na k - atpase insertion into liposomes , and therefore measured incorporation , function and orientation of na k - atpase in liposomes made from lipids extracted from the head plasma membrane ( hpm ) of bull sperm , and then altered the lipid composition of the proteoliposomes to determine the effect of lipid composition on the nature of the incorporation of na k - atpase into lipid vesicles . adenosine 5-triphosphate ( atp ) , sodium chloride ( nacl ) , hepes , ouabain octahydrate , -amylase ( type ii - a from bacillus species ) , bovine serum albumin ( bsa ) , dog kidney na k - atpase , osmium tetroxide , imidazole , ammonium molybdate l - histidine , carbonyl cyanide 3-chlorophenylhydrazone ( cccp ) , valinomycin , phosphatidylcholine ( pc ; 16 : 0 , 18 : 0 , 18 : 1 , and 18 : 2 ) , phosphatidylethanolamine ( pe ) , sphingomyelin ( sph ; 18 : 0 ) , and phosphatidylserine ( ps ; 16 : 0 ) were purchased from sigma - aldrich ( mississauga , on , canada ) . phosphatidylcholine ( 20 : 4 ) , sph ( 16 : 0 ) , ps ( 18 : 0 and 18 : 2 ) , and phosphatidylinositol ( pi ; 18 : 1 ) were purchased from avanti ( alabaster , al ) , and pc ( 22 : 6 ) and pi ( 16 : 0 ) were from matreya ( pleasant gap , pa ) . lipids were extracted from the head plasma membrane ( hpm ) of whole bull sperm ( 33 ejaculates from 14 different bulls ) using established methods [ 8 , 9 ] , pooled , dried , weighed , resolubilized in chloroform methanol ( 2 : 1 , v / v ) , divided into equal aliquots containing 1.5 mg lipids , and stored at 70c under nitrogen gas to provide identical lipid compositions for all liposomes . dog kidney na k - atpase was purified using an ouabain affinity column , freeze dried and stored at 20c . protein ( purified na k - atpase , -amylase , bsa ) was diluted in ice - cold hbs ( final concentration , 0.075 or 0.15 mg protein / ml ) , added to the cooled lipid suspension and vortexed ( 15 seconds ) . liposomes or proteoliposomes were created using high pressure nitrogen filtration , which was the best of five methods for consistency of producing small , unilamellar liposomes using bull sperm hpm lipids . planchettes were made conductive by sputter coating with argon to 15 nm thick using emscope k550 sputter coater , ( ashford , kent , uk ) and were viewed using a hitachi s-570 scanning electron microscope ( tokyo , japan ) . proteoliposomes were settled on polished carbon planchettes , fixed ( 2 hours , 0.5% ( w / v ) oso4 in 0.05 m veronal acetate and 0.2 m imidazole ) , rinsed ( 3x , 0.2 m imidazole ) , and then dehydrated in increasing concentrations of ethanol ( 50% , 70% , 90% , then 3x in 100% ; ) . planchettes were made conductive by sputter coating with argon to 15 nm thick using emscope k550 sputter coater , ( ashford , kent , uk ) and were viewed using a hitachi s-570 scanning electron microscope ( tokyo , japan ) . fluorescence resonance energy transfer ( fret ) tested incorporation of protein into the bilayer , assessing the fluorescence emission of the fret probe n-(dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , triethylammonium salt ( dansyl dhpe ) with a spectrofluorometer ( photon technology international , model a-1010 , london , on , canada ) . each replicate ( n = 3 ) measured fluorescence emission on 4 samples : buffer , liposomes ( no protein ) , atpase proteoliposomes , and amylase proteoliposomes ; all contained 2 mol% dansyl dhpe . each sample within each replicate the function of na k - atpase was determined ( n = 4 ) as the time - dependent release of inorganic phosphate ( po4 ) from atp ( adapted from [ 31 , 32 ] ) in the presence of atpase proteoliposomes , liposomes ( no protein ) , and also na k - atpase alone ( no liposomes present ) ; all samples were prepared as per liposome / proteoliposome methods . the assay was first optimized in 96-well plates for liposome / proteoliposome testing : sample volume ( 10 , 20 , 30 , 40 , 50 , 75 , and 100 l / well ; n = 3 ) , lipid : protein ratio ( 20 : 1 or 10 : 1 ; wt : wt ; n = 4 ) , assay incubation times ( 20 , 30 , 40 , and 60 minutes ; n = 4 ) and optimal atp concentration ( 6 mg / ml or 12 mg / ml ; n = 1 ) . the reaction was stopped by addition of a stop reagent ( equal parts of 1% ( w / v ) ammonium molybdate in 12% sds ( w / v in distilled h2o ) and 6% ( w / v ) ascorbic acid in 1 n hcl ) , incubated ( 58 minutes , room temperature ) , stabilized ( 2% sodium citrate/2% sodium arsenite/2% acetic acid ) , and colour allowed to develop ( 20 minutes , room temperature ) . absorbance was then read at 750 nm using a powerwave x 340 microplate scanning spectrophotometer ( bio - tek instruments inc . the orientation of na k - atpase within the proteoliposomes was assessed with an adapted sidedness assay . sidedness expresses whether the protein is located in the proteoliposome bilayer in one of three orientations : inside - out ( subunit facing outwards ) , right - side - out ( subunit facing outwards ) , or not incorporated ( not incorporated into the bilayer ) . so that k was on the inside and outside of the proteoliposomes ( k ) 3 mm ouabain , and liposomes opened with detergent ( dodecyloctaethylene glycol monoether ( c12e8 ) , 0.2 mg / ml ) in the presence of k ( open k ) 3 mm ouabain . orientation of na k - atpase is thus determined as : ( 1)inside out : ( a - b)(f - g ) or ( c - d)(f - g),right side out : ( f a)(f - g),not incorporated : ( b g)(f - g ) . to determine the impact of lipid composition on na k - atpase incorporation and orientation in proteoliposomes , atpase proteoliposomes were composed of 100% hpm lipids 50% select lipids ( , hpm : sl 1 : 1 ( w / w ) ; n = 3 ) . for the function assay ( po4 production ) , general linear model ( glm ) evaluated differences in po4 production among liposomes , proteoliposomes , and na k - atpase . least squares means ( lsm ) analysis assessed specific differences where type by method interactions were significant , and contrast statements assessed differences among main effects . a glm analyzed the fret raw emission values , determining differences within each replicate using a tukey 's test . glm also analyzed the ratios calculated for emission from atpase proteoliposomes and amylase proteoliposomes expressed relative to the emission intensity of the pure lipid liposomes ( set to 100% ) within each replicate . calculated amounts of na k - atpase in each orientation ( inside out , right side out and not incorporated ) were analyzed using glm , and compared by lsm . overall effect of ouabain was analyzed with glm , comparing pooled ouabain treatments with lsm of log - transformed data . dynamic light scattering showed that the diameters of proteoliposomes created using high pressure nitrogen filtration ranged from 5872 nm and were not statistically different than those of the lipid - only liposomes ( 5482 nm ) . the dansyl absorbed this light and the tryptophan - excited dansyl emitted at the empirically measured 500 nm ( similar to the published maxima of 497 nm ; ) . emission intensity differed overall among different vesicles ( p .001 ; figure 2 ) and specifically when emission of liposomes with no protein was set to 100% ( 100 0% < 114.9 2.9% < 161.6 29.3% for liposomes , atpase , and amylase , respectively ; p = .052 ) . it is interesting to note that -amylase proteoliposomes had a greater emission at 345 nm , the wavelength of tryptophan emission , than the atpase liposomes ( figure 2 ) , in accordance with the greater tryptophan content in the amylase compared to atpase [ 12 , 35 ] . proteoliposomes and enzyme - only produced more po4 with increasing protein concentration in a linear fashion ( p < .001 ) , while liposomes did not ( figure 3 ) . although , as before the na k - atpase in both hpm and hpm : sl proteoliposomes was largely not incorporated ( 84.4% ) , inclusion of sl lipids caused all atpase that was incorporated to be inside out ( 15.6% ) , and none to be right side out , differing significantly from the exclusively right side out atpase in the pure hpm liposomes ( p = .038 ; figure 6 ) . these results convincingly demonstrate that atpase enzyme can be functionally incorporated into an artificial lipid bilayer and that the lipid composition impacts the orientation and functional ability of amphipathic proteins , consistent with raft theory . small , unilamellar proteoliposomes , such as those produced by high pressure filtration methods established here , are therefore appropriate for the study of protein function . a measurement of the proximity of lipid and protein molecules in relation to each other using fluorescence resonance energy transfer can be employed to determine if a protein is incorporated into a lipid bilayer . the key is that the energy transfer can only occur over very short distance , so the donor and acceptor must be in close enough physical proximity to interact in this way . optimal conditions were first established to detect resonance energy transfer , using 2 mol% dansyl ( energy acceptor ) , a lipid : protein ratio of 10 : 1 ( hpm : na k - atpase ) which also was the optimum lipid : protein ratio to measure atpase function . this is similar to the lipid : protein ratios commonly used for na k - atpase function studies . emission from atpase proteoliposomes was consistently significantly higher than in liposomes alone , indicating that atpase was incorporated into the lipid bilayer and its tryptophan excited . the -amylase proteoliposomes showed a large peak at the tryptophan emission wavelength , which both corresponds with the reported high level of tryptophan present and suggests that the tryptophan emission exceeded the absorbance capacity of the dansyl molecules in the membrane . proteoliposomes containing na k - atpase produced significantly more po4 than bsa or -amylase proteoliposomes , or liposomes alone , indicating that the incorporated na k - atpase enzyme is indeed functional under the optimal assay conditions of 10 : 1 lipid : protein incubated for 60 minutes incubation time , which is in the range used by others . having demonstrated a reliable method to produce proteoliposomes with functional incorporated atpase in the bilayer , it was important to know the orientation of the enzyme . furthermore , since hpm lipids from boar sperm form distinct domains of differing fluidities , which change over time , or with cryopreservation and/or with the medium in which sperm are preserved , the hpm lipids in the liposomes here were expected to similarly organize in a nonrandom manner . others have found that the sidedness of na k - atpase in artificial membrane differs depending on the source of protein , the method of reconstitution , and especially on the lipid composition of the vesicles . vesicles of pc made by detergent dialysis caused 50% of atpase to orient inside out , while in liposomes made of pe , pc , pi , and cholesterol by bio bead detergent removal , the ratio for na k - atpase right side out : inside out : not oriented was 65 : 15 : 20 . altering the lipid composition of the vesicles did not affect the amount of protein that was not incorporated but did affect the orientation of na k - atpase in the membrane . when the bilayers contained endogenous native lipids , all the atpase was oriented with its subunit in the outer leaflet , but replacing the hpm with 50% selected mixed lipids significantly increased the amount of na k - atpase oriented with its subunit in the inner leaflet . na k - atpase orientation in reconstituted vesicles is affected by lipid composition , more specifically by the acyl chain lengths of phospholipids , the degree of saturation of the phospholipids , and the amount of cholesterol present [ 47 , 48 ] . bilayers that are too thick or too thin to accommodate the hydrophobic region of na k - atpase may result in improper folding , altered function , and differing levels of inclusion of atpase . increasing the acyl chain length of monounsaturated pc results in an increase in the amount of na k - atpase that is not incorporated into liposomes , and the optimal amount of protein incorporated into liposomes occurs at 14- and 20-carbon chain length for unsaturated and saturated pc , respectively . lipid rafts are enriched in cholesterol , sphingomyelin , lipids with saturated long chain acyl chains , and in sperm , the plasma membrane which lays over the acrosome displays rafts enriched in gangliosides and cholesterol [ 53 , 54 ] . while the addition of 40 mol% cholesterol into pc liposomes had no effect on the amount of na k - atpase that was not incorporated into liposomes , it doubled the amount of enzyme that was inside out ( right - side - out enzyme was not measured ; ) . the extracted hpm lipids were measured and consisted of 45.2% pc , 9.7% pe , 5.7% sph , 5.4% pi , 4.2% ps , and 29.8% cholesterol , with 29.3% of the total phospholipids being saturated . although reducing the cholesterol did not affect the amount of atpase incorporated into the liposomes , either here or for cornelius , the lower cholesterol content could have caused the altered orientation , since removal of cholesterol from sperm membranes alters raft structure , changing the size , distribution , and content of glycoproteins [ 5456 ] . regardless of how rafts change , it is clear that the lipid rafts in sperm membranes change during capacitation . seminolipid , a sperm - specific glycolipid concentrated in the outer leaflet of the plasma membrane on the apical ridge of the sperm head [ 56 , 58 ] , may have the ability to stabilize the plasma membrane , preventing the acrosome reaction [ 56 , 59 ] . consistent with activation of na k - atpase by raft - mediated relocation in other cells [ 19 , 20 ] , atpase in sperm may be relocated in the plasma membrane by the efflux of cholesterol during capacitation so that it is accessible to bind ouabain and initiate signalling cascade . cholesterol efflux from sperm membranes during capacitation occurs more readily in nonraft or fluid fractions of the membrane . certainly the current results demonstrate that na k - atpase is sensitive to its lipid environment , and that lipid environment alters its orientation , and therefore its accessibility to external stimuli such as ouabain . high pressure nitrogen filtration applied to native lipids extracted from sperm membranes reliably creates consistently sized liposomes and proteoliposomes . fret elegantly proves that the proteoliposomes have actually incorporated the amylase or atpase proteins into the lipid bilayer . alteration of the lipid composition changes the orientation of na k - atpase in the lipid bilayer , confirming that specific lipids effectively influence integral membrane proteins . these results support recent results suggesting that inclusion of raft - associated lipids and proteins may increase the quantity and stabilize the orientation of na k - atpase in model , or cellular , membranes .

a commonly occurring systemic autoimmune disorder , antiphospholipid syndrome ( aps or hughes syndrome ) , is characterized by arterial and/or venous thrombosis , pregnancy morbidity , or recurrent miscarriage . it is also characterized by thrombocytopenia , increased levels of antiphospholipid antibodies ( apl ) , such as positive lupus anticoagulant ( lac ) , which is the most common , and/or anticardiolipin antibodies ( acl ) and 2-glycoprotein i antibodies . the mechanism of actions proposed for thrombosis includes endothelial injury mediated by free radicals , coagulation malfunction , and compliment activation [ 3 , 4 ] . for the first hit , a prothrombotic state is created by the presence of antiphospholipid antibodies . the second hit happens after a prolonged sustenance of antiphospholipid antibodies , following which the thrombosis condition becomes evident [ 5 , 6 ] . however , not all individuals with high levels of apl develop thrombosis , and hence it can not be assumed that all antiphospholipid antibodies are thrombogenic [ 7 , 8 ] . various clinical studies have shown that apl - related thrombosis is accompanied by irreversible functional loss of organs and lethality in systemic lupus erythematosus ( sle ) . aps was first detected in individuals suffering from sle and was later recognized in patients with other autoimmune diseases , although at a lower frequency compared to sle patients . most of the case studies have shown that patients who have elevated levels of antiphospholipids and were affected by thrombosis also had other common habitual risk factors , such as smoking and a high bmi ; allied risk factors included hyperlipidaemia , hypertension , high triglycerides , and type ii diabetes [ 1013 ] . while it is still unclear if the presence of antiphospholipid antibodies is an additional risk factor for individuals who are not affected by autoimmune disorders , it can be hypothesized that the extent of endothelial injury intensified over time caused by the abovementioned common risk factors is reflected in increased levels of antiphospholipid antibody and the presence of lac [ 4 , 14 ] . nevertheless , this does not provide a clear understanding of the role of traditional risk factors and thrombosis . moreover , it can also be argued that aps is not dependent on any other disease that may already be present . these patients will fall into two categories : those who have experienced a thrombosis event previously and those who have not . if the patient has already had a thrombosis event , it is called secondary thromboprophylaxis . the second category will include asymptomatic patients , sle patients , and female patients who have obstetric antiphospholipid syndrome ( primary thromboprophylaxis ) . considering that apl - related manifestation of the syndrome is associated with a high morbidity and lethality rate , preventing the incidence of primary and secondary thrombosis is imperative [ 15 , 16 ] . at present , patients affected with thrombosis as a result of high apl levels are recommended for secondary prophylactic measures to reduce mortality rates . however , the time , methods , and target of drugs are highly debated [ 1719 ] . nevertheless , several effective therapeutic methods have been documented in clinical studies . it has been understood that multiple pathological processes are involved in aps while analyzing the nature of the clinical manifestations of this disease . while 70% were found to show positive results , this leaves a substantial number of patients with no effective treatment . furthermore , patients may experience bleeding complications with conventional anticoagulation therapy ( 2 - 3% ) . the optimal treatment in aps patients resistant or intolerant to long - term anticoagulation remains unclear . recent improvement in the understanding of the pathogenic mechanisms , including apl - induced activation of platelets , endothelial cells , monocytes , complement , and coagulation cascade , has led to the identification of potential targets and future therapies for aps . such evidence - based therapeutic methods are not well known for primary thrombosis ; hence , controlled and potential clinical trials are underway [ 15 , 21 , 22 ] . due to a lack of evidence - based therapeutic measures , patients with sle and high apl levels not affected by thrombosis are generally not recommended for any prophylactic measures , although some patients are administered aspirin and hydroxychloroquine ( hcq ) [ 23 , 24 ] . aspirin is commonly prescribed for the prevention of primary thrombosis in populations exposed to traditional risk factors and secondary venous thrombosis . again , the preventive ability of aspirin in apl patients has not been proven yet due to conflicting results obtained in clinical studies . further , hcq has been used in some clinical trials as a therapeutic drug for sle patients and for aps in animal models [ 2628 ] . in fact , there has been a lack of conclusive evidence as to whether to choose aspirin , hcq , or a combination of these two in patients positive for apls with other risk factors for thrombosis and with no additional risk factors . although plenty of data is available on aps , evidence - based studies on the efficacy of therapeutic methods are not sufficient . there are no extensive studies on the effectiveness and safety of primary prophylaxis in apl patients who are identified with other risk factors for thrombosis or apl patients who have no risk factors for the same . however , the efficacy of primary prophylaxis in these two groups has been analyzed and compared in this study . following this comparison , this paper represents and discusses the benefits in considering hcq for primary prophylaxis . it also examines the literature on hcq and compares the results obtained from the trials to arrive at a conclusion . through this systematic review , we intend to collate information on the effectiveness of treatment methods available for the prevention of primary thrombosis in apl patients with and without risk factors . with this background , the first step with either methodology is to develop a question , which is paraphrased as formulating review question for systematic reviews ( table 1 ) . this program of research is defined by two key research questions.is primary prophylaxis safe and effective in preventing thrombosis for patients both with and without other risk factors for thrombosis in antiphospholipids syndrome apls?what is the best treatment to prevent recurrent thrombosis in apl patients with and without additional risk factors for thrombosis and venous and arterial events not fulfilling and fulfilling criteria for aps ? is primary prophylaxis safe and effective in preventing thrombosis for patients both with and without other risk factors for thrombosis in antiphospholipids syndrome apls ? what is the best treatment to prevent recurrent thrombosis in apl patients with and without additional risk factors for thrombosis and venous and arterial events not fulfilling and fulfilling criteria for aps ? the review will predominantly focus on primary prophylaxis as a treatment for patients with elevated apl , with and without additional risk factors for thrombosis . for the systematic review pubmed , the cochrane library , medline , and allied health literature were searched for studies that examined the efficacy and safety of primary prophylaxis in apl patients . to reflect current apl practices and techniques , the literature search was limited to studies published from january 1990 to february 2013 . searches were conducted using a combination of three terms from the following sets : ( 1 ) antiphospholipid syndrome , antiphospholipid antibodies , thrombosis , and/or beta 2-glycoprotein i ; with ( 2 ) thrombosis not children , not review , not recurrent , antiphospholipid syndrome , or lupus anticoagulant or primary prevention , primary prophylaxis , or primary therapy or drug therapy , antiphospholipid syndrome / therapy , antibodies ; and ( 3 ) thrombosis therapy . in addition to this , the references that were mentioned in the articles chosen were reviewed in the hope that they might reveal further insights into this subject area . studies assessing the effect of primary prophylaxis in preventing thrombosis in antiphospholipid syndrome apls were explored . subsequently , studies that assessed efficacy and safety levels of primary prophylaxis , followed by identification of the best treatment to prevent recurrent thrombosis in apl patients and venous and arterial events not fulfilling and fulfilling criteria for apl , were included . the review focused only on a main objective of primary prophylaxis as treatment for individuals with apl who had not yet suffered any thrombosis . our search strategy was used to identify a set of potentially relevant studies . using predetermined selection criteria , each study identified during the search process was independently assessed by the researcher to determine its suitability for data extraction . in this study , a total of 101 citations were retrieved and reviewed for inclusion . a total of 25 studies were identified , and 2 further studies were identified by manual review of the reference list of relevant review articles . however , only 21 were found to be eligible . a total of 22 studies ( randomized controlled trials = 7 ; retrospective = 7 ; prospective = 8) were therefore included in this review . among the articles identified , only one rct ( n = 1 ) was eligible for the present study . the study compared the efficacy of aspirin 81 mg daily versus placebo for the prevention of thrombotic complications among 98 asymptomatic patients with persistently positive apl . the study participants were predominantly female , of whom more than 60% had sle . in a separate parallel observation study , patients who were apl - positive and taking aspirin were declined randomization and followed prospectively . the acute thrombosis incidence rates in aspirin - treated subjects were 2.75 per 100 patient - years and , for placebo - treated , 0 per 100 patient - years ( hazard ratio : 1.04 ; 95% ci : 0.691.56 , p = 0.83 ) ; in the observational study , the incidence rates were 2.70 per 100 for aspirin treated and 0 per 100 for not treated . however , this study was terminated due to an unexpectedly low rate of thrombotic events ( arterial or venous ) ( n = 3 ) occurring in the aspirin group and none in placebo . although this study observed infrequent outcome events and was limited by the small number of patients enrolled , in the patients with persistently positive apl aspirin was not effective in comparison to placebo for the primary prevention of thrombotic events . thus , the study revealed no benefit from low - dose aspirin for primary thrombosis prophylaxis . arterial and venous thrombotic manifestations can be prevented by using a prophylactic treatment with aspirin in the case of sle patients . using a markov decision analysis , wahl et al . the study also discovered that patients who tested positive for acl or la , or patients who had lupus , were found to have prevented bleeding episodes by keeping a good balance between thrombotic events . however , another clinical trial ( aplasa ) that was conducted on patients with sle showed that there were not many differences among apl - positive patients treated with either low - dose aspirin or placebo . the authors noted that the zero event rate in the case of the placebo group can be attributed to a number of factors , such as the inclusion of individuals ( over 40% ) with a low - risk apl profile , a short followup , and good control of vascular risk factors . in the case of asymptomatic individuals with apl , the additive clinical thrombotic risk factors , along with the likely preventive treatments , were analysed in a cross - sectional study that surveyed 77 aps patients . the results from the study were compared to the results that were obtained by patients who were asymptomatic apl - positive and who had no history of pregnancy morbidity or vascular thrombosis . the analysis of the results indicated that pregnancy ( p = 0.005 ) and surgical procedures ( p = 0.04 ) were significantly more frequent in group a , whereas aspirin ( p < 0.001 ) , hcq ( p < 0.001 ) , and corticosteroids ( p = 0.002 ) were used extensively more frequently in group b. surgical procedures and pregnancy , along with thrombocytopenia , raised the risk of an event in the case of women . the study also showed an increased risk of hypertension along with a number of other factors , especially in the case of arterial thrombosis ; however , these factors were not shown to be associated with venous thrombosis . the risk of thrombosis was also increased by certain traditional factors that were similar among various groups . however , there is still a lack of sufficient research that has dealt with the question of whether or not the efficacy of primary thromboprophylaxis in this group of patients with sle and apl increases for the combination therapy of antimalarials plus low - dose aspirin . another research trial by finazzi et al . evaluated 109 patients who suffered from antiphospholipid antibody syndrome . the trial attempted to determine the optimal intensity of oral anticoagulation for the prevention of recurrent thrombosis . in order to determine conclusively that intensive anticoagulation is better than standard treatment for preventing symptomatic thromboembolism without increasing the bleeding risk , the study gave two possibilities . one was to use either standard antithrombotic therapy ( warfarin , inr range 2.0 - 3.0 in 52 patients or aspirin alone , 100 mg day(1 ) in 3 patients ) ; the other was to use a high - intensity warfarin ( inr range 3.04.5 , 54 patients ) with a follow - up median time of 3.6 years . the study 's results showed that when conventional treatment was being given to the patients , recurrent thrombosis could be seen in 3 out of 55 patients ( 5.5% ) . in the case of patients who were given high - intensity warfarin , recurrent thrombosis could be seen in 6 of 54 patients ( 11.1% ) [ hazard ratio for the high - intensity group , 1.97 ; 95% confidence interval ( ci ) 0.497.89 ] . in addition to this , out of the 15 patients who received high - intensity warfarin , major bleeding was observed in 2 of them while the rest showed minor bleeding . in the case of patients who received conventional treatment , thus , it is clearly evident from the trial results that high - intensity warfarin is not a better treatment than conventional treatment when it comes to preventing recurrent thrombosis in patients with aps , as high - intensity warfarin - based treated patients were more prone to minor hemorrhagic complications . in a randomized control study , one group of asymptomatic antiphospholipid antibody carriers was administered a low - dose aspirin and the other group was given a placebo . the results of this study have shown no considerable differences between these two groups , but the placebo group showed 0% of incidence for thrombosis ; hence there was a necessity to under - power this trial for analyzing the role of aspirin . as a result of the low possibilities for risks , followup has become simple , vascular risk factors can be controlled effectively , and the immunological status of the patients can be preserved . however , the study did not provide the exact count of the lupus anticoagulant and obstetric antiphospholipid patients . on the contrary , the effect of aspirin on asymptomatic antiphospholipid antibody carriers who have sle has been shown in the outcomes of the observational studies [ 10 , 32 , 33 ] . as a result of the benefits associated with low - dose aspirin , the patients who have sle or anticardiolipin or both have been treated with the combination of low - dose aspirin and hcq by the experts . low - dose aspirin has been found to be useful in providing long - term treatment for the female patients who have obstetric antiphospholipid syndrome . however , this treatment will be recommended based on the immunological profile of the patients . it is necessary to individualize the treatment for healthy carriers of antiphospholipid antibodies as well as for patients who have a wide range of antiphospholipid antibodies such as lupus anticoagulant . in addition , the patients who have antiphospholipid antibodies should be treated by giving considerable importance to other vascular risk factors . it has been understood from the literature that both anticoagulation and extremely concentrated steroids can be used for the treatment of patients with less severe cases of caps . contrastingly , if the cases are extremely severe , it is necessary to use plasma intravenous immunoglobulins or plasma exchange . examined the influence of hcq on the synthesis of apl-2gpi complexes on phospholipid bilayers using ellipsometry and atomic force microscopy ( afm ) . the interaction between apl-2gpi complexes and thp-1 ( human acute monocytic leukemia cell line ) monocytes can be prevented with 1 g / ml concentration of hcq . the interaction between the individual proteins and bilayers can also be prevented by the same means . if the hcq protein is dialysed against a buffer , then the aforementioned reductions in binding can be reversed . it is also possible to obtain statistically significant reductions of clinical antiphospholipid assays through hcq . hence , the synthesis of apl-2gpi complexes has been found to be reduced with hcq . ( 2012 ) studied the interactions of hcq with proinflammatory / prothrombotic markers in 35 sle patients who were examined in a multiethnic , multicenter cohort ( lumina ) using slam - r scores . a multiplex immunoassay assessed the level of interferon- ( ifn- ) 2 , interleukin- ( il- ) 1 , il-6 , il-8 , inducible protein- ( ip- ) 10 , monocyte chemotactic protein-1 , tumor necrosis factor- ( tnf- ) , and soluble cd40 ligand ( scd40l ) levels . a positive correlation between slam - r scores at baseline and levels of ifn- ( p = 0.0546 ) was observed . hydroxychloroquine therapy resulted in a significant decrease in slam - r scores ( p = 0.0157 ) , and the decrease in slam - r after hcq therapy strongly correlated with decreases in ifn- ( p = 0.0087 ) . of all the articles that were researched , 6 studies were found to be relevant to the current research study . numerous risk factors such as sle - related factors and traditional or apl - profile factors were evaluated by a study conducted by tektonidou et al . the selected patients were divided amongst those that did or did not have apl , and the follow - up times were 104 and 112 months , respectively . in the case of apl - positive patients , the study demonstrated that 20.1% of them showed high thrombosis rates and about 7.6% ( p = 0.003 ) of the negative apl patients showed high thrombosis rates . in the case of apl - positive patients , a low dose of aspirin helped in protecting the patients against thrombosis ( hr per month 0.98 , p = 0.05 ) . similarly , the duration of hydroxychloroquine in both apl - positive ( hr per month 0.99 , p = 0.05 ) and apl - negative patients ( hr per month 0.98 , p = 0.04 ) also appeared to have protected the patients against thrombosis . patient records were obtained retrospectively from their previous medical records , even if the study was longitudinal in nature . even so , the results from the study are quite beneficial as the data relates to a number of sle patients with high - risk apl . it is also relevant to the evaluation of coexisting non - apl thrombosis risk factors and the age- and gender - matched control group used , and it offers the opportunity to have detailed information on the patients ' characteristics during a regular followup . another study by ruffatti et al . evaluated the overall risk factors associated with a first thrombotic event in apl - positive carriers in people 1865 years of age ; it also explored the effectiveness of prophylactic treatments . the study evaluated 370 people that were analysed retrospectively for a mean ( sd ) followup of 59.3 ( 45.5 ) months . during the follow - up checks , some of the risk factors identified were hypertension and medium / high levels of igg anticardiolipins . thromboprophylaxis during high - risk and long - term periods was significantly protective . in the case of asymptomatic apl carriers , once again medium / high titres of igg acl along with hypertension were determined to be risk factors , and it was also determined that primary prophylaxis is protective . the study by mok et al . had a very diverse sample group , with a total of 625 patients , among whom 258 were chinese , 140 were african american , and 227 were caucasian . the study primarily sought to compare the incidence and risk factors associated with thromboembolic events in systemic lupus erythematosus ( sle ) . in the case of 83 patients , the study made observations over a period of 3,094 patient - years , 40 venous events , and 48 arterial events . the incidence of arterial thromboembolism was 16/1,000 patient - years , and the incidence of venous thromboembolism was 13/1,000 patient - years . sixty months from the time of diagnosis of sle , the study discovered that 8.5% of the chinese were at risk of arterial events , followed by 8.1% of african americans , and 5.1% of caucasians . similarly , the study also determined that 60 months from the time of diagnosis of sle , the risk of venous events was 3.7% , 6.6% , and 10.3% , respectively ( p = 0.008 for chinese versus caucasians , by log - rank test ) . arterial events were predicted in a cox regression model with the help of chinese ethnicity , serositis , low levels of high - density lipoprotein ( hdl ) cholesterol , and oral ulcers . similarly , venous events were predicted in a cox regression model with the help of non - chinese ethnicity , hemolytic anemia , low levels of hdl cholesterol , obesity , male sex , renal disease , and antiphospholipid antibodies . thus , some of the differences and factors associated with arterial and venous thromboembolism in patients with sle can not be understood or explained . apl - positive patients who have no symptoms of sle have been assessed for the effect of hcq on anxa5 resistance assay ( 1997 ) . comparing the results of anxa5 resistance assay before and after the administration of hcq is the major aim of this study . the results from other tests such as la test , acl elisa , elisa , and anti - domain - i 2gp1 elisa ) have also been compared . broder and putterman identified a link between hcq apl and lac levels in 90 sle patients who are older than 21 . here , the major outcome variable is lac+ and/or at least 1 apl 40 u [ immunoglobulin a ( iga ) , igg , or igm ] . nineteen percent of the study population showed persistent lac+ and/or at least 1 apl 40 u. however , this level was found to be low with the administration of hcq ( or 0.21 , 95% ci 0.05 , 0.79 , p = 0.02 ) . the current study analysed clinical and analytical findings in 404 individuals among apl - positive patients with sle and included a followup for 36 months . the sample population was grouped into two categories : asymptomatic carrier of apl ( n = 178);patients with primary or secondary antiphospholipid syndrome . asymptomatic carrier of apl ( n = 178 ) ; patients with primary or secondary antiphospholipid syndrome . dicumarine was used to treat patients with aps and an international normalized ratio at or near 3.0 was targeted . meanwhile , when the patients were at higher risk of thrombosis without treating asymptomatic carriers , then specific prophylaxis with low - molecular weight heparin or aspirin was used . the study revealed that 31% of the patients with aps showed arterial thrombosis and/or 46.9% of the patients with aps showed venous thrombosis or feta loss ( 51.8% ) . according to ( 2004 ) , aspirin or low - molecular weight heparins were found to be the best treatments for aps patients who suffered from thrombotic complications . a study by tarr et al . outlined and grouped all of the risk factors associated with thrombotic events in 272 lupus patients . three groups were classified at the baseline into an apl+ group with 81 apl - positive patients without clinical thrombosis ; an apl- group with 107 apl negative patients ; and a secondary antiphospholipid syndrome ( aps ) group with 84 apl+ patients who met the sapporo criteria . another study by hereng et al . evaluated whether or not aspirin could provide primary prevention of antiphospholipid syndrome ( aps ) symptoms . in the study , a total of 103 individuals were taken as the sample , 75 of which were taking aspirin . the results indicated that , among apl - positive patients , 16 had autoimmune thrombocytopenia ( ait ) , 20 had prolonged activated partial thromboplastin times , 11 had diverse diseases , 37 had systemic lupus erythematosus ( sle ) , and 19 had other connective tissue diseases . aspects such as hcq use , clinical features , and biological parameters were similar across groups ; however , there was a distinct difference regarding thrombosis ( log - rank test ; p = 0.02 ) . thrombosis developed among 4 of the 10 sle patients who were not taking aspirin and among 3 of 27 patients who were taking aspirin ( log - rank test ; p = 0.03 ) . therefore , it can be stated that aspirin should be taken by apl - positive sle and ait patients in order to prevent aps manifestations . another study evaluated the risk factors for thrombosis in a large , multiethnic sle cohort . the study involved 1930 sle patients from a variety of ethnic backgrounds , including caucasians , african americans , asian americans , and hispanics . some of the factors that were considered to be at risk for developing thrombosis were apl positivity ( or 3.22 , p < 10(9 ) , or 1.26 per 5 years , and p = 0.027 10(7 ) ) , immunomodulating medication use ( or 1.40 , p = 0.011 ) , nephritis ( or 1.35 , p = 0.036 ) , and smoking ( or 1.26 , p = 0.011 ) . sle onset at younger ages was protective ( or 0.52 for age 20 , p = 0.001 ) , the use of hcq was found to be the best treatment against thrombosis . thus , the study confirms immunomodulating medication use , longer disease duration , history of nephritis , and older age at onset , along with apl positivity , as risk factors for thrombosis in sle . miscellaneous thrombosis risk factors , including apl - profile , sle - related , and traditional risk factors , have been analysed by tektonidou et al . . this study questions how thrombosis can be prevented in 144 patients with sle ( follow - up period 104 and 112 months ) and those who do not have apl . in apl patients , thrombosis was predicted with male sex ( hazard ratio [ hr ] 6.25 , p < 0.01 ) , lac ( hr 3.48 , p < 0.04 ) , and consistently positive acl ( hr 5.87 , p 0.01 ) . in apl - negative patients , male sex ( hr 7.14 , p 0.03 ) and hypertension were markers . apl - positive patients were found to be protected with the administration of lda ( hr per month 0.98 , p < 0.05 ) . in addition , both apl - positive and -negative patients had been treated with hcq ( hr per month 0.99 , p < 0.05 ) ( hr per month 0.98 , p 0.04 ) , respectively . in a cohort study conducted by rubenstein et al . on 1795 sle patients who had apl antibodies , the capability of hcq in reducing thrombosis was proven . the effect of hcq on the anxa5-ra in apl - positive patients has been studied in a prospective study conducted by rand et al . . here , anxa5-ra is the primary measure whereas la , acl , a2gpi , d - dimer , and anti - domain - i 2gpi antibody [ adi-2gpi ] are the secondary measures . this study involves the comparison of baseline data from 15 apl - positive patients ( mean age 43.3 12.0 years [ range 2761 ] , 10 [ 67% ] female , 13 [ 87% ] caucasian ; 10 had antiphospholipid syndrome , 5 asymptomatic apl ) and 7 apl - negative sle patients ( mean age 40.0 16.3 years [ range 2264 ] , 7 [ 100% ] female , 4 [ 57% ] caucasian ) . positive anxa5-ra , triple apl - positive , double apl - positive , and single apl - positive were observed in 87% , 69% , 15% , and 15% of the apl - positive patients , respectively . in this study , vasculitis , premature atherosclerosis , and hypercoagulability are the major causes of thrombosis in sle patients . in a hopkins cohort study of 100 patients , it was identified that thrombosis can be predicted with high anti - dsdna and low c3 , atherosclerosis ( hypertension , hyperlipidemia , and elevated homocysteine ) , and antiphospholipid antibodies ( lupus anticoagulant or anticardiolipin ) and could be treated with hcq . conducted a prospective study on a cohort of 232 patients to identify the effect of antimalarial drugs in preventing thrombosis in sle patients through a cox regression - multiple - failure time survival analysis model . in this study , the antimalarial drug was protective against thrombosis ( hr 0.28 , 95% ci 0.080.90 ) , while apl positivity ( hr 3.16 , 95% ci 1.456.88 ) and previous thrombosis ( hr 3.85 , 95% ci 1.509.91 ) increased the risk of thrombotic events . this systematic review comprehensively evaluated the efficacy , safety , and treatment methods for primary prophylaxis of therapeutic measures among patients positive for apls with other risk factors for thrombosis and apls with no additional risk factors for thrombosis . the study specifically compares the two groups of apl with other risk factors for thrombosis and with no additional risk factors . further , the study also focuses on primary prophylaxis to prevent thrombosis in antiphospholipid syndrome ( aps ) . moreover , by searching the literature and extant studies covering populations where hydroxychloroquine ( hcq ) was effective and comparing them to other studies where hcq was not effective as a primary prophylaxis , this review concludes that hcq is an effective primary approach when compared to aspirin . along with hcq , aspirin 's effects as a preventive therapy have been analyzed in this review . based on an assessment of the literature collected , it is clear that primary thrombosis prophylaxis strategies in asymptomatic apl - positive with other risk factors for thrombosis and apls with no additional risk factors for thrombosis are limited . in a study conducted on antiphospholipids antibodies patients , an aspirin of 81 mg , was administered to asymptomatic , persistently apl - positive individuals and another group was left on placebo ( mean sd followup period 2.30 0.95 years ) . both of the groups were followed up prospectively , and the results confirmed that aspirin administration does not prevent thrombosis but that the net annual incidence was low . wahl et al . showed that in markov decision analysis when patients with sle and apl - positive are given a prophylactic treatment with aspirin , arterial and venous thrombosis is prevented . to add to this , a study performed by ( 2009 ) also clearly demonstrated that incidence of thrombosis decreased in individuals with elevated apl but was otherwise asymptomatic . erkan et al . also conducted a prospective study on the effect of low - dose aspirin on apl - positive individuals without thrombosis . in subsequent study , the results suggested that , while low - dose aspirin did not prove beneficial , it did lower the overall annual rate of thrombosis incidence . this study also included patients affected by other autoimmune disorders and asymptomatic individuals who did not contract autoimmune disease . similar to sle patients , these individuals also showed low incidence of autoimmune disease on administering low - dose aspirin . future followups showed that more than 50% of the patients who later developed thrombosis were diagnosed with sle . further , the study also revealed that 42% of the examined patients exhibited low - risk rates of thrombosis due to high apl levels , which indicated lesser benefits from aspirin administration [ 45 , 46 ] . the limitation of this study was that the follow - up time was only 2.3 years , which is brief compared to other related studies [ 4749 ] . in a randomized clinical study , it was found that the overall incidence rate of thrombosis was 1.33 per 100 patient - years ; for aspirin treated cases , the incidence rate was 2.75 per 100 patient - years , and , in the placebo group , the rate was 0 per 100 patient - years . accordingly , in the observational study carried out , the overall incidence rate of thrombosis was 2.2 per 100 patient - years ; in aspirin treated cases , the incidence rate was 2.70 per 100 patient - years and 0 per 100 patient - years in the placebo group of subjects . in this study , all patients ( except one ) had thrombosis risk factors along with the incidence of thrombosis . all of the above results indicate that persistently apl - positive individuals receive little to no benefit from aspirin treatment for primary prophylaxis , but the overall annual incidence rate of acute thrombosis is minimized . however , in the presence of other traditional risk factors , vascular disorders may occur . the limitations of this research are inadequate sample size , low frequency of the disease ( which lowered the number of subjects studied ) , and the challenge in identifying asymptomatic patients . despite these drawbacks , the aplasa study represents the first randomized study carried out with persistently apl - positive subjects that was successful and consistent with many of the clinical and epidemiological studies conducted . not all asymptomatic patients with an apl - positive diagnosis were given aspirin for three reasons.the unselected cases displayed a predicted thrombosis incidence rate of 1% patient-years.it is believed that aspirin could cause acute bleeding . considering this level of thrombotic risk , the benefits did not outweigh the potential risks.the expected benefits of aspirin have been proven in at least one randomized clinical trial , even though there may have been few methodological limitations . it is believed that aspirin could cause acute bleeding . considering this level of thrombotic risk the expected benefits of aspirin have been proven in at least one randomized clinical trial , even though there may have been few methodological limitations . various risk factors of thrombosis other than high apl levels such as habitual risks ( e.g. , smoking ) are alterable factors . modifying these factors would be a manageable and safer approach to lower the rate of thrombosis . pengo et al . questioned : does aspirin prove to be beneficial for patients at particularly high risk of thrombosis including high risk for other autoimmune diseases and syndromes occurring with undocumented patterns of antibodies ( triple positivity ) ? currently , no substantial information is available to recommend aspirin as a primary prophylaxis treatment for thrombosis as the drug has demonstrated risk of bleeding . however , the expert committee of european league against rheumatism ( eular ) has recommended guidelines for effective management of sle . according to eular , low - dose aspirin can be recommended for primary thrombosis patients , despite the lack of substantial evidence . have shown that hcq effectively lowers the risk of thrombosis in both apl - positive and -negative subjects . in addition , hcq was administered in the 1970s and 1980s to treat deep vein thrombosis and pulmonary embolisms , which commonly affected patients who underwent hip replacement surgery . several studies have shown results favourable to thrombosis prevention with the use of hcq , even though its mechanism of action has been demonstrated only in animal models [ 2628 , 44 ] . hcq has also been proven to have optimal effects on treating high lipid profiles [ 27 , 53 ] . although the protective effects of antimalarials in preventing thrombosis and its advantag against lupus activity are well documented , its beneficial effects in sle patients are only minimally known [ 44 , 54 ] . the past literature review indicates that the presence of apl along with sle increases the risk associated with thrombosis significantly [ 29 , 36 , 37 , 55 , 56 ] . therefore , in the case of patients who are apl - positive the highest percentage of thrombosis occurs in the subgroup with sle [ 32 , 36 , 37 , 39 , 49 , 5762 ] . according to a study by ruiz - irastorza et al . , aps has been seen to have an adverse effect on the survival rate of sle patients and to cause irreversible organ damage . in addition to this , no significant differences were discovered between low - dose aspirin and placebo in the aplasa clinical trials . however , most of the clinical trials included patients who tested positive for sle , and sle itself is known to have its own set of influences over thrombosis ; hence , the results obtained can not be considered applicable to non - sle populations [ 6466 ] . thromboembolic events can be effectively treated with or without antiphospholipid antibodies by means of hcq in patients who have lupus [ 6770 ] and in patients with apl . primary prophylaxes with hcq and aspirin have been observed to reduce the frequency of thrombotic events in the case of asymptomatic apl - positive patients with sle . in a markov decision analysis , oral anticoagulant therapy and observation , along with prophylactic aspirin , there is still a lack of sufficient research that has dealt with the question of whether or not the efficacy of primary thromboprophylaxis in this group of patients with sle and apl increases when both therapies ( antimalarials plus low - dose aspirin ) are combined . in the case of asymptomatic apl carriers , medium / high titres of igg acl , along with hypertension , were determined to be the risk factors , and it was also determined that primary prophylaxis is protective . for the clinician who is faced with an apl - positive patient with no history of pregnancy morbidity or thrombosis , it is better that an individual assessment of the patient 's thrombotic risk be done . aspirin by itself is still not considered to be the best treatment for primary thromboprophylaxis , considering that aspirin is a blood thinner . primary prophylaxis with aspirin , either alone or in combination with hcq , is not advisable if the patient does not show any signs of sle , cardiovascular , or thrombotic risk factors . . the annual incidence rate of thrombosis events in anti - cl - positive patients who have sle was found to be 3.8% . there has also been a study on the prophylactic role of lda in apl - positive sle patients . the possibilities for reducing the thrombosis events have been discussed in this study . in this study , it was proven that treatment - caused bleeding can be effectively treated with lda . on the other hand , independent cardiovascular risk factors include autoimmune defects such as sle , rheumatoid arthritis , and atherosclerosis , where overall venous thrombosis will be induced by systemic inflammation . more than 1/3 of sle patients ' deaths were due to thrombosis events , and in these cases thrombosis can be predicted based on the status of apl . in addition , female apl - positive patients who have pregnancy morbidity will show high risks for thrombosis , yet they can not fulfill the eligibility criteria for undergoing aps diagnostic procedures . the possibilities for vascular thrombosis after pregnancy can be reduced by using lda . for example , fetal loss was observed in a single female apl - positive patient only in a retrospective study . in this study , the control group showed 59% of incidence of thrombosis event , whereas the lda group showed 10% of incidence . it has also been identified from surveys that fetal loss will occur among the female patients who show strong apl . for example , it has been identified that thrombosis events , common in patients with sle , can be managed effectively with hcq [ 32 , 36 , 41 ] . with its ability to inhibit the inflammatory cytokines such as tnf , il1 , il2 , and il6 , hcq can effectively prevent thrombosis . in addition , platelet aggregation and arachidonic acid release from stimulated platelets can also be inhibited with hcq [ 72 , 73 ] . further , the interaction between anti-2gp1 antibodies and the phospholipid bilayers , thrombus size , and total time of thrombus formation can prevent thromboembolic events in sle patients and primary thrombotic events in asymptomatic apl - positive patients through hcq . however , discovering the most effective therapeutic targets requires elucidation of the pathogenesis process involved in aps . to date , there is no in - depth knowledge on the advantages associated with therapeutic agents such as rituximab or alternative antiplatelet drugs ; more researches and evidence are required .

context . antiphospholipid antibodies syndrome is an autoimmune disorder that is characterized by the association between presence of antiphospholipid antibodies and risk of thrombosis and/or pregnancy morbidity . objectives . to systematically review the evidence for primary prophylaxis in patients with antiphospholipids antibodies syndrome or aps with or without other traditional risk factors of thrombosis when they did not have any thrombotic event yet . methods . pubmed , the cochrane library , and allied health literature were searched for studies that examined the efficacy and safety of primary prophylaxis in apl patients from 1990 to february 2013 . we examined literature looking at patients with apls with other risk factors for thrombosis and apls with no additional risk factors for thrombosis . conclusion . we concluded that , in patients with apls , primary prophylaxes with hcq and aspirin have been observed to reduce the frequency of thrombotic events in the case of asymptomatic apl - positive patients with sle . we also in this study concluded that lda was effective in patients with autoimmune diseases . independent cardiovascular risk factors include autoimmune defects such as sle , rheumatoid arthritis , and atherosclerosis , where overall venous thrombosis will be induced by systemic inflammation . this review concludes that hcq is an effective primary approach when compared to aspirin .

1. Introduction 2. Research Methodology 3. Results 4. Discussion 5. Conclusion

a commonly occurring systemic autoimmune disorder , antiphospholipid syndrome ( aps or hughes syndrome ) , is characterized by arterial and/or venous thrombosis , pregnancy morbidity , or recurrent miscarriage . it is also characterized by thrombocytopenia , increased levels of antiphospholipid antibodies ( apl ) , such as positive lupus anticoagulant ( lac ) , which is the most common , and/or anticardiolipin antibodies ( acl ) and 2-glycoprotein i antibodies . for the first hit , a prothrombotic state is created by the presence of antiphospholipid antibodies . aps was first detected in individuals suffering from sle and was later recognized in patients with other autoimmune diseases , although at a lower frequency compared to sle patients . most of the case studies have shown that patients who have elevated levels of antiphospholipids and were affected by thrombosis also had other common habitual risk factors , such as smoking and a high bmi ; allied risk factors included hyperlipidaemia , hypertension , high triglycerides , and type ii diabetes [ 1013 ] . while it is still unclear if the presence of antiphospholipid antibodies is an additional risk factor for individuals who are not affected by autoimmune disorders , it can be hypothesized that the extent of endothelial injury intensified over time caused by the abovementioned common risk factors is reflected in increased levels of antiphospholipid antibody and the presence of lac [ 4 , 14 ] . recent improvement in the understanding of the pathogenic mechanisms , including apl - induced activation of platelets , endothelial cells , monocytes , complement , and coagulation cascade , has led to the identification of potential targets and future therapies for aps . aspirin is commonly prescribed for the prevention of primary thrombosis in populations exposed to traditional risk factors and secondary venous thrombosis . in fact , there has been a lack of conclusive evidence as to whether to choose aspirin , hcq , or a combination of these two in patients positive for apls with other risk factors for thrombosis and with no additional risk factors . there are no extensive studies on the effectiveness and safety of primary prophylaxis in apl patients who are identified with other risk factors for thrombosis or apl patients who have no risk factors for the same . however , the efficacy of primary prophylaxis in these two groups has been analyzed and compared in this study . through this systematic review , we intend to collate information on the effectiveness of treatment methods available for the prevention of primary thrombosis in apl patients with and without risk factors . this program of research is defined by two key research questions.is primary prophylaxis safe and effective in preventing thrombosis for patients both with and without other risk factors for thrombosis in antiphospholipids syndrome apls?what is the best treatment to prevent recurrent thrombosis in apl patients with and without additional risk factors for thrombosis and venous and arterial events not fulfilling and fulfilling criteria for aps ? is primary prophylaxis safe and effective in preventing thrombosis for patients both with and without other risk factors for thrombosis in antiphospholipids syndrome apls ? what is the best treatment to prevent recurrent thrombosis in apl patients with and without additional risk factors for thrombosis and venous and arterial events not fulfilling and fulfilling criteria for aps ? the review will predominantly focus on primary prophylaxis as a treatment for patients with elevated apl , with and without additional risk factors for thrombosis . for the systematic review pubmed , the cochrane library , medline , and allied health literature were searched for studies that examined the efficacy and safety of primary prophylaxis in apl patients . to reflect current apl practices and techniques , the literature search was limited to studies published from january 1990 to february 2013 . searches were conducted using a combination of three terms from the following sets : ( 1 ) antiphospholipid syndrome , antiphospholipid antibodies , thrombosis , and/or beta 2-glycoprotein i ; with ( 2 ) thrombosis not children , not review , not recurrent , antiphospholipid syndrome , or lupus anticoagulant or primary prevention , primary prophylaxis , or primary therapy or drug therapy , antiphospholipid syndrome / therapy , antibodies ; and ( 3 ) thrombosis therapy . subsequently , studies that assessed efficacy and safety levels of primary prophylaxis , followed by identification of the best treatment to prevent recurrent thrombosis in apl patients and venous and arterial events not fulfilling and fulfilling criteria for apl , were included . the study compared the efficacy of aspirin 81 mg daily versus placebo for the prevention of thrombotic complications among 98 asymptomatic patients with persistently positive apl . however , this study was terminated due to an unexpectedly low rate of thrombotic events ( arterial or venous ) ( n = 3 ) occurring in the aspirin group and none in placebo . although this study observed infrequent outcome events and was limited by the small number of patients enrolled , in the patients with persistently positive apl aspirin was not effective in comparison to placebo for the primary prevention of thrombotic events . arterial and venous thrombotic manifestations can be prevented by using a prophylactic treatment with aspirin in the case of sle patients . however , another clinical trial ( aplasa ) that was conducted on patients with sle showed that there were not many differences among apl - positive patients treated with either low - dose aspirin or placebo . the authors noted that the zero event rate in the case of the placebo group can be attributed to a number of factors , such as the inclusion of individuals ( over 40% ) with a low - risk apl profile , a short followup , and good control of vascular risk factors . in the case of asymptomatic individuals with apl , the additive clinical thrombotic risk factors , along with the likely preventive treatments , were analysed in a cross - sectional study that surveyed 77 aps patients . the results from the study were compared to the results that were obtained by patients who were asymptomatic apl - positive and who had no history of pregnancy morbidity or vascular thrombosis . the analysis of the results indicated that pregnancy ( p = 0.005 ) and surgical procedures ( p = 0.04 ) were significantly more frequent in group a , whereas aspirin ( p < 0.001 ) , hcq ( p < 0.001 ) , and corticosteroids ( p = 0.002 ) were used extensively more frequently in group b. surgical procedures and pregnancy , along with thrombocytopenia , raised the risk of an event in the case of women . the study also showed an increased risk of hypertension along with a number of other factors , especially in the case of arterial thrombosis ; however , these factors were not shown to be associated with venous thrombosis . however , there is still a lack of sufficient research that has dealt with the question of whether or not the efficacy of primary thromboprophylaxis in this group of patients with sle and apl increases for the combination therapy of antimalarials plus low - dose aspirin . in the case of patients who were given high - intensity warfarin , recurrent thrombosis could be seen in 6 of 54 patients ( 11.1% ) [ hazard ratio for the high - intensity group , 1.97 ; 95% confidence interval ( ci ) 0.497.89 ] . in the case of patients who received conventional treatment , thus , it is clearly evident from the trial results that high - intensity warfarin is not a better treatment than conventional treatment when it comes to preventing recurrent thrombosis in patients with aps , as high - intensity warfarin - based treated patients were more prone to minor hemorrhagic complications . numerous risk factors such as sle - related factors and traditional or apl - profile factors were evaluated by a study conducted by tektonidou et al . in the case of apl - positive patients , the study demonstrated that 20.1% of them showed high thrombosis rates and about 7.6% ( p = 0.003 ) of the negative apl patients showed high thrombosis rates . in the case of apl - positive patients , a low dose of aspirin helped in protecting the patients against thrombosis ( hr per month 0.98 , p = 0.05 ) . evaluated the overall risk factors associated with a first thrombotic event in apl - positive carriers in people 1865 years of age ; it also explored the effectiveness of prophylactic treatments . in the case of asymptomatic apl carriers , once again medium / high titres of igg acl along with hypertension were determined to be risk factors , and it was also determined that primary prophylaxis is protective . in the case of 83 patients , the study made observations over a period of 3,094 patient - years , 40 venous events , and 48 arterial events . sixty months from the time of diagnosis of sle , the study discovered that 8.5% of the chinese were at risk of arterial events , followed by 8.1% of african americans , and 5.1% of caucasians . similarly , the study also determined that 60 months from the time of diagnosis of sle , the risk of venous events was 3.7% , 6.6% , and 10.3% , respectively ( p = 0.008 for chinese versus caucasians , by log - rank test ) . thus , some of the differences and factors associated with arterial and venous thromboembolism in patients with sle can not be understood or explained . apl - positive patients who have no symptoms of sle have been assessed for the effect of hcq on anxa5 resistance assay ( 1997 ) . the current study analysed clinical and analytical findings in 404 individuals among apl - positive patients with sle and included a followup for 36 months . the results indicated that , among apl - positive patients , 16 had autoimmune thrombocytopenia ( ait ) , 20 had prolonged activated partial thromboplastin times , 11 had diverse diseases , 37 had systemic lupus erythematosus ( sle ) , and 19 had other connective tissue diseases . thus , the study confirms immunomodulating medication use , longer disease duration , history of nephritis , and older age at onset , along with apl positivity , as risk factors for thrombosis in sle . miscellaneous thrombosis risk factors , including apl - profile , sle - related , and traditional risk factors , have been analysed by tektonidou et al . this study questions how thrombosis can be prevented in 144 patients with sle ( follow - up period 104 and 112 months ) and those who do not have apl . in apl patients , thrombosis was predicted with male sex ( hazard ratio [ hr ] 6.25 , p < 0.01 ) , lac ( hr 3.48 , p < 0.04 ) , and consistently positive acl ( hr 5.87 , p 0.01 ) . in addition , both apl - positive and -negative patients had been treated with hcq ( hr per month 0.99 , p < 0.05 ) ( hr per month 0.98 , p 0.04 ) , respectively . the effect of hcq on the anxa5-ra in apl - positive patients has been studied in a prospective study conducted by rand et al . this study involves the comparison of baseline data from 15 apl - positive patients ( mean age 43.3 12.0 years [ range 2761 ] , 10 [ 67% ] female , 13 [ 87% ] caucasian ; 10 had antiphospholipid syndrome , 5 asymptomatic apl ) and 7 apl - negative sle patients ( mean age 40.0 16.3 years [ range 2264 ] , 7 [ 100% ] female , 4 [ 57% ] caucasian ) . positive anxa5-ra , triple apl - positive , double apl - positive , and single apl - positive were observed in 87% , 69% , 15% , and 15% of the apl - positive patients , respectively . in this study , vasculitis , premature atherosclerosis , and hypercoagulability are the major causes of thrombosis in sle patients . in a hopkins cohort study of 100 patients , it was identified that thrombosis can be predicted with high anti - dsdna and low c3 , atherosclerosis ( hypertension , hyperlipidemia , and elevated homocysteine ) , and antiphospholipid antibodies ( lupus anticoagulant or anticardiolipin ) and could be treated with hcq . in this study , the antimalarial drug was protective against thrombosis ( hr 0.28 , 95% ci 0.080.90 ) , while apl positivity ( hr 3.16 , 95% ci 1.456.88 ) and previous thrombosis ( hr 3.85 , 95% ci 1.509.91 ) increased the risk of thrombotic events . this systematic review comprehensively evaluated the efficacy , safety , and treatment methods for primary prophylaxis of therapeutic measures among patients positive for apls with other risk factors for thrombosis and apls with no additional risk factors for thrombosis . the study specifically compares the two groups of apl with other risk factors for thrombosis and with no additional risk factors . moreover , by searching the literature and extant studies covering populations where hydroxychloroquine ( hcq ) was effective and comparing them to other studies where hcq was not effective as a primary prophylaxis , this review concludes that hcq is an effective primary approach when compared to aspirin . along with hcq , aspirin 's effects as a preventive therapy have been analyzed in this review . based on an assessment of the literature collected , it is clear that primary thrombosis prophylaxis strategies in asymptomatic apl - positive with other risk factors for thrombosis and apls with no additional risk factors for thrombosis are limited . showed that in markov decision analysis when patients with sle and apl - positive are given a prophylactic treatment with aspirin , arterial and venous thrombosis is prevented . in subsequent study , the results suggested that , while low - dose aspirin did not prove beneficial , it did lower the overall annual rate of thrombosis incidence . in a randomized clinical study , it was found that the overall incidence rate of thrombosis was 1.33 per 100 patient - years ; for aspirin treated cases , the incidence rate was 2.75 per 100 patient - years , and , in the placebo group , the rate was 0 per 100 patient - years . accordingly , in the observational study carried out , the overall incidence rate of thrombosis was 2.2 per 100 patient - years ; in aspirin treated cases , the incidence rate was 2.70 per 100 patient - years and 0 per 100 patient - years in the placebo group of subjects . in this study , all patients ( except one ) had thrombosis risk factors along with the incidence of thrombosis . all of the above results indicate that persistently apl - positive individuals receive little to no benefit from aspirin treatment for primary prophylaxis , but the overall annual incidence rate of acute thrombosis is minimized . however , in the presence of other traditional risk factors , vascular disorders may occur . considering this level of thrombotic risk , the benefits did not outweigh the potential risks.the expected benefits of aspirin have been proven in at least one randomized clinical trial , even though there may have been few methodological limitations . considering this level of thrombotic risk the expected benefits of aspirin have been proven in at least one randomized clinical trial , even though there may have been few methodological limitations . various risk factors of thrombosis other than high apl levels such as habitual risks ( e.g. have shown that hcq effectively lowers the risk of thrombosis in both apl - positive and -negative subjects . therefore , in the case of patients who are apl - positive the highest percentage of thrombosis occurs in the subgroup with sle [ 32 , 36 , 37 , 39 , 49 , 5762 ] . thromboembolic events can be effectively treated with or without antiphospholipid antibodies by means of hcq in patients who have lupus [ 6770 ] and in patients with apl . primary prophylaxes with hcq and aspirin have been observed to reduce the frequency of thrombotic events in the case of asymptomatic apl - positive patients with sle . in a markov decision analysis , oral anticoagulant therapy and observation , along with prophylactic aspirin , there is still a lack of sufficient research that has dealt with the question of whether or not the efficacy of primary thromboprophylaxis in this group of patients with sle and apl increases when both therapies ( antimalarials plus low - dose aspirin ) are combined . in the case of asymptomatic apl carriers , medium / high titres of igg acl , along with hypertension , were determined to be the risk factors , and it was also determined that primary prophylaxis is protective . for the clinician who is faced with an apl - positive patient with no history of pregnancy morbidity or thrombosis , it is better that an individual assessment of the patient 's thrombotic risk be done . primary prophylaxis with aspirin , either alone or in combination with hcq , is not advisable if the patient does not show any signs of sle , cardiovascular , or thrombotic risk factors . the annual incidence rate of thrombosis events in anti - cl - positive patients who have sle was found to be 3.8% . there has also been a study on the prophylactic role of lda in apl - positive sle patients . the possibilities for reducing the thrombosis events have been discussed in this study . on the other hand , independent cardiovascular risk factors include autoimmune defects such as sle , rheumatoid arthritis , and atherosclerosis , where overall venous thrombosis will be induced by systemic inflammation . in addition , female apl - positive patients who have pregnancy morbidity will show high risks for thrombosis , yet they can not fulfill the eligibility criteria for undergoing aps diagnostic procedures . in this study , the control group showed 59% of incidence of thrombosis event , whereas the lda group showed 10% of incidence . for example , it has been identified that thrombosis events , common in patients with sle , can be managed effectively with hcq [ 32 , 36 , 41 ] . further , the interaction between anti-2gp1 antibodies and the phospholipid bilayers , thrombus size , and total time of thrombus formation can prevent thromboembolic events in sle patients and primary thrombotic events in asymptomatic apl - positive patients through hcq .

the term acoustic neuroma defines a benign tumor of the schwann cell neurilemma , which grows mostly in the lower vestibular nerve of the 8 cranial nerve7 . from 1991 on , vestibular schwannoma ( vs ) became the most appropriate term , representing the real situation for the majority of cases . vestibular schwannomas are among the most commonly diagnosed tumors and account for approximately 6% of all brain tumors34 . vestibular schwannomas are characterized by the slow progression of hearing loss , with or without balance loss . this happens because these tumors grow slowly , causing such a gradual decrease in labyrinth stimulation , that the central compensation mechanisms are able to reduce the impact of these symptoms27 . on the other hand , this pain frequently resembles trigeminal neuralgia26,34 . in schwannomas reaching the trigeminal nerve , although the facial nerve is anatomically impaired by the tumor growing adjacent to it , seldom does facial paralysis appear in clinical cases , which is a sign of the remarkable resistance to compression exhibited by the 7 nerve35 . the afferent root of the trigeminal nerve , which links the gasserian ganglion and the pons , may be a site for schwannomas , with the tumor growing in the cerebellopontine angle7,34,35 . the majority of tumors growing in this region is comprised by vestibular schwannomas . at times , the trigeminal nerve can be partly affected by schwannomas , as the latter occurs adjacent to the former . small neuromas are characterized by appearing only in the pons , where nerves responsible for auditory performance , equilibrium and movements of motor muscles as well as some vessels of the inner ear are found . the medium - sized neuromas stretch from the pons to the cranial cavity , yet without compressing any brain structure . on the other hand , larger neuromas stretch outside the internal canal towards the cranial cavity producing some pressure on the brain and thus altering important vital centers2 . it is described by the international association for the study of pain ( 2004 ) as ' unilateral facial pain , resembling an electric discharge , limited to one or more branches of the trigeminal nerve path'13 . the pain is triggered by ordinary sensorial stimuli such as washing the face , shaving , applying make up , drinking water , speaking , brushing the teeth , among others24 . the pain lasts only a few seconds3 , and the clinical diagnosis is based on the patient 's history40 . in a study using magnetic resonance imaging , yang , et al.28 observed that in 51 patients suffering from trigeminal neuralgia , 16% ( 8) presented vestibular schwannoma38 . similarly , samii and matthies28 reported that between approximately 1% and 3% of vestibular schwannoma patients presented trigeminal neuralgia symptoms . nevertheless , selesnick , et al.30 reported that in a group of 126 patients with vestibular schwannoma , none of them presented neuralgia . of every 100 temporal bones selected and submitted to post - mortem anatomy studies , one presents scwannoma on the vestibular nerve . the prevalence of this disorder , considering the current diagnosis conditions , is from 7 to 9 in 1,000,000 subjects36 . it happens independently of ethnicity and is more frequently diagnosed in men within the 50 - 60 year old age group ( 61%)7 . it is estimated that between 2,000 and 3,000 new unilateral vestibular neuroma cases are diagnosed yearly ( incidence ) in the us , which characterizes a 1:100,000 occurrence1 . research has shown that unilateral neuroma is not hereditary , and only one in 1,000 cases occurred in which mother and daughter suffered from the disorder7,14 . as a rule , two kinds of microscopic findings can be observed according to the description elaborated by antoni in 1920 . first , vestibular schwannoma of type a is defined by a more closely packed tissue in which cells form a palisade pattern and in a bipolar , parallel cell arrangements disposed in interlacing bundles that alternate with enucleate fibrilar zones composed of cellular processes . second , type b occurs as a more loosely organized tissue , with a more noticeable reticulated appearance and interstitial edema7 . neoplastic cells having condensed nuclei and indistinguishable cytoplasm are observed , which may look like lymphocytes at first sight4 . using mri ( t2 ) the vestibular signs , the effect on the trigeminal nerve , and the cerebellar and intracranial pressure signs may all become noticeable with tumor growth , thus enabling the delineation of a sequential and evolutionary pattern7 . the clinical signs may be characterized as : a ) progressive unilateral or asymmetrical sensorineural dysacusis , b ) intermittent hearing loss , c ) tinnitus and sensation of fullness in the ear , d ) lasting positional vertigo , e ) difficulties in walking , f ) visual blurring , g ) trigeminal neuralgia , and e ) headaches7,10,19,20,40 . hearing ability is normal in at least 8% of cases , and tumor is suspected in the case of unusual or bizarre complaints that are investigated by mri7 . matsuka , et al.20 listed the following symptoms related to vestibular schwannoma : hearing deficits ( 60 to 97% ) , tinnitus ( 50 to 66% ) , vestibular disturbances ( 46 to 59% ) , numbness or tingling of the face ( 33% ) , headaches ( 19 to 29% ) , dizziness ( 23% ) , bell 's palsy ( 17% ) , and trigeminal disturbances hyperesthesia , paresthesia , and neuralgia ( 12 to 45% ) . differential diagnosis between acoustic and trigeminal neuromas is fundamentally based on early impairment of auditory acuity ipsilateral to the lesion in patients with 8 nerve schwannoma7,34,35 . mri is more advantageous as compared to ct scans and to any other imaging technique when identifying lesions in the trigeminal nerve path , and it is considered the technique of choice in the diagnosis of vestibular schwannomas12 . although the schwannoma is benign because of growing in the vestibule , morbidity is high due to the compression of vital structures such as the cranial nerves and the brain1 . a 59-year - old woman was referred to the center for post graduation studies on pain and temporomandibular disorders ( tmd ) of the brazilian dental association state of rio grande do sul ( abo - rs ) with acute pain around the lower lip , jaw , and right temporomandibular joint ( tmj ) , which impaired mouth opening . the main complaint was the difficulty to open the mouth due to shooting pain attacks on the right side of the face . pain happened along the trigeminal nerve path , yet not reaching beyond the median line . the patient 's previous history , assessed by the had questionnaire ( hospital anxiety and depression scale ) , revealed the clinical signs characteristic of depression and anxiety22 . the patient also described persistent and localized episodes of bilateral pain in the left and right masseter as well as in the temporalis muscles which increased with function , and the presence of painful hypertrophic bands . when palpating the right masseter , pain was felt in the insertion region and in the muscle itself and was graded as 3 by the patient on a four - point scale ( 0 = no pain , 1 = report of discomfort , 2 = report of pain , 3 = report of pain with withdrawal reflex ) . on the left side , pain happened in the masseter itself , and was graded as 1 . as for right and left temporal muscles , the patient reported grade 1 pain in all muscle sheaths . pterygoid muscles were also reported to be affected by the pain on both sides , graded as 3 . the patient characterized facial pain episodes as grade 10 , while muscle pain was graded as 7 . the patient 's everyday chores as well as professional work was impaired by the pain , which also affected her social and family life . furthermore , the patient exhibited clicking and popping sounds in both tmjs and reported displaced bite , a history of mandible locking , both at night and during the day , teeth grinding , and a certain degree of discomfort in the morning . the research diagnostic criteria for temporomandibular disorders ( rdc / tmd ) was used to assess the patient 's physical characteristics8 . on the first occasion , this test was not completed , because of the very small mouth opening ( 26 mm ) . the patient was instructed to make use of moist heat on the affected muscle area for 20 min , 3 times a day , for fourteen days . a non - steroid antiinflammatory drug selective cyclooxygenase 2 was prescribed , three times a day , for the same period . on a second assessment , the rdc / tmd questionnaire with assistance , the patient was able to further open the mouth 44 mm with intense pain . an overbite of 3 mm was observed , along with a lateral centric slide of 2 mm to the left of the median line . the opening pattern was s - shaped , deviated to the right side , presenting a 4-mm maximum lateral excursive movement to the right and 12 mm to the left . concerning articular sounds , a click was observed on the right side , at 23 mm opening , and on the left , at 25 mm . on closing , clicking was heard at 21 mm on the right and 25 mm on the left . reciprocal sounds ceased in protrusive movement on both sides , but they were present in lateral movements . in this particular case , there were signs and symptoms indicative of neuralgic pain of the fifth branch ( trigeminal division ) , probably related to brain tumor . a physician , under the recommendation of the dentists supervising the case , requested a mri evaluation , which was performed forty days after the clinical evaluation . mri revealed a slight disk displacement , with a decrease on the right side and an extensive tumoral lesion in the right acoustic channel stretching to cerebellopontine angle ( figure 1a ) , suggesting a vestibular schwannoma . initially , the treatment proposed was to reduce localized muscle pain using anti - inflammatory drugs , muscle relaxation drugs , and moist heat in the affected muscular region , for four days . concomitantly , a michigan - type bite plane was manufactured to stabilize occlusion and to rule out dental influence as a trigger to central pain , to decrease abnormal muscle activity , and to diminish muscle pain sensitivity . in the follow - up period , the splint underwent adjustments , and from that moment on , the patient started using the splint both at night and during the day , removing it only to eat and clean the teeth . with sequential adjustments and frequent controls , the patient reached a favorable response to the use of the splint , improving the pain prospects . the splint was indicated due to the presence of muscle and skeletal pain indicative of tmd in conjunction with stabilizing agents . there was an improvement not only in the muscle and skeletal pain , including maximum mouth opening , but also in the neuralgic pain . the patient was referred to a psychiatrist for a more comprehensive evaluation of her psychological condition , which revealed symptoms that were suggestive of anxiety and depression . the patient was advised to also see a brain surgeon to evaluate the tumoral lesion ( figures 1b and 1c ) and to investigate dizziness , vertigo and neuralgia . after medical assessment , the patient began to take sertraline , carbamazepine and clonazepam , with subsequent decrease in pain crises . the facial nerve was damaged causing hearing loss and bell 's palsy on the affected side . of every 100 temporal bones selected and submitted to post - mortem anatomy studies , one presents scwannoma on the vestibular nerve . the prevalence of this disorder , considering the current diagnosis conditions , is from 7 to 9 in 1,000,000 subjects36 . it happens independently of ethnicity and is more frequently diagnosed in men within the 50 - 60 year old age group ( 61%)7 . it is estimated that between 2,000 and 3,000 new unilateral vestibular neuroma cases are diagnosed yearly ( incidence ) in the us , which characterizes a 1:100,000 occurrence1 . research has shown that unilateral neuroma is not hereditary , and only one in 1,000 cases occurred in which mother and daughter suffered from the disorder7,14 . as a rule , two kinds of microscopic findings can be observed according to the description elaborated by antoni in 1920 . first , vestibular schwannoma of type a is defined by a more closely packed tissue in which cells form a palisade pattern and in a bipolar , parallel cell arrangements disposed in interlacing bundles that alternate with enucleate fibrilar zones composed of cellular processes . second , type b occurs as a more loosely organized tissue , with a more noticeable reticulated appearance and interstitial edema7 . neoplastic cells having condensed nuclei and indistinguishable cytoplasm are observed , which may look like lymphocytes at first sight4 . using mri ( t2 ) the vestibular signs , the effect on the trigeminal nerve , and the cerebellar and intracranial pressure signs may all become noticeable with tumor growth , thus enabling the delineation of a sequential and evolutionary pattern7 . the clinical signs may be characterized as : a ) progressive unilateral or asymmetrical sensorineural dysacusis , b ) intermittent hearing loss , c ) tinnitus and sensation of fullness in the ear , d ) lasting positional vertigo , e ) difficulties in walking , f ) visual blurring , g ) trigeminal neuralgia , and e ) headaches7,10,19,20,40 . hearing ability is normal in at least 8% of cases , and tumor is suspected in the case of unusual or bizarre complaints that are investigated by mri7 . matsuka , et al.20 listed the following symptoms related to vestibular schwannoma : hearing deficits ( 60 to 97% ) , tinnitus ( 50 to 66% ) , vestibular disturbances ( 46 to 59% ) , numbness or tingling of the face ( 33% ) , headaches ( 19 to 29% ) , dizziness ( 23% ) , bell 's palsy ( 17% ) , and trigeminal disturbances differential diagnosis between acoustic and trigeminal neuromas is fundamentally based on early impairment of auditory acuity ipsilateral to the lesion in patients with 8 nerve schwannoma7,34,35 . mri is more advantageous as compared to ct scans and to any other imaging technique when identifying lesions in the trigeminal nerve path , and it is considered the technique of choice in the diagnosis of vestibular schwannomas12 . although the schwannoma is benign because of growing in the vestibule , morbidity is high due to the compression of vital structures such as the cranial nerves and the brain1 . a 59-year - old woman was referred to the center for post graduation studies on pain and temporomandibular disorders ( tmd ) of the brazilian dental association state of rio grande do sul ( abo - rs ) with acute pain around the lower lip , jaw , and right temporomandibular joint ( tmj ) , which impaired mouth opening . the main complaint was the difficulty to open the mouth due to shooting pain attacks on the right side of the face . pain happened along the trigeminal nerve path , yet not reaching beyond the median line . the patient 's previous history , assessed by the had questionnaire ( hospital anxiety and depression scale ) , revealed the clinical signs characteristic of depression and anxiety22 . the patient also described persistent and localized episodes of bilateral pain in the left and right masseter as well as in the temporalis muscles which increased with function , and the presence of painful hypertrophic bands . when palpating the right masseter , pain was felt in the insertion region and in the muscle itself and was graded as 3 by the patient on a four - point scale ( 0 = no pain , 1 = report of discomfort , 2 = report of pain , 3 = report of pain with withdrawal reflex ) . on the left side , pain happened in the masseter itself , and was graded as 1 . as for right and left temporal muscles , the patient reported grade 1 pain in all muscle sheaths . pterygoid muscles were also reported to be affected by the pain on both sides , graded as 3 . the patient characterized facial pain episodes as grade 10 , while muscle pain was graded as 7 . the patient 's everyday chores as well as professional work was impaired by the pain , which also affected her social and family life . furthermore , the patient exhibited clicking and popping sounds in both tmjs and reported displaced bite , a history of mandible locking , both at night and during the day , teeth grinding , and a certain degree of discomfort in the morning . the research diagnostic criteria for temporomandibular disorders ( rdc / tmd ) was used to assess the patient 's physical characteristics8 . on the first occasion , this test was not completed , because of the very small mouth opening ( 26 mm ) . the patient was instructed to make use of moist heat on the affected muscle area for 20 min , 3 times a day , for fourteen days . a non - steroid antiinflammatory drug selective cyclooxygenase 2 was prescribed , three times a day , for the same period . on a second assessment , the rdc / tmd questionnaire with assistance , the patient was able to further open the mouth 44 mm with intense pain . an overbite of 3 mm was observed , along with a lateral centric slide of 2 mm to the left of the median line . the opening pattern was s - shaped , deviated to the right side , presenting a 4-mm maximum lateral excursive movement to the right and 12 mm to the left . concerning articular sounds , a click was observed on the right side , at 23 mm opening , and on the left , at 25 mm . on closing , clicking was heard at 21 mm on the right and 25 mm on the left . reciprocal sounds ceased in protrusive movement on both sides , but they were present in lateral movements . in this particular case , there were signs and symptoms indicative of neuralgic pain of the fifth branch ( trigeminal division ) , probably related to brain tumor . a physician , under the recommendation of the dentists supervising the case , requested a mri evaluation , which was performed forty days after the clinical evaluation . mri revealed a slight disk displacement , with a decrease on the right side and an extensive tumoral lesion in the right acoustic channel stretching to cerebellopontine angle ( figure 1a ) , suggesting a vestibular schwannoma . initially , the treatment proposed was to reduce localized muscle pain using anti - inflammatory drugs , muscle relaxation drugs , and moist heat in the affected muscular region , for four days . concomitantly , a michigan - type bite plane was manufactured to stabilize occlusion and to rule out dental influence as a trigger to central pain , to decrease abnormal muscle activity , and to diminish muscle pain sensitivity . in the follow - up period , the splint underwent adjustments , and from that moment on , the patient started using the splint both at night and during the day , removing it only to eat and clean the teeth . with sequential adjustments and frequent controls , the patient reached a favorable response to the use of the splint , improving the pain prospects . the splint was indicated due to the presence of muscle and skeletal pain indicative of tmd in conjunction with stabilizing agents . there was an improvement not only in the muscle and skeletal pain , including maximum mouth opening , but also in the neuralgic pain . the patient was referred to a psychiatrist for a more comprehensive evaluation of her psychological condition , which revealed symptoms that were suggestive of anxiety and depression . the patient was advised to also see a brain surgeon to evaluate the tumoral lesion ( figures 1b and 1c ) and to investigate dizziness , vertigo and neuralgia . after medical assessment , the patient began to take sertraline , carbamazepine and clonazepam , with subsequent decrease in pain crises . the facial nerve was damaged causing hearing loss and bell 's palsy on the affected side . as a rule , vestibular schwannomas grow slowly20 . in the present study , mri showed that there was a 0.5-cm growth in 8 months . due to this slow growth and to the neurological adaptations undergone by patients , clinical symptoms are concealed and at times only superficially observed and investigated8,18,30 . facial pain caused by tumors is often related to neurological abnormalities such as : a ) sensory changes , b ) loss of reflexes , and c ) constant pain . patients place little value on these symptoms , because they are not very intense and non - debilitating and because they ignore the need for more specific treatment . according to matsuka , et al.20 similarly , the case study presented here revealed that the patient had felt myofascial pain as early as at 8 years of age . regarding differential diagnosis , some of the symptoms described in the literature , which are associated to the internal auditory canal , are otovestibular dysfunctions , such as hearing loss , tinnitus , vertigo and dizziness6 . in the present case report according to fricton , et al.11 ( 1985 ) , patients with temporomandibular disorders may also describe similar otologic and neurological symptoms . for example , 42.1% of 164 tmd patients presented tinnitus , 41.5% perceived ear pain , 23.1% vertigo , and 17.7% perceived hearing loss . in addition , 27.4% of the subjects presented tingling , 26.2% numbness , 14% blurred vision , 12.2% twitching , 7.9% tremors , and 7.3% lacrimation . these symptoms might be explained by peripheral and central sensitization due to ongoing inflammatory process in the masticatory muscles and/or temporomandibular joint or may be in fact medical conditions with associated and reflex muscular and skeletal symptoms , similar to tmd31 . at the present time , medical imaging , particularly mri , as well as a multidisciplinary assessment , are probably the greatest tools in differentiating the two possible sources of pain as well as otologic and neurological symptoms . the mri is indicated when trigeminal neuralgia is suspected , considering that 10% of trigeminal neuralgias are caused by intracranial tumors28,30,38 . regarding treatment , the differential diagnosis described above and if tmd is suspected to be the major etiology after negative confirmation of neoplasia ( particularly vestibular schwannoma ) , an interocclusal michigan bite appliance may be sufficient to decrease all symptoms . in the clinical case presented here , the patient did improve after the use of a michigan - type bite splint , but this treatment was aimed towards the secondary muscle pain ( tmd type ) caused by the vestibular schwannoma . in other words , in this case , the tmd signs and symptoms were a consequence of the tumor ( referred pain and muscle splinting ) rather than its cause . for a neuralgic pain secondary to a brain tumor , anticonvulsants are the drugs used at the beginning of treatment to prevent and treat dizziness as well as in migraine prophylaxis . this class of drugs is also used in the management of chronic pain ( neuropathies and neuralgias)25 . for trigeminal neuralgia , carbamazepine is the drug of first choice , controlling pain in 70 - 98% of cases . bullit , et al.3 reported that carbamazepine is an efficient drug in the treatment of trigeminal neuralgia associated to intracranial tumors . other studies have also proved the efficiency of other drugs as gabapentine in trigeminal neuralgia therapy5,33 . the action of gabapentine has not been fully established and may involve the modulation of gama - aminobutiric acid and glutamate , or even have an effect on calcium channels25 . with the prescription of such drugs , the patient reported a decrease in the occurrence and duration of crises . however , yang , et al.38 reported an increase in the number of patients suffering from trigeminal neuralgia associated with intracranial tumors which are refractory to these drugs . other drugs may be useful in the control of neuralgic pain , such as baclofen , lamotrigine and tricyclic antidepressants . in the case presented here , the pain attacks were controlled , but crises still persisted due to the delay of surgery . tumors that involve the trigeminal nerve are uncommon , but are an important cause of trigeminal neuralgia and myofascial pain3 . because of the similar characteristics localized myofascial pain increase in intensity when the affected region is palpated , and that one which occurs with hypersensitive bands and trigger points were all symptoms reported by the patient reported here . neuropathic pain is generated in the central nervous system and may simulate a superficial somatic pain , similar to the myofascial pain1 . the use of anti - inflammatory and central muscle relaxation drugs together with moist heat becomes effective in the control of somatic myofascial pain . the neuralgic pain must always be investigated in detail , as it yields debilitation and also because such pain may at times come from intracranial tumors3 . it has advantages over ct and any other type of imaging in the identification of soft tissues lesions , nerve paths and presence of tumors , as mentioned above15,17 . the improved resolution for soft tissues and the capacity to visualize multilayer sections contribute to a better assessment of intracranial anatomic segments of the trigeminal nerve . some authors have reported that conservative treatment may be a choice for patients with few symptoms , small lesions , and who are not able to undergo surgery , due to the slow tumor growth . surgical procedures are the best choice for patients who did not respond favorably to conservative treatment39 . the prognosis of the vestibular schwannoma surgery is good , although all surgical procedures are under a certain morbidity risk16,21 . hearing impairment in the affected side is a likely outcome as well as a lesion in the trigeminal nerve . samii and matthies28 observed that after complete removal of 979 vestibular schwannomas , in 93% of cases , there was preservation of the facial nerve . however , these two authors found major neurological side effects which included paresthesia , hematomas , brain and spinal fistulas , hydrocephalus , bacterial meningitis . the literature reveals that there might be a need for a clinical assessment when an intracranial tumor , such as the vestibular schwannoma , is diagnosed and when the patient initially reports unilateral hearing loss , apart from tinnitus . a clinical diagnosis must be carried out , together with an investigation of the patient 's history and imaging techniques such as mri .

approximately 6 to 16% of patients with trigeminal neuralgia symptoms present intracranial tumors , the most common being the vestibular schwannoma ( acoustic neuroma ) . some symptoms reported by patients include hearing loss , tinnitus , headaches , vertigo and trigeminal disturbances . an increased muscle response in the surrounding head and neck musculature may also be observed , which mimics signs and symptoms of temporomandibular disorders . in these cases , magnetic resonance imaging ( mri ) has proved to be a useful tool in tumor diagnosis . the differential diagnosis between myofascial and neuralgic pain is important , as both may present similar characteristics , while being of different origin , and demanding special treatment approaches . the purpose of this paper is to demonstrate the relationship among trigeminal neuralgia symptoms , intracranial tumors and temporomandibular dysfunction by presenting a clinical case .

INTRODUCTION Prevalence Microscopic Findings Diagnosis and Prognosis Case report DISCUSSION

the term acoustic neuroma defines a benign tumor of the schwann cell neurilemma , which grows mostly in the lower vestibular nerve of the 8 cranial nerve7 . from 1991 on , vestibular schwannoma ( vs ) became the most appropriate term , representing the real situation for the majority of cases . vestibular schwannomas are among the most commonly diagnosed tumors and account for approximately 6% of all brain tumors34 . vestibular schwannomas are characterized by the slow progression of hearing loss , with or without balance loss . in schwannomas reaching the trigeminal nerve , although the facial nerve is anatomically impaired by the tumor growing adjacent to it , seldom does facial paralysis appear in clinical cases , which is a sign of the remarkable resistance to compression exhibited by the 7 nerve35 . the afferent root of the trigeminal nerve , which links the gasserian ganglion and the pons , may be a site for schwannomas , with the tumor growing in the cerebellopontine angle7,34,35 . at times , the trigeminal nerve can be partly affected by schwannomas , as the latter occurs adjacent to the former . the pain is triggered by ordinary sensorial stimuli such as washing the face , shaving , applying make up , drinking water , speaking , brushing the teeth , among others24 . the pain lasts only a few seconds3 , and the clinical diagnosis is based on the patient 's history40 . in a study using magnetic resonance imaging , yang , et al.28 observed that in 51 patients suffering from trigeminal neuralgia , 16% ( 8) presented vestibular schwannoma38 . similarly , samii and matthies28 reported that between approximately 1% and 3% of vestibular schwannoma patients presented trigeminal neuralgia symptoms . nevertheless , selesnick , et al.30 reported that in a group of 126 patients with vestibular schwannoma , none of them presented neuralgia . of every 100 temporal bones selected and submitted to post - mortem anatomy studies , one presents scwannoma on the vestibular nerve . the prevalence of this disorder , considering the current diagnosis conditions , is from 7 to 9 in 1,000,000 subjects36 . it is estimated that between 2,000 and 3,000 new unilateral vestibular neuroma cases are diagnosed yearly ( incidence ) in the us , which characterizes a 1:100,000 occurrence1 . research has shown that unilateral neuroma is not hereditary , and only one in 1,000 cases occurred in which mother and daughter suffered from the disorder7,14 . first , vestibular schwannoma of type a is defined by a more closely packed tissue in which cells form a palisade pattern and in a bipolar , parallel cell arrangements disposed in interlacing bundles that alternate with enucleate fibrilar zones composed of cellular processes . neoplastic cells having condensed nuclei and indistinguishable cytoplasm are observed , which may look like lymphocytes at first sight4 . using mri ( t2 ) the vestibular signs , the effect on the trigeminal nerve , and the cerebellar and intracranial pressure signs may all become noticeable with tumor growth , thus enabling the delineation of a sequential and evolutionary pattern7 . the clinical signs may be characterized as : a ) progressive unilateral or asymmetrical sensorineural dysacusis , b ) intermittent hearing loss , c ) tinnitus and sensation of fullness in the ear , d ) lasting positional vertigo , e ) difficulties in walking , f ) visual blurring , g ) trigeminal neuralgia , and e ) headaches7,10,19,20,40 . hearing ability is normal in at least 8% of cases , and tumor is suspected in the case of unusual or bizarre complaints that are investigated by mri7 . matsuka , et al.20 listed the following symptoms related to vestibular schwannoma : hearing deficits ( 60 to 97% ) , tinnitus ( 50 to 66% ) , vestibular disturbances ( 46 to 59% ) , numbness or tingling of the face ( 33% ) , headaches ( 19 to 29% ) , dizziness ( 23% ) , bell 's palsy ( 17% ) , and trigeminal disturbances hyperesthesia , paresthesia , and neuralgia ( 12 to 45% ) . differential diagnosis between acoustic and trigeminal neuromas is fundamentally based on early impairment of auditory acuity ipsilateral to the lesion in patients with 8 nerve schwannoma7,34,35 . mri is more advantageous as compared to ct scans and to any other imaging technique when identifying lesions in the trigeminal nerve path , and it is considered the technique of choice in the diagnosis of vestibular schwannomas12 . although the schwannoma is benign because of growing in the vestibule , morbidity is high due to the compression of vital structures such as the cranial nerves and the brain1 . a 59-year - old woman was referred to the center for post graduation studies on pain and temporomandibular disorders ( tmd ) of the brazilian dental association state of rio grande do sul ( abo - rs ) with acute pain around the lower lip , jaw , and right temporomandibular joint ( tmj ) , which impaired mouth opening . the patient also described persistent and localized episodes of bilateral pain in the left and right masseter as well as in the temporalis muscles which increased with function , and the presence of painful hypertrophic bands . when palpating the right masseter , pain was felt in the insertion region and in the muscle itself and was graded as 3 by the patient on a four - point scale ( 0 = no pain , 1 = report of discomfort , 2 = report of pain , 3 = report of pain with withdrawal reflex ) . on the left side , pain happened in the masseter itself , and was graded as 1 . as for right and left temporal muscles , the patient reported grade 1 pain in all muscle sheaths . the patient 's everyday chores as well as professional work was impaired by the pain , which also affected her social and family life . furthermore , the patient exhibited clicking and popping sounds in both tmjs and reported displaced bite , a history of mandible locking , both at night and during the day , teeth grinding , and a certain degree of discomfort in the morning . the research diagnostic criteria for temporomandibular disorders ( rdc / tmd ) was used to assess the patient 's physical characteristics8 . on a second assessment , the rdc / tmd questionnaire with assistance , the patient was able to further open the mouth 44 mm with intense pain . in this particular case , there were signs and symptoms indicative of neuralgic pain of the fifth branch ( trigeminal division ) , probably related to brain tumor . mri revealed a slight disk displacement , with a decrease on the right side and an extensive tumoral lesion in the right acoustic channel stretching to cerebellopontine angle ( figure 1a ) , suggesting a vestibular schwannoma . initially , the treatment proposed was to reduce localized muscle pain using anti - inflammatory drugs , muscle relaxation drugs , and moist heat in the affected muscular region , for four days . concomitantly , a michigan - type bite plane was manufactured to stabilize occlusion and to rule out dental influence as a trigger to central pain , to decrease abnormal muscle activity , and to diminish muscle pain sensitivity . in the follow - up period , the splint underwent adjustments , and from that moment on , the patient started using the splint both at night and during the day , removing it only to eat and clean the teeth . with sequential adjustments and frequent controls , the patient reached a favorable response to the use of the splint , improving the pain prospects . there was an improvement not only in the muscle and skeletal pain , including maximum mouth opening , but also in the neuralgic pain . the patient was referred to a psychiatrist for a more comprehensive evaluation of her psychological condition , which revealed symptoms that were suggestive of anxiety and depression . the patient was advised to also see a brain surgeon to evaluate the tumoral lesion ( figures 1b and 1c ) and to investigate dizziness , vertigo and neuralgia . after medical assessment , the patient began to take sertraline , carbamazepine and clonazepam , with subsequent decrease in pain crises . it is estimated that between 2,000 and 3,000 new unilateral vestibular neuroma cases are diagnosed yearly ( incidence ) in the us , which characterizes a 1:100,000 occurrence1 . research has shown that unilateral neuroma is not hereditary , and only one in 1,000 cases occurred in which mother and daughter suffered from the disorder7,14 . as a rule , two kinds of microscopic findings can be observed according to the description elaborated by antoni in 1920 . first , vestibular schwannoma of type a is defined by a more closely packed tissue in which cells form a palisade pattern and in a bipolar , parallel cell arrangements disposed in interlacing bundles that alternate with enucleate fibrilar zones composed of cellular processes . neoplastic cells having condensed nuclei and indistinguishable cytoplasm are observed , which may look like lymphocytes at first sight4 . using mri ( t2 ) the vestibular signs , the effect on the trigeminal nerve , and the cerebellar and intracranial pressure signs may all become noticeable with tumor growth , thus enabling the delineation of a sequential and evolutionary pattern7 . the clinical signs may be characterized as : a ) progressive unilateral or asymmetrical sensorineural dysacusis , b ) intermittent hearing loss , c ) tinnitus and sensation of fullness in the ear , d ) lasting positional vertigo , e ) difficulties in walking , f ) visual blurring , g ) trigeminal neuralgia , and e ) headaches7,10,19,20,40 . hearing ability is normal in at least 8% of cases , and tumor is suspected in the case of unusual or bizarre complaints that are investigated by mri7 . matsuka , et al.20 listed the following symptoms related to vestibular schwannoma : hearing deficits ( 60 to 97% ) , tinnitus ( 50 to 66% ) , vestibular disturbances ( 46 to 59% ) , numbness or tingling of the face ( 33% ) , headaches ( 19 to 29% ) , dizziness ( 23% ) , bell 's palsy ( 17% ) , and trigeminal disturbances differential diagnosis between acoustic and trigeminal neuromas is fundamentally based on early impairment of auditory acuity ipsilateral to the lesion in patients with 8 nerve schwannoma7,34,35 . mri is more advantageous as compared to ct scans and to any other imaging technique when identifying lesions in the trigeminal nerve path , and it is considered the technique of choice in the diagnosis of vestibular schwannomas12 . although the schwannoma is benign because of growing in the vestibule , morbidity is high due to the compression of vital structures such as the cranial nerves and the brain1 . a 59-year - old woman was referred to the center for post graduation studies on pain and temporomandibular disorders ( tmd ) of the brazilian dental association state of rio grande do sul ( abo - rs ) with acute pain around the lower lip , jaw , and right temporomandibular joint ( tmj ) , which impaired mouth opening . the patient also described persistent and localized episodes of bilateral pain in the left and right masseter as well as in the temporalis muscles which increased with function , and the presence of painful hypertrophic bands . when palpating the right masseter , pain was felt in the insertion region and in the muscle itself and was graded as 3 by the patient on a four - point scale ( 0 = no pain , 1 = report of discomfort , 2 = report of pain , 3 = report of pain with withdrawal reflex ) . on the left side , pain happened in the masseter itself , and was graded as 1 . as for right and left temporal muscles , the patient reported grade 1 pain in all muscle sheaths . the patient characterized facial pain episodes as grade 10 , while muscle pain was graded as 7 . the patient 's everyday chores as well as professional work was impaired by the pain , which also affected her social and family life . furthermore , the patient exhibited clicking and popping sounds in both tmjs and reported displaced bite , a history of mandible locking , both at night and during the day , teeth grinding , and a certain degree of discomfort in the morning . the research diagnostic criteria for temporomandibular disorders ( rdc / tmd ) was used to assess the patient 's physical characteristics8 . an overbite of 3 mm was observed , along with a lateral centric slide of 2 mm to the left of the median line . the opening pattern was s - shaped , deviated to the right side , presenting a 4-mm maximum lateral excursive movement to the right and 12 mm to the left . in this particular case , there were signs and symptoms indicative of neuralgic pain of the fifth branch ( trigeminal division ) , probably related to brain tumor . a physician , under the recommendation of the dentists supervising the case , requested a mri evaluation , which was performed forty days after the clinical evaluation . mri revealed a slight disk displacement , with a decrease on the right side and an extensive tumoral lesion in the right acoustic channel stretching to cerebellopontine angle ( figure 1a ) , suggesting a vestibular schwannoma . initially , the treatment proposed was to reduce localized muscle pain using anti - inflammatory drugs , muscle relaxation drugs , and moist heat in the affected muscular region , for four days . concomitantly , a michigan - type bite plane was manufactured to stabilize occlusion and to rule out dental influence as a trigger to central pain , to decrease abnormal muscle activity , and to diminish muscle pain sensitivity . in the follow - up period , the splint underwent adjustments , and from that moment on , the patient started using the splint both at night and during the day , removing it only to eat and clean the teeth . there was an improvement not only in the muscle and skeletal pain , including maximum mouth opening , but also in the neuralgic pain . the patient was referred to a psychiatrist for a more comprehensive evaluation of her psychological condition , which revealed symptoms that were suggestive of anxiety and depression . the patient was advised to also see a brain surgeon to evaluate the tumoral lesion ( figures 1b and 1c ) and to investigate dizziness , vertigo and neuralgia . after medical assessment , the patient began to take sertraline , carbamazepine and clonazepam , with subsequent decrease in pain crises . the facial nerve was damaged causing hearing loss and bell 's palsy on the affected side . facial pain caused by tumors is often related to neurological abnormalities such as : a ) sensory changes , b ) loss of reflexes , and c ) constant pain . patients place little value on these symptoms , because they are not very intense and non - debilitating and because they ignore the need for more specific treatment . according to matsuka , et al.20 similarly , the case study presented here revealed that the patient had felt myofascial pain as early as at 8 years of age . regarding differential diagnosis , some of the symptoms described in the literature , which are associated to the internal auditory canal , are otovestibular dysfunctions , such as hearing loss , tinnitus , vertigo and dizziness6 . in the present case report according to fricton , et al.11 ( 1985 ) , patients with temporomandibular disorders may also describe similar otologic and neurological symptoms . for example , 42.1% of 164 tmd patients presented tinnitus , 41.5% perceived ear pain , 23.1% vertigo , and 17.7% perceived hearing loss . these symptoms might be explained by peripheral and central sensitization due to ongoing inflammatory process in the masticatory muscles and/or temporomandibular joint or may be in fact medical conditions with associated and reflex muscular and skeletal symptoms , similar to tmd31 . regarding treatment , the differential diagnosis described above and if tmd is suspected to be the major etiology after negative confirmation of neoplasia ( particularly vestibular schwannoma ) , an interocclusal michigan bite appliance may be sufficient to decrease all symptoms . in the clinical case presented here , the patient did improve after the use of a michigan - type bite splint , but this treatment was aimed towards the secondary muscle pain ( tmd type ) caused by the vestibular schwannoma . in other words , in this case , the tmd signs and symptoms were a consequence of the tumor ( referred pain and muscle splinting ) rather than its cause . for a neuralgic pain secondary to a brain tumor , anticonvulsants are the drugs used at the beginning of treatment to prevent and treat dizziness as well as in migraine prophylaxis . this class of drugs is also used in the management of chronic pain ( neuropathies and neuralgias)25 . bullit , et al.3 reported that carbamazepine is an efficient drug in the treatment of trigeminal neuralgia associated to intracranial tumors . with the prescription of such drugs , the patient reported a decrease in the occurrence and duration of crises . however , yang , et al.38 reported an increase in the number of patients suffering from trigeminal neuralgia associated with intracranial tumors which are refractory to these drugs . other drugs may be useful in the control of neuralgic pain , such as baclofen , lamotrigine and tricyclic antidepressants . in the case presented here , the pain attacks were controlled , but crises still persisted due to the delay of surgery . because of the similar characteristics localized myofascial pain increase in intensity when the affected region is palpated , and that one which occurs with hypersensitive bands and trigger points were all symptoms reported by the patient reported here . neuropathic pain is generated in the central nervous system and may simulate a superficial somatic pain , similar to the myofascial pain1 . the use of anti - inflammatory and central muscle relaxation drugs together with moist heat becomes effective in the control of somatic myofascial pain . the neuralgic pain must always be investigated in detail , as it yields debilitation and also because such pain may at times come from intracranial tumors3 . it has advantages over ct and any other type of imaging in the identification of soft tissues lesions , nerve paths and presence of tumors , as mentioned above15,17 . some authors have reported that conservative treatment may be a choice for patients with few symptoms , small lesions , and who are not able to undergo surgery , due to the slow tumor growth . the prognosis of the vestibular schwannoma surgery is good , although all surgical procedures are under a certain morbidity risk16,21 . the literature reveals that there might be a need for a clinical assessment when an intracranial tumor , such as the vestibular schwannoma , is diagnosed and when the patient initially reports unilateral hearing loss , apart from tinnitus . a clinical diagnosis must be carried out , together with an investigation of the patient 's history and imaging techniques such as mri .

the face is a frequent target for trauma in motorvehicle accidents , especially when seat belts are not used . the high rate of motorvehicle accidents leads to economic and social problems and causes harm to the country , including increased costs for hospital care , pension expenses , and changes in family structure.1 further , individuals lose their strength and ability to work at their most productive , given that most traumas due to car accidents occur among individuals who are under the age of 45 years . results of epidemiological studies show that the etiology of facial fractures is variable across the world . however , motorvehicle accidents are among the most frequent causes of facial fractures.12,13 in brazil , rates of hospitalization due to motorvehicle accidents are high . consequently , authorities made changes in the brazilian traffic code,2 such as the so paulo municipal law of 1994.3 ultimately adopted by most brazilian cities , this law regulated the use of seat belts in november 1994 . more recently , the dry law ( number 11,705)4 also gained prominence in epidemiological indices and effectively reduced the number of accident victims attended in hospitals due to car accidents.5 the purpose of this paper is to highlight the importance of these laws the brazilian our health system and demonstrate their effectiveness . , this number is lower than only four other countries : india ( 1,057,000 deaths ) , china ( 966,000 deaths ) , the united states ( 426,000 deaths ) , and russia ( 359,000 deaths ) ( see figure 1 ) . however , brazil occupies an intermediate position in a ranking among countries , with 18 deaths for every 100,000 inhabitants . therefore , the rate of traffic deaths is higher than that of the united states ( 13/100 ) and lower than russia ( 25/100).6,7 in 2002 , motorvehicle accidents claimed the lives of 1.18 million people and injured 20 to 50 million more worldwide . it is estimated that by 2020 , if this frequency is maintained , the annual proportion of people killed and disabled as a result of motorvehicle accidents will rise to more than 60% . consequently , motor vehicle accidents will be third on the world health organization 's ( who ) list of the main causes of disease and traumas.8 motorvehicle accidents have become a significant cause of morbidity and mortality among the brazilian population as a result of the increase in the number of vehicles circulating and the high frequency of inappropriate behavior , coupled with ineffective monitoring and poor public road conditions.9 in addition , the number of vehicles on the road continues to grow . so paulo 's fleet of vehicles , which makes up more than 90% of the national fleet,10 was 6,558,463 vehicles in the capital and 19,499,430 vehicles in the state in june 2009.11 according to who data , traumas are among the main causes of death and morbidity in the world . every day , 16,000 people die as a result of some trauma.14 among the various types of trauma , facial trauma is notable due to its emotional and functional consequences and the possibility of permanent deformities.15 the increased exposure and little protection increases the likelihood of serious injuries to this part of the body in car accidents . head and face injuries represent about 50% of all traumatic deaths.16 recognizing which etiologies are most prevalent and which population is most affected by face traumas is a fundamental part of structuring services . with the population growth , increasing traffic of motor vehicles , and socioeconomic discrepancies , the number of traumatic facial fractures has increased,17 although fractures from sports activities are less frequent.18 in general , facial traumas are associated with other factors , such as assaults or falls.13,18 results of a study of 450 individuals with facial fracture in the city of so paulo in the 1980s showed that the main cause of facial fracture was motorvehicle accidents.19 a study carried out more recently in a hospital in the south zone of so paulo20 found that the most frequent etiology of facial trauma was motorvehicle accidents ( 25% of cases ) and motorcycle accidents ( 25% ) , whereas less frequent etiologies were physical assaults ( 15% ) and work accidents ( 1% ) . results of a retrospective study of 323 individuals with facial fractures due to motorvehicle accidents at the so paulo clinics hospital between february 2001 and july 2006 showed that the average age of the patients was 30.9 years and the proportion of men to women was 3.3 to 1 ( see table 1).21 the findings are consistent with the results of a minas gerais study , which found that 23% of facial injuries were due to accidents involving bicycles and motorcycles , whereas 21% were due to interpersonal violence . in addition , trauma to the face from motorvehicle accidents was found to be more common among men ( 80% ) than women ( 20%).13 in recife , motorvehicle accidents account for 31.8% , followed by physical assault ( 22.2% ) and assaults with firearms ( 18.7%).22 on the other hand , in porto alegre , approximately 36% of facial fractures occur as a result of of urban violence.23 traffic accidents ( 19% ) were reported to be the second most common cause , together with sports accidents ( 19% ) . in so paulo , a municipal law was passed that mandated the use of seat belts on november 4 , 1994.3 reactions for and against the law were immediate because of the difficulties and adjustments required for its implementation . however , all of this loses importance when it comes to lives and traumas.1 according to the traffic engineering company ( tec),24 after two years of the municipal act 's requiring the use of seat belts , the measure was found to be beneficial to the physical safety of both drivers and front seat passengers of vehicles , as well as the entire population . the tec conducted a survey of victims from the vehicles who received treatment at the emergency room at santa casa de misericordia in so paulo ( see table 2 ) . as shown in table 2 , there was a reduction in head injuries following the application of the law . soriano et al.25 also reported a decrease of 4.9% in the frequency of ocular perforations caused by motorvehicle accidents after the implementation of the law . in 1997 , the new national traffic code ( ntc ) was approved.4 beginning in april 1998 , individuals were punished for failing to use their seat belt . individuals caught driving without wearing a seat belt or allowing a passenger to ride in their vehicle without wearing one received a fine of r$127.69 and five points on their driver 's license , constituting a serious violation . in brazil , the mortality rate from motorvehicle accidents has declined from 29.8% in 1996 to 25.3% in 1999.26 this decline can be attributed to several factors , including lower speed limits , more surveillance , greater use of seat belts , the implementation of electronic speed bumps , and an increase in the car fleet.26 between 1996 and 1998 , there was a 30% reduction in the number of traffic deaths in so paulo because of the introduction of the new brazilian traffic code and the installation of electronic radar cameras for speed surveillance in the city.27 according to results of a survey conducted in the region of pelotas ( rs ) , 26% of oral and maxillofacial fractures occur as a result of motorvehicle accidents.28 a similar rate of 29% was found in a study of jaw fractures in a hospital in pernambuco.29 significant facial impact occurs during a motorvehicle accident . even with the use of a safety belt and an airbag , simple or multiple fractures can occur that crush the face . researchers affirm that the major problem is not treating patients with multiple traumas , but their reentry into society . many of these individuals lose their teeth and can not afford dental implants , a treatment that is currently done only in the private sector.30 the seat belt is considered to be the most effective device in reducing the severity of trauma in motorvehicle accidents and the most available safety equipment for vehicles currently operating in brazil.31 using a seat belt reduces the risk of death in individuals sitting in the front seat of a vehicle by 4065% and may reduce deaths in rear seat passengers by 2575%.6 however , results of other studies show that only 57% of countries require the use of seat belts in vehicles for all passengers , both seated in the front and back of the vehicle , with only 38% of lowincome countries having such laws12 ( see table 3 ) . research evaluating young drivers at the catholic university of so paulo showed that only 74% of university students always use a seat belt ; therefore , one quarter of the population studied does not use frequently use one . falls and traffic accidents have been reported , respectively , as the first and second leading cause of hospitalizations from external causes in brazil.32 in both brazil and so paulo , external causes account for increased length of hospital stay , more than natural causes , despite the lower proportion of hospital admissions and shorter average hospital stay of the latter.33 in so paulo in 2005 , the proportion of hospital admissions due to external causes was 48% for falls and 17% for motorvehicle accidents , although the proportion of expenditures was 41% for falls and 22% for motorvehicle accidents.32 a study of hospitalizations for external causes paid for by the unified health system ( sistema nico de sade , sus ) at the municipal hospital in so jos dos campos conducted between january 1 and june 30 , 2003 found that motorvehicle accidents were not only the leading cause of hospitalization ( 32% ) , but also the most expensive to treat ( 41.2%).34 ( see table 4 ) . in brazil , approximately twothirds of hospital beds in the orthopedics and traumatology sectors are occupied by victims of motorvehicle accidents they have an average hospital stay of 20 days , generating an average cost of $ 20,000 per serious injury . the estimated annual cost exceeds $ 3 billion.35 throughout the world , between onequarter and onehalf of motorvehicle accidents with fatalities are related to alcohol use by some of those responsible for the event.36 in a study conducted between december 2005 and december 2006 , on friday and saturday nights between 10 p.m. and 3 a.m. , it was found that 334 ( 36.6% ) respondents stated that they had been involved in traffic accidents as drivers . among these individuals , 200 ( 60% ) had a pattern of alcohol consumption two days per week , and 185 ( 55.4% ) were between the ages of 18 and 30 years.37 the positive results of municipal law 11,705/08.4 reverberated throughout the brazilian press.4 this law , which became known as the dry law,38 established zero limit for alcohol in drivers ' blood . as a result of this new law , some cities saw a reduction of up to 40% in the number of motorvehicle deaths in the first 30 days of enforcement . the work of the emergency care system , known as the urgent mobile care service ( servio de atendimento mvel de urgncia , samu ) , was reduced by an average of 25% . the city of sao paulo , for example , saved more than $ 4 million reais in hospital expenses.39 brazil has the fifth highest number of traffic deaths in the world . in 2007 , 35,100 thousand resulted from motorvehicle accidents in brazil . in absolute terms , this number is lower than only four other countries : india ( 1,057,000 deaths ) , china ( 966,000 deaths ) , the united states ( 426,000 deaths ) , and russia ( 359,000 deaths ) ( see figure 1 ) . however , brazil occupies an intermediate position in a ranking among countries , with 18 deaths for every 100,000 inhabitants . therefore , the rate of traffic deaths is higher than that of the united states ( 13/100 ) and lower than russia ( 25/100).6,7 in 2002 , motorvehicle accidents claimed the lives of 1.18 million people and injured 20 to 50 million more worldwide . it is estimated that by 2020 , if this frequency is maintained , the annual proportion of people killed and disabled as a result of motorvehicle accidents will rise to more than 60% . consequently , motor vehicle accidents will be third on the world health organization 's ( who ) list of the main causes of disease and traumas.8 motorvehicle accidents have become a significant cause of morbidity and mortality among the brazilian population as a result of the increase in the number of vehicles circulating and the high frequency of inappropriate behavior , coupled with ineffective monitoring and poor public road conditions.9 in addition , the number of vehicles on the road continues to grow . so paulo 's fleet of vehicles , which makes up more than 90% of the national fleet,10 was 6,558,463 vehicles in the capital and 19,499,430 vehicles in the state in june 2009.11 according to who data , traumas are among the main causes of death and morbidity in the world . every day , 16,000 people die as a result of some trauma.14 among the various types of trauma , facial trauma is notable due to its emotional and functional consequences and the possibility of permanent deformities.15 the increased exposure and little protection increases the likelihood of serious injuries to this part of the body in car accidents . head and face injuries represent about 50% of all traumatic deaths.16 recognizing which etiologies are most prevalent and which population is most affected by face traumas is a fundamental part of structuring services . with the population growth , increasing traffic of motor vehicles , and socioeconomic discrepancies , the number of traumatic facial fractures has increased,17 although fractures from sports activities are less frequent.18 in general , facial traumas are associated with other factors , such as assaults or falls.13,18 results of a study of 450 individuals with facial fracture in the city of so paulo in the 1980s showed that the main cause of facial fracture was motorvehicle accidents.19 a study carried out more recently in a hospital in the south zone of so paulo20 found that the most frequent etiology of facial trauma was motorvehicle accidents ( 25% of cases ) and motorcycle accidents ( 25% ) , whereas less frequent etiologies were physical assaults ( 15% ) and work accidents ( 1% ) . results of a retrospective study of 323 individuals with facial fractures due to motorvehicle accidents at the so paulo clinics hospital between february 2001 and july 2006 showed that the average age of the patients was 30.9 years and the proportion of men to women was 3.3 to 1 ( see table 1).21 the findings are consistent with the results of a minas gerais study , which found that 23% of facial injuries were due to accidents involving bicycles and motorcycles , whereas 21% were due to interpersonal violence . in addition , trauma to the face from motorvehicle accidents was found to be more common among men ( 80% ) than women ( 20%).13 in recife , motorvehicle accidents account for 31.8% , followed by physical assault ( 22.2% ) and assaults with firearms ( 18.7%).22 on the other hand , in porto alegre , approximately 36% of facial fractures occur as a result of of urban violence.23 traffic accidents ( 19% ) were reported to be the second most common cause , together with sports accidents ( 19% ) . in so paulo , a municipal law was passed that mandated the use of seat belts on november 4 , 1994.3 reactions for and against the law were immediate because of the difficulties and adjustments required for its implementation . however , all of this loses importance when it comes to lives and traumas.1 according to the traffic engineering company ( tec),24 after two years of the municipal act 's requiring the use of seat belts , the measure was found to be beneficial to the physical safety of both drivers and front seat passengers of vehicles , as well as the entire population . the tec conducted a survey of victims from the vehicles who received treatment at the emergency room at santa casa de misericordia in so paulo ( see table 2 ) . as shown in table 2 , there was a reduction in head injuries following the application of the law . soriano et al.25 also reported a decrease of 4.9% in the frequency of ocular perforations caused by motorvehicle accidents after the implementation of the law . in 1997 , the new national traffic code ( ntc ) was approved.4 beginning in april 1998 , individuals were punished for failing to use their seat belt . individuals caught driving without wearing a seat belt or allowing a passenger to ride in their vehicle without wearing one received a fine of r$127.69 and five points on their driver 's license , constituting a serious violation . in brazil , the mortality rate from motorvehicle accidents has declined from 29.8% in 1996 to 25.3% in 1999.26 this decline can be attributed to several factors , including lower speed limits , more surveillance , greater use of seat belts , the implementation of electronic speed bumps , and an increase in the car fleet.26 between 1996 and 1998 , there was a 30% reduction in the number of traffic deaths in so paulo because of the introduction of the new brazilian traffic code and the installation of electronic radar cameras for speed surveillance in the city.27 according to results of a survey conducted in the region of pelotas ( rs ) , 26% of oral and maxillofacial fractures occur as a result of motorvehicle accidents.28 a similar rate of 29% was found in a study of jaw fractures in a hospital in pernambuco.29 significant facial impact occurs during a motorvehicle accident . even with the use of a safety belt and an airbag , simple or multiple fractures can occur that crush the face . researchers affirm that the major problem is not treating patients with multiple traumas , but their reentry into society . many of these individuals lose their teeth and can not afford dental implants , a treatment that is currently done only in the private sector.30 the seat belt is considered to be the most effective device in reducing the severity of trauma in motorvehicle accidents and the most available safety equipment for vehicles currently operating in brazil.31 using a seat belt reduces the risk of death in individuals sitting in the front seat of a vehicle by 4065% and may reduce deaths in rear seat passengers by 2575%.6 however , results of other studies show that only 57% of countries require the use of seat belts in vehicles for all passengers , both seated in the front and back of the vehicle , with only 38% of lowincome countries having such laws12 ( see table 3 ) . research evaluating young drivers at the catholic university of so paulo showed that only 74% of university students always use a seat belt ; therefore , one quarter of the population studied does not use frequently use one . falls and traffic accidents have been reported , respectively , as the first and second leading cause of hospitalizations from external causes in brazil.32 in both brazil and so paulo , external causes account for increased length of hospital stay , more than natural causes , despite the lower proportion of hospital admissions and shorter average hospital stay of the latter.33 in so paulo in 2005 , the proportion of hospital admissions due to external causes was 48% for falls and 17% for motorvehicle accidents , although the proportion of expenditures was 41% for falls and 22% for motorvehicle accidents.32 a study of hospitalizations for external causes paid for by the unified health system ( sistema nico de sade , sus ) at the municipal hospital in so jos dos campos conducted between january 1 and june 30 , 2003 found that motorvehicle accidents were not only the leading cause of hospitalization ( 32% ) , but also the most expensive to treat ( 41.2%).34 ( see table 4 ) . in brazil , approximately twothirds of hospital beds in the orthopedics and traumatology sectors are occupied by victims of motorvehicle accidents . they have an average hospital stay of 20 days , generating an average cost of $ 20,000 per serious injury . throughout the world , between onequarter and onehalf of motorvehicle accidents with fatalities are related to alcohol use by some of those responsible for the event.36 in a study conducted between december 2005 and december 2006 , on friday and saturday nights between 10 p.m. and 3 a.m. , it was found that 334 ( 36.6% ) respondents stated that they had been involved in traffic accidents as drivers . among these individuals , 200 ( 60% ) had a pattern of alcohol consumption two days per week , and 185 ( 55.4% ) were between the ages of 18 and 30 years.37 the positive results of municipal law 11,705/08.4 reverberated throughout the brazilian press.4 this law , which became known as the dry law,38 established zero limit for alcohol in drivers ' blood . as a result of this new law , some cities saw a reduction of up to 40% in the number of motorvehicle deaths in the first 30 days of enforcement . the work of the emergency care system , known as the urgent mobile care service ( servio de atendimento mvel de urgncia , samu ) , was reduced by an average of 25% . the city of sao paulo , for example , saved more than $ 4 million reais in hospital expenses.39 facial traumas are of great significance in modern society because they have emotional and functional influences on people 's lives and can cause permanent deformities . further , they frequently involve serious injuries.15,16,40 various researchers have described the etiologies of facial trauma . however , these causes change according to location and year , as evidence by the marked change in the epidemiological profile of the population.18 - 20,22,23 nevertheless , facial traumas continue to be associated with motorvehicle accidents , given that the number of vehicles has increased with the population . in addition , the negligence of the population regarding the dangers of traffic does not seem to have diminished . brazil faces millions of deaths every year from motorvehicle crashes . in the comparision with other countries , brazil is one of the worldwide leaders in terms of the number of motorvehicle accidents.6,9,10,12 in order to curb this type of trauma , authorities have taken extreme measures in relation to traffic safety laws . this year , the seat belt law has been enforced for 15 years in so paulo . after the law mandating the use of seat belts in so paulo was approved in 1994 , authorities encountered a significant decrease in facial traumas . several studies were conducted , and all of the authors agreed that facial trauma rates decreased after the implementation of this law . in 1996 , just two years after the implementation of the new law , tec confirmed that there was a decrease from 44.2% to 30.6% in head injuries in so paulo . the same year , soriano et al.25 reported a decrease of 4.9% in ocular perforations . mello jorge et al.26 reported that traffic accidents decreased from 29.8% in 1996 to 25.3% in 1999 . according to results of a study published in 1996 by the tec , there was a 30% reduction in the number of traffic deaths in so paulo with the use of the seat belt.24 between 2007 and 2008 , after the seat belt law first came into effect , in a public hospital in the northern part of so paulo , accidents accounted for only 3% of the causes of facial trauma , which suggested that the epidemiological profile of the population was changing.41 the most frequent cause of facial trauma in this study was physical assault.41 in 2007 , the who6 reported that the use of seat belts reduced deaths to 40% from 65% in patients sitting in the front seat and to 25% from 75% in the back seat . despite all of the reported benefits , pine et al.42 revealed in 2009 that onequarter individuals in a university population were not using their seat belt . this is an alarming figure , considering that the age group most affected by deaths and facial injuries related to the use of seat belts is 20 to 40yearolds.21,28 this has a significant impact on families and face traumas put these patients away from social interaction . in addition , the treatments represent heavy expenditures for the public health system because hospitalizations for external causes , such as motorvehicle accidents , have the largest average expense and daily costs . they also require greater spending on health care , accounting for up to 41% of the causes of public spending . after the implementation of the law , we noted that the expenses decreased to 22%.7,32 - 34 the seat belt law provided numerous benefits to so paulo and other cities in brazil . as a result , similar laws were created to reduce the accident rate , such as the dry law , which restricts the consumption of alcoholic beverages and has a greater impact on the population of individuals between the ages of 18 and 40 years , who are most at risk for this type of accident . after the implementation of the dry law , the number of deaths in so paulo was reduced by 40% . in addition , it accounted for only 25% of the mobile service 's work and saved 4 million reais on hospital costs.37,39 there is no doubt that all of these measures have changed the epidemiological indices of brazil 's largest city and had impacts in the population 's life . in addition , epidemiological data allows a better planning of the public health services , reducing its costs . the system now provides less assistance to accident victims due to the reduction in the number of accidents and of facial traumas . there is still a need for greater awareness among the population , especially young people , in relation to the laws adopted by the authorities . however , it is clear that it has brought only benefits and moved brazil toward a better future .

traffic accidents are a reality throughout brazil . the face is one of the anatomic parts most affected by these accidents , especially when a seat belt is not used . these accidents are costly for the public health system and have a significant impact on society and the lives of families involved . the compulsory use of seat belts in brazil , especially in so paulo , has decreased the rate of facial trauma . this suggests that the public health policies and measures adopted by the brazilian authorities have benefited the population 15 years after the enactment of the law of compulsory seat belts in the city of so paulo .

INTRODUCTION Traffic accidents Traumas Seat belts Costs for the health care system Dry Law DISCUSSION CONCLUSIONS

the face is a frequent target for trauma in motorvehicle accidents , especially when seat belts are not used . however , motorvehicle accidents are among the most frequent causes of facial fractures.12,13 in brazil , rates of hospitalization due to motorvehicle accidents are high . consequently , authorities made changes in the brazilian traffic code,2 such as the so paulo municipal law of 1994.3 ultimately adopted by most brazilian cities , this law regulated the use of seat belts in november 1994 . more recently , the dry law ( number 11,705)4 also gained prominence in epidemiological indices and effectively reduced the number of accident victims attended in hospitals due to car accidents.5 the purpose of this paper is to highlight the importance of these laws the brazilian our health system and demonstrate their effectiveness . therefore , the rate of traffic deaths is higher than that of the united states ( 13/100 ) and lower than russia ( 25/100).6,7 in 2002 , motorvehicle accidents claimed the lives of 1.18 million people and injured 20 to 50 million more worldwide . consequently , motor vehicle accidents will be third on the world health organization 's ( who ) list of the main causes of disease and traumas.8 motorvehicle accidents have become a significant cause of morbidity and mortality among the brazilian population as a result of the increase in the number of vehicles circulating and the high frequency of inappropriate behavior , coupled with ineffective monitoring and poor public road conditions.9 in addition , the number of vehicles on the road continues to grow . so paulo 's fleet of vehicles , which makes up more than 90% of the national fleet,10 was 6,558,463 vehicles in the capital and 19,499,430 vehicles in the state in june 2009.11 according to who data , traumas are among the main causes of death and morbidity in the world . every day , 16,000 people die as a result of some trauma.14 among the various types of trauma , facial trauma is notable due to its emotional and functional consequences and the possibility of permanent deformities.15 the increased exposure and little protection increases the likelihood of serious injuries to this part of the body in car accidents . with the population growth , increasing traffic of motor vehicles , and socioeconomic discrepancies , the number of traumatic facial fractures has increased,17 although fractures from sports activities are less frequent.18 in general , facial traumas are associated with other factors , such as assaults or falls.13,18 results of a study of 450 individuals with facial fracture in the city of so paulo in the 1980s showed that the main cause of facial fracture was motorvehicle accidents.19 a study carried out more recently in a hospital in the south zone of so paulo20 found that the most frequent etiology of facial trauma was motorvehicle accidents ( 25% of cases ) and motorcycle accidents ( 25% ) , whereas less frequent etiologies were physical assaults ( 15% ) and work accidents ( 1% ) . results of a retrospective study of 323 individuals with facial fractures due to motorvehicle accidents at the so paulo clinics hospital between february 2001 and july 2006 showed that the average age of the patients was 30.9 years and the proportion of men to women was 3.3 to 1 ( see table 1).21 the findings are consistent with the results of a minas gerais study , which found that 23% of facial injuries were due to accidents involving bicycles and motorcycles , whereas 21% were due to interpersonal violence . in addition , trauma to the face from motorvehicle accidents was found to be more common among men ( 80% ) than women ( 20%).13 in recife , motorvehicle accidents account for 31.8% , followed by physical assault ( 22.2% ) and assaults with firearms ( 18.7%).22 on the other hand , in porto alegre , approximately 36% of facial fractures occur as a result of of urban violence.23 traffic accidents ( 19% ) were reported to be the second most common cause , together with sports accidents ( 19% ) . in so paulo , a municipal law was passed that mandated the use of seat belts on november 4 , 1994.3 reactions for and against the law were immediate because of the difficulties and adjustments required for its implementation . however , all of this loses importance when it comes to lives and traumas.1 according to the traffic engineering company ( tec),24 after two years of the municipal act 's requiring the use of seat belts , the measure was found to be beneficial to the physical safety of both drivers and front seat passengers of vehicles , as well as the entire population . soriano et al.25 also reported a decrease of 4.9% in the frequency of ocular perforations caused by motorvehicle accidents after the implementation of the law . in brazil , the mortality rate from motorvehicle accidents has declined from 29.8% in 1996 to 25.3% in 1999.26 this decline can be attributed to several factors , including lower speed limits , more surveillance , greater use of seat belts , the implementation of electronic speed bumps , and an increase in the car fleet.26 between 1996 and 1998 , there was a 30% reduction in the number of traffic deaths in so paulo because of the introduction of the new brazilian traffic code and the installation of electronic radar cameras for speed surveillance in the city.27 according to results of a survey conducted in the region of pelotas ( rs ) , 26% of oral and maxillofacial fractures occur as a result of motorvehicle accidents.28 a similar rate of 29% was found in a study of jaw fractures in a hospital in pernambuco.29 significant facial impact occurs during a motorvehicle accident . even with the use of a safety belt and an airbag , simple or multiple fractures can occur that crush the face . many of these individuals lose their teeth and can not afford dental implants , a treatment that is currently done only in the private sector.30 the seat belt is considered to be the most effective device in reducing the severity of trauma in motorvehicle accidents and the most available safety equipment for vehicles currently operating in brazil.31 using a seat belt reduces the risk of death in individuals sitting in the front seat of a vehicle by 4065% and may reduce deaths in rear seat passengers by 2575%.6 however , results of other studies show that only 57% of countries require the use of seat belts in vehicles for all passengers , both seated in the front and back of the vehicle , with only 38% of lowincome countries having such laws12 ( see table 3 ) . research evaluating young drivers at the catholic university of so paulo showed that only 74% of university students always use a seat belt ; therefore , one quarter of the population studied does not use frequently use one . falls and traffic accidents have been reported , respectively , as the first and second leading cause of hospitalizations from external causes in brazil.32 in both brazil and so paulo , external causes account for increased length of hospital stay , more than natural causes , despite the lower proportion of hospital admissions and shorter average hospital stay of the latter.33 in so paulo in 2005 , the proportion of hospital admissions due to external causes was 48% for falls and 17% for motorvehicle accidents , although the proportion of expenditures was 41% for falls and 22% for motorvehicle accidents.32 a study of hospitalizations for external causes paid for by the unified health system ( sistema nico de sade , sus ) at the municipal hospital in so jos dos campos conducted between january 1 and june 30 , 2003 found that motorvehicle accidents were not only the leading cause of hospitalization ( 32% ) , but also the most expensive to treat ( 41.2%).34 ( see table 4 ) . in brazil , approximately twothirds of hospital beds in the orthopedics and traumatology sectors are occupied by victims of motorvehicle accidents they have an average hospital stay of 20 days , generating an average cost of $ 20,000 per serious injury . the city of sao paulo , for example , saved more than $ 4 million reais in hospital expenses.39 brazil has the fifth highest number of traffic deaths in the world . therefore , the rate of traffic deaths is higher than that of the united states ( 13/100 ) and lower than russia ( 25/100).6,7 in 2002 , motorvehicle accidents claimed the lives of 1.18 million people and injured 20 to 50 million more worldwide . consequently , motor vehicle accidents will be third on the world health organization 's ( who ) list of the main causes of disease and traumas.8 motorvehicle accidents have become a significant cause of morbidity and mortality among the brazilian population as a result of the increase in the number of vehicles circulating and the high frequency of inappropriate behavior , coupled with ineffective monitoring and poor public road conditions.9 in addition , the number of vehicles on the road continues to grow . so paulo 's fleet of vehicles , which makes up more than 90% of the national fleet,10 was 6,558,463 vehicles in the capital and 19,499,430 vehicles in the state in june 2009.11 according to who data , traumas are among the main causes of death and morbidity in the world . every day , 16,000 people die as a result of some trauma.14 among the various types of trauma , facial trauma is notable due to its emotional and functional consequences and the possibility of permanent deformities.15 the increased exposure and little protection increases the likelihood of serious injuries to this part of the body in car accidents . with the population growth , increasing traffic of motor vehicles , and socioeconomic discrepancies , the number of traumatic facial fractures has increased,17 although fractures from sports activities are less frequent.18 in general , facial traumas are associated with other factors , such as assaults or falls.13,18 results of a study of 450 individuals with facial fracture in the city of so paulo in the 1980s showed that the main cause of facial fracture was motorvehicle accidents.19 a study carried out more recently in a hospital in the south zone of so paulo20 found that the most frequent etiology of facial trauma was motorvehicle accidents ( 25% of cases ) and motorcycle accidents ( 25% ) , whereas less frequent etiologies were physical assaults ( 15% ) and work accidents ( 1% ) . results of a retrospective study of 323 individuals with facial fractures due to motorvehicle accidents at the so paulo clinics hospital between february 2001 and july 2006 showed that the average age of the patients was 30.9 years and the proportion of men to women was 3.3 to 1 ( see table 1).21 the findings are consistent with the results of a minas gerais study , which found that 23% of facial injuries were due to accidents involving bicycles and motorcycles , whereas 21% were due to interpersonal violence . in addition , trauma to the face from motorvehicle accidents was found to be more common among men ( 80% ) than women ( 20%).13 in recife , motorvehicle accidents account for 31.8% , followed by physical assault ( 22.2% ) and assaults with firearms ( 18.7%).22 on the other hand , in porto alegre , approximately 36% of facial fractures occur as a result of of urban violence.23 traffic accidents ( 19% ) were reported to be the second most common cause , together with sports accidents ( 19% ) . in so paulo , a municipal law was passed that mandated the use of seat belts on november 4 , 1994.3 reactions for and against the law were immediate because of the difficulties and adjustments required for its implementation . however , all of this loses importance when it comes to lives and traumas.1 according to the traffic engineering company ( tec),24 after two years of the municipal act 's requiring the use of seat belts , the measure was found to be beneficial to the physical safety of both drivers and front seat passengers of vehicles , as well as the entire population . the tec conducted a survey of victims from the vehicles who received treatment at the emergency room at santa casa de misericordia in so paulo ( see table 2 ) . soriano et al.25 also reported a decrease of 4.9% in the frequency of ocular perforations caused by motorvehicle accidents after the implementation of the law . in brazil , the mortality rate from motorvehicle accidents has declined from 29.8% in 1996 to 25.3% in 1999.26 this decline can be attributed to several factors , including lower speed limits , more surveillance , greater use of seat belts , the implementation of electronic speed bumps , and an increase in the car fleet.26 between 1996 and 1998 , there was a 30% reduction in the number of traffic deaths in so paulo because of the introduction of the new brazilian traffic code and the installation of electronic radar cameras for speed surveillance in the city.27 according to results of a survey conducted in the region of pelotas ( rs ) , 26% of oral and maxillofacial fractures occur as a result of motorvehicle accidents.28 a similar rate of 29% was found in a study of jaw fractures in a hospital in pernambuco.29 significant facial impact occurs during a motorvehicle accident . many of these individuals lose their teeth and can not afford dental implants , a treatment that is currently done only in the private sector.30 the seat belt is considered to be the most effective device in reducing the severity of trauma in motorvehicle accidents and the most available safety equipment for vehicles currently operating in brazil.31 using a seat belt reduces the risk of death in individuals sitting in the front seat of a vehicle by 4065% and may reduce deaths in rear seat passengers by 2575%.6 however , results of other studies show that only 57% of countries require the use of seat belts in vehicles for all passengers , both seated in the front and back of the vehicle , with only 38% of lowincome countries having such laws12 ( see table 3 ) . research evaluating young drivers at the catholic university of so paulo showed that only 74% of university students always use a seat belt ; therefore , one quarter of the population studied does not use frequently use one . falls and traffic accidents have been reported , respectively , as the first and second leading cause of hospitalizations from external causes in brazil.32 in both brazil and so paulo , external causes account for increased length of hospital stay , more than natural causes , despite the lower proportion of hospital admissions and shorter average hospital stay of the latter.33 in so paulo in 2005 , the proportion of hospital admissions due to external causes was 48% for falls and 17% for motorvehicle accidents , although the proportion of expenditures was 41% for falls and 22% for motorvehicle accidents.32 a study of hospitalizations for external causes paid for by the unified health system ( sistema nico de sade , sus ) at the municipal hospital in so jos dos campos conducted between january 1 and june 30 , 2003 found that motorvehicle accidents were not only the leading cause of hospitalization ( 32% ) , but also the most expensive to treat ( 41.2%).34 ( see table 4 ) . in brazil , approximately twothirds of hospital beds in the orthopedics and traumatology sectors are occupied by victims of motorvehicle accidents . throughout the world , between onequarter and onehalf of motorvehicle accidents with fatalities are related to alcohol use by some of those responsible for the event.36 in a study conducted between december 2005 and december 2006 , on friday and saturday nights between 10 p.m. and 3 a.m. , it was found that 334 ( 36.6% ) respondents stated that they had been involved in traffic accidents as drivers . the city of sao paulo , for example , saved more than $ 4 million reais in hospital expenses.39 facial traumas are of great significance in modern society because they have emotional and functional influences on people 's lives and can cause permanent deformities . however , these causes change according to location and year , as evidence by the marked change in the epidemiological profile of the population.18 - 20,22,23 nevertheless , facial traumas continue to be associated with motorvehicle accidents , given that the number of vehicles has increased with the population . in the comparision with other countries , brazil is one of the worldwide leaders in terms of the number of motorvehicle accidents.6,9,10,12 in order to curb this type of trauma , authorities have taken extreme measures in relation to traffic safety laws . this year , the seat belt law has been enforced for 15 years in so paulo . after the law mandating the use of seat belts in so paulo was approved in 1994 , authorities encountered a significant decrease in facial traumas . several studies were conducted , and all of the authors agreed that facial trauma rates decreased after the implementation of this law . in 1996 , just two years after the implementation of the new law , tec confirmed that there was a decrease from 44.2% to 30.6% in head injuries in so paulo . according to results of a study published in 1996 by the tec , there was a 30% reduction in the number of traffic deaths in so paulo with the use of the seat belt.24 between 2007 and 2008 , after the seat belt law first came into effect , in a public hospital in the northern part of so paulo , accidents accounted for only 3% of the causes of facial trauma , which suggested that the epidemiological profile of the population was changing.41 the most frequent cause of facial trauma in this study was physical assault.41 in 2007 , the who6 reported that the use of seat belts reduced deaths to 40% from 65% in patients sitting in the front seat and to 25% from 75% in the back seat . despite all of the reported benefits , pine et al.42 revealed in 2009 that onequarter individuals in a university population were not using their seat belt . this is an alarming figure , considering that the age group most affected by deaths and facial injuries related to the use of seat belts is 20 to 40yearolds.21,28 this has a significant impact on families and face traumas put these patients away from social interaction . in addition , the treatments represent heavy expenditures for the public health system because hospitalizations for external causes , such as motorvehicle accidents , have the largest average expense and daily costs . after the implementation of the law , we noted that the expenses decreased to 22%.7,32 - 34 the seat belt law provided numerous benefits to so paulo and other cities in brazil . after the implementation of the dry law , the number of deaths in so paulo was reduced by 40% . in addition , it accounted for only 25% of the mobile service 's work and saved 4 million reais on hospital costs.37,39 there is no doubt that all of these measures have changed the epidemiological indices of brazil 's largest city and had impacts in the population 's life . in addition , epidemiological data allows a better planning of the public health services , reducing its costs . there is still a need for greater awareness among the population , especially young people , in relation to the laws adopted by the authorities .

for the past decade , scientists , institutions , governments , and policymakers have warned the general public about serious health hazards associated with chemicals known as endocrine disruptors ( eds ) . eds are exogenous compounds that interfere with the synthesis , secretion , transport , metabolism , and/or action of endogenous hormones that are responsible for normal homeostasis , reproduction , and development . chemicals with hormonal activity can be divided into three main groups : ( i ) synthetic compounds used in industry and agriculture as well as in consumer products , ( ii ) synthetic compounds used in pharmaceutical drugs , and ( iii ) natural compounds present in the food chain ( i.e. , phytoestrogens , compounds that are structurally similar to estrogen ( e2 ) ) . only ( i ) synthetic compounds used in industry and agriculture and consumer products will be discussed in presented paper . within this class , compounds can be further subcategorized into those that are persistent in all elements of the environment , bio - accumulative , transportable over long distances , and capable of adversely affecting life forms that reside within short and long distances from the site of contamination . these compounds include dichlorodiphenyltrichloroethane ( ddt , a pesticide ) , polychlorinated biphenyls ( pcbs ) , polychlorinated naphthalenes ( pcns ) , polychlorinated dibenzodioxins ( pcdds ) , polychlorinated dibenzofurans ( pcdfs ) , and polybrominated diphenyl ethers ( pbdes ) ( figure 1 ) . because xenobiotics can accumulate in the body for an extremely long period of time ( i.e. , decades ) for instance , pesticides and other synthetic compounds used in the 1950s have polluted the air that we breathe , the water that we drink , and the soil that we grow our food in . in addition , many of these compounds are nonbiodegradable . studies have shown the presence of eds both in adipose tissue and other organs , in almost all humans and many animals . human exposure to environmental toxicants is mediated via food and water chains , breathing , and dermal absorption , with approximately 90% of the exposure coming from food . moreover , several mammalian organs ( e.g. , ovary , breast , uterus , cervix , bone , muscle , and skin ) rely on sex steroids for normal function , and these organs are especially vulnerable to endocrine disruption by environmental toxicants . much of the information on the impact of eds on human health has come from in vivo studies where animals were exposed to a single compound , usually at an acute ( i.e. , pharmacological ) dose ; however , humans are exposed to several compounds at unknown concentrations for unknown durations . for example , hospital patients receive an average of six drugs daily ( i.e. , aspirin , antihistamines , antibiotics , anti - cough syrup , etc . ) . food and water may also contain low levels of organic and inorganic compounds ( e.g. , pesticides and heavy metals ) and solvents ( e.g. , benzene , toluene , and chloroform ) , and air is filled with hundreds of chemicals ( e.g. , industrial pollutants , smoke , gasoline vapors , etc . ) . the ability of these environmental toxicants to affect human health depends on several factors , including interactions between compounds ; absorption , metabolism , accumulation , and excretion of compounds ; and the ability of some compounds bind to cell receptors which can affect hormone action . unfortunately , little is known about how these environmental toxicants interact with each other existing in a mixture . it is known that such xenobiotic acts in different ways depending on whether their action is observed on the fetus , newborn babies , or adult individuals , depending on the period during whose body has been exposed to these factors . in many cases , the effect of their actions may be undetected until sexual maturity , especially when exposure occurs during a period of embryonic development or shortly after birth . the female reproductive cycle is a complex process comprised of gametogenesis , embryogenesis , menstruation , ovulation , possible pregnancy , endometrial , and mammary gland changes . women are born with all the oocytes they will ever have through their life , and therefore oocytes are more vulnerable to toxic chemicals than germ cells in men who continue to make more germ cells . oocyte maturation , the final step of germ cell differentiation , determines reproductive capacity . follicles are especially susceptible to the adverse effects of environmental toxicants , and developing oocytes may be damaged directly or indirectly by action on follicular cells ( i.e. , granulosa cells ) that may also be vulnerable to endocrine disruption , thereby affecting oocyte function indirectly . a previous study illustrated that human oocytes harvested from follicles with elevated levels of polycyclic aromatic hydrocarbons ( pcahs ) had fewer cell divisions after in vitro fertilization . chloroorganic mixtures such as pcbs and ddt , as well as its metabolites , have also been reported to affect puberty , development , and oocyte viability adversely . some of these studies point to unpredictable changes of translational regulation within the oocyte under the influence of pcbs . polyspermia ( secretion of an excessive amount of semen ) has also been demonstrated in cattle exposed to an environmentally relevant mixture of more than 15 organochlorines . presently , additional research is needed to better understand the molecular mechanisms behind mammalian ovotoxicity caused by exposure to environmental toxicants . hormone - mimicking compounds can bind to cell receptors , interfere with hormone action , and affect ovarian function . how eds affect ovarian function is not yet clear , but a disruption in gonadotropin ( i.e. , follicle stimulating hormone ( fsh ) and luteinizing hormone ( lh ) ) secretion and feedback mechanisms involving e2 and progesterone ( p4 ) may be involved . damaged oocytes can affect overall hormone production and follicular function , resulting in an endocrinological imbalance ( i.e. , a decrease in e2 and p4 , but an increase in fsh and lh ) and ovarian failure . fertility in sexually mature women depends largely on the maintenance of healthy follicles , and their steady production ensures that an adequate number of follicles reach the antral stage . the stage of development at which the follicles are destroyed determines the influence of these factors on the fertility of women . complete depletion of the follicle reserve results in irreversible infertility ; partial depletion of the follicle reserve results in moderate effects on periodicity . damage to large follicles can also lead to reversible acyclic disorders that affect hormone production and ovulation . nevertheless , the effects of pops on the hypothalamic - pituitary - ovarian ( hpo ) axis are generally reversible because they do not permanently affect the follicle reserve . tcdd ( 2 , 3 , 7 , 8-teterachlorodibenzo - para - dioxin ) is an isomer of pcdd , and one of the most toxic man - made compounds to pollute our environment . studies on the mechanism of tcdd action during ovulation indicate a dysfunction in the hpo axis . misregulation of fsh and lh secretion before ovulation was noted in rats exposed to tcdd . moreover , gonadotropin - releasing hormone ( gnrh ) inhibited the surge in fsh and lh secretion , thereby restoring ovulation [ 5 , 6 ] . for instance , gore showed methoxychlor to affect gnrh expression in hypothalamic gt1 - 7 cells . in a study from our laboratory , tcdd inhibited e2 secretion by follicular cells and p4 secretion by luteal cells dose - dependently . these adverse effects on hormone production were mediated in part by enzymes involved in steroidogenic biosynthesis . in luteal cells , tcdd action was independent of e2 receptor stimulation , instead involving the aryl hydrocarbon receptor ( ahr ) . on a final note , other environmental toxicants ( i.e. , pcdfs , biphenyls , ddt , and methoxychlor ) can also block the surge in lh and fsh secretion during the female reproductive cycle [ 10 , 11 ] . polychlorinated biphenyls ( pcbs ) are the man - made chemicals that may disrupt follicular steroidogenesis either by mimicking natural hormones as agonist or antagonist , altering the pattern of hormone synthesis , modulating hormone receptor affinities or numbers , or by altering enzymes involved in hormone secretion . in our previous study , we showed that the orthosubstituted pcb 153 congener accumulated preferentially in the follicular wall when compared to the nonorthosubstituted pcb 126 congener . 71% , 71.4% , and 30.4% of the total exposure for pcb 153 were in small , medium , and large follicles , respectively ( figure 1 ) . interestingly , about 70% of pcb153 accumulated in early antral and antral follicles and only 30% in preovultory follicles . the consequence was a reduction in estradiol secretion by early antral and antral follicles and lack of influence on estradiol secretion by preovulatory follicles . moreover , moreover , it has been showed that action on estradiol secretion was correlated with action on aromatase activity [ 1214 ] . a similar dose - responsive relationship was reported after exposure of follicular cells to a mixture of organic pollutants or a mixture of pbdes . polybrominated dibenzoethers ( pbdes ) are persistent and ubiquitous environmental toxicants found at increasing levels in humans and animals . despite recent bans by the european union , united states , and china on the production of penta- and octa - bde as well as on the diminished use of pbde in japan , 2,2,4,4-tetra - bde ( bde-47 ) , 2,2,4,4,5-penta - bde ( bde-99 ) , and 2,2,4,4,6-penta - bde ( bde-100 ) are the major pbde congeners present in humans and animals . the literary data related to the effects of pbde mixture are limited predominantly to commercial mixture de-71 which contains mainly penta - bdes ( bde-99 and bde-100 ) and tetra - bde ( bde-47 ) . indicated that de-71 ( a mixture of bde-99 , bde-100 , and tetra - bde ) affected the production of thyroid and sex steroids and the development of reproductive organs [ 20 , 21 ] . results from our laboratory have shown an increase in the p4/testosterone ( t ) ratio but a decrease in the t / e2 ratio , in ovarian follicles suggesting premature luteinization of antral follicles . removal of the pbde mixture from cell cultures did not reverse adverse effects . in a follow - up study , we reported changes in the levels of steroidogenic enzymes ( e.g. , 17-hydroxysteroid dehydrogenase ( 17-hsd ) , cytochrome p450 , family 17 , subfamily a , polypeptide 1 ( cyp17 ) , and aromatase ( cyp19 ) ) by pbde congeners 47 , 99 , and 100 . last published data showed fast activation of cyp2b1/2 , and late activation of comt ( with a very low basal sult1a activity ) in ovarian follicles by bde-47 indicates a possible action of locally produced hydroxylated metabolites prior to their detoxification . additionally , it have been showed that 5-oh - bde-47 and 6-oh - bde-47 have a different mechanism of action in ovarian follicles from their parent compound and lead to an increase in estradiol secretion . the metabolites stimulate aromatase expression and activity , while the parent compound increases androgen production and stimulation of 17-hsd protein expression and activity . polychlorinated naphthalenes ( pcns ) are members of a large and diverse group of compounds with several industrial applications . pcns occur as mixtures of congeners sold under various trade names ( e.g. , halowax , nibren wax , and seekay wax ) . the toxicological characteristics of pcns are similar to those caused by pcbs , pcdds , and pcdfs [ 27 , 28 ] . presently , there are little data on the toxicity of pcns in experimental animals , and our recently published data was the first showed direct action on ovarian function . we showed an increase in basal testosterone secretion in all doses used , with the highest stimulatory action of the smallest dose , which was accompanied by a parallel decrease in basal estradiol secretion , induced by halowax 1051 suggesting androgenic properties of halowax 1051 . as a mechanism we propose direct stimulatory action on 17-hsd activity and protein expression , enzyme responsible for testosterone synthesis and inhibitory action on cyp19 activity , and enzyme responsible for conversion testosterone to estradiol . it should be taken into consideration that as in the case of bde-47 , the effects of exposure to pcns may be due to the side effects of pcn metabolites . examining the effects of the halowax 1051 on phase i ( cyp1a1 ) and phase ii ( sult1a and comt ) enzyme activities and expression in cultured ovarian follicles we showed fast activation of enzymes involved in phase i and concurrent inhibition of enzymes involved in phase ii metabolism confirming our suggestion that the observed effects of halowax 1051 are partially result from the action of metabolites formed locally in ovarian follicles . while hcbz is toxic to humans [ 31 , 32 ] and animals , information on the effects of hcbz in ovarian steroidogenesis is limited despite data that showed an increase in the p4 serum level in superovulated rats exposed to hcbz . treatment of cynomolgus monkeys with hcbz , on the other hand , decreased the p4 serum level and unaltered the e2 serum level during the luteal phase . in a recently published study , we determined in vitro accumulation of hexachlorobenzene ( hcbz ) and pentachlorobenzene ( pecbz ) in porcine ovarian follicles , the effect on steroidogenesis , and the expression of enzymes responsible for steroid synthesis . we showed that sixty percent of the hcbz and almost 100% of the pecbz that was added to the culture medium accumulated in ovarian tissue , and only 1% of each was found in the medium . moreover , we showed inhibitory hcbz effect and stimulatory pecbz effect on testosterone and estradiol secretion . as a conclusion we mentioned that the greater exposure to an estrogenic action of pecbz than antiestrogenic hcbz would be a consequence of the preferential accumulation of pecbz in the ovarian follicles we showed that hcbz by fast activation of i and ii phase metabolism is probably metabolized to pecbz in placental tissue ( gregoraszczuk et al . , unpublished data ) . toxicants usually occur in a mixture of several toxicants , making it difficult to predict adverse effects on human health . demonstrated human follicular fluid obtained from women to contain 1 , 1-dichloro-2 , 2-bis ( p - chlorophenyl)-ethylene ( p , p-dde ) , mirex ( a pesticide ) , hexachloro - ethane 1 , 2 , 4-trichlorobenzene , and numerous pcbs ( i.e. , pcbs 49 , 153 , and 180 ) . of these , p , p-dde was the most frequently detected ed in follicular fluid and serum . several environmental toxicants are er agonists , and they include estrogenic steroids ( natural and synthetic ) , phyto- and mycoestrogens , and xenoestrogens ( e.g. , pesticides , plasticizers , and alkylphenols ) . in addition to simple additive effects , interactions between different chemicals in a mixture may result in either a weaker ( antagonistic ) or stronger ( synergistic , potentiated ) combined effect than would be expected from knowledge about the toxicity and mode of action of each individual compound . these interactions may occur during toxicant uptake , distribution , metabolism , and/or excretion ( i.e. , toxicokinetic phase ) , or during toxicant binding to receptors and cellular targets ( i.e. , toxicodynamic phase ) [ 39 , 40 ] . moreover , some estrogenic compounds exert their effects , not by binding to er but rather by binding to estrogen plasma transport proteins , resulting in an increase in free endogenous e2 . nevertheless , mixtures containing both xenoestrogens and endoantiestrogens may have no adverse effects . in our previously published data , to determine which compounds within a pcb - ddt - dde mixture stimulated e2 secretion , we exposed cells to pcbs 118 , 138 , 153 , and 180 , ddt , and dde alone or in different combinations . interestingly , ddt and dde affected e2 secretion , and these results are in agreement with another series of experiments that used mixtures of pbdes . in the next experiments , using western blot analysis indicated that pcbs mixture ( marine mix ) is an inducer of ahr and mixed - type cyp inducer ( cyp1a1 and cyp2b1 ) while pbdes mixture ( mjosa mix ) is an inducer of er and cyp2b . early puberty is a recent growing concern as there are reports of many girls reaching their first menstruation and developing breasts earlier in life than was the case 40 years ago . early puberty associates with polycystic ovarian syndrome ( pcos ) , obesity , breast cancer , depression , and a number of social challenges such as experimentation with sex , alcohol , or drugs at a younger age . moreover , earlier menarche and thelarche ages have been reported in girls after exposure to pcbs , pbbs , ddt , and/or phthalate esters [ 46 , 47 ] , and precocious puberty has been observed following exposure to ddt metabolites . the acyclicity of the syndrome is linked with the hyperfunctioning of theca and hypofunctioning of granulosa cells . pcos also associates with other processes ( e.g. , neuroendocrine function and ovarian steroidogenesis ) and diseases ( e.g. , insulin resistance , and obesity ) that are regulated by hormonal and metabolic factors . for instance , women with pcos have higher levels of bisphenol a ( bpa , a plastics additive ) and testosterone which is consistent with the decreased clearance of bpa that is often observed . although a cause - and - effect relationship has not been established , the role of environmental toxicants in the pathogenesis of pcos is worthy of further consideration . pof , the cessation of normal ovarian function before the age of 40 , occurs in approximately 1% of women of reproductive age . the underlying causes of pof are largely known in most cases , and any factor that can decrease the ovarian reserve can result in pof . for instance , disruption of germ cell migration from the genital ridge to the developing gonad results in ovarian dysgenesis and pof . in addition , adult and in utero exposure of mice to bpa resulted in oocyte damage [ 52 , 53 ] , whereas exposure of women to cigarette smoke decreased fertility , in vitro fertilization ( ivf ) success rates , and the ovarian reserve resulted in earlier menopause and increased miscarriage rate . in another study , exposure of rats to tcdd in utero and throughout reproductive life resulted in premature reproductive senescence . endocrine disruption caused by acute exposure to environmental toxicants such as the ahr agonist tcdd suggests that ahr - mediated apoptosis of oocytes may be involved . breast cancer is the most frequent neoplasm affecting women residing in western countries and is the second leading cause of death . the general population is exposed to several hormonally active compounds on a daily basis . the majority of these compounds are xenoestrogens ( e.g. , pcahs , pesticides , pcbs , pbdes , ddt , selected drugs , fungicides , phytoestrogens , mycotoxins , bpa , and phthalates ) , and they can possess estrogenic action , affect estrogen levels , and/or bind to ers . the role of pcbs in breast cancer has been investigated intensively . data suggest that a correlation may exist between high levels of pcbs in mammary tissues or sera and breast cancer risk , while another study reported no association . high pcb levels upregulated cyp expression , suggesting that pcb metabolites may be critical for the pathogenesis of breast cancer . likewise , pang and colleagues reported that exposure of mcf-7 human breast cancer cells to pcbs 81 , 126 , and 39 increased cyp1a1 and cyp1b1 mrna levels , resulting in the formation of e2 metabolites . we demonstrated pcb 3 to induce and to be a substrate for cyp1a1 in mcf-7 cells . on the other hand , others have shown pcbs 52 and 77 to induce oxidative damage ( i.e. , dna strand breaks ) in er ( )/mda breast cancer cells but not in er ( + ) /mcf-7 cells , suggesting that the er receptor may play a protective role in breast cancer . moreover radice et al . demonstrated those pcb congeners such as pcbs 101 , 118 , 138 , 153 , and 180 increased proliferations of mcf-7 cells . our published data demonstrated that from investigated congeners ( 118 , 138 , 153 , and 180 ) , pcb138 and 153 had the highest stimulatory effects on basal mcf-7 cell proliferation as well as the highest inhibitory actions on basal caspase-9 activity . moreover , we showed that pcbs 138 and 153 contribute to the action of endogenous 17-estradiol on cell proliferation and apoptosis in the breast cancer cell line mcf-7 . in vivo experiments have also shown pcbs to increase metastasis by triggering the production of reactive oxygen species ( ros ) , thereby activating the rho - associated protein kinase ( rock ) signaling pathway or vascular endothelial growth factor ( vegf ) overexpression that stimulates endothelial hyperpermeability and transendothelial migration of cancer cells . an increase in mcf-7 cell proliferation by de-71 ( a mixture of bde-47 , -99 , -100 , -153 , and -154 ) was also reported by mercado - feliciano and bigsby . pbde-209 also induced mcf-7 cell proliferation by affecting critical steps of the cell cycle . at the molecular level , on the other hand , kwieciska et al . have shown no changes in mcf-7 cell proliferation following exposure to bde-47 , -99 , -100 , and -209 ; however , apoptosis was inhibited by decreasing caspase-9 activity in these cells . resistance to apoptosis associates with tumorigenesis as it enables tumorigenic cells to expand even in a stressful environment . thus , additional studies are needed to determine if exposure to pbdes can indeed cause breast cancer . when rodents were exposed to bpa early in life , there were changes in mammary gland morphogenesis and tumor susceptibility [ 7274 ] . these findings are supported by in vitro data which demonstrated bpa to induce transformation of mcf-10f human breast cancer cells . these cells formed tubule - like structures when cultured in 3d collagen matrix , but spherical masses were noted after bpa treatment , leading the authors to conclude that bpa produces adducts or ros which can inadvertently introduce a variety of dna modifications and cause breast cancer . others have shown bpa to stimulate proliferation but to inhibit apoptosis in mcf-7 cells [ 7678 ] . bpa may also induce breast cancer cell proliferation by upregulating cell cycle genes and downregulating antiproliferative genes , especially genes that control the g1/s transition via er signaling . ovarian cancer is the most prevalent type of gynecological cancer affecting women residing in western countries . as more than 60% of tumors are diagnosed at stage iii and certain forms of cancer are very aggressive , ovarian cancers are associated with a high mortality . while most cells undergo neoplastic transformation , including germ cells , granulose , and stromal cells , approximately 90% of tumors are derived from the ovarian surface epithelium ( ose ) . similar to breast cancer , hormonal factors such as estrogen and xenoestrogens have been linked to ovarian cancer [ 80 , 81 ] ; however , the role of environmental toxicants in ovarian cancer requires further study . studies in adult c57bl mice showed that orally administered pbdes-47 , -85 , and -99 to accumulate in the liver , adrenal cortex , and ovary in adult c57bl mice suggest a possible carcinogenic activity , especially in light of research showing that exposure of cho and ovcar-3 cells to pbde-209 initiated s and g2/m phases of the cell cycle , respectively . several in vitro studies have shown bpa to induce chromosomal aberrations in cho cells [ 84 , 85 ] , a common genetic alteration in cancer . moreover , ovarian cyst formation was observed in mice treated neonatally with bpa . ovarian cancer may stem from incessant ovulation , which may be linked to the formation of cysts that are frequently found in perimenopausal women . in a study from our laboratory , we demonstrated an increase in proliferation in ovcar- 3 cells treated with bpa . specifically , we showed bpa to promote the cell cycle by upregulating the expression of cyclin d1 , cdk4 , e2f1 , e2f3 , and pcna ( a mediator of g1 to s - phase progression ) and cyclin a ( a mediator of g2-phase progression to mitosis ) , but by downregulating the expression of p21waf1/cip1 , weel-1 , and gadd45 . additionally , we demonstrated a decrease in the expression of proapoptotic genes ( i.e. , fas , fadd , raidd , caspase-8 , -10 , -3 , -6 , and 7 , cad , bax , bak , bok , and apaf-1 ) but an increase in the expression of prosurvival genes ( i.e. , bcl - x and mcl-1 ) . bpa also activates a caspases - independent apoptotic pathway by inducing endonuclease g gene expression . also , hwang et al . using microarray analysis increased mrna levels of e2-responsive genes in er - positive ovarian cancer bg-1 cells under the influence of bpa . in a subsequent study , we showed bpa to trigger phosphorylation of stat3 , erk1/2 , and akt in ovcar-3 cells . cited results of research indicated that bpa acting as a mitogen as well as an antiapoptotic factor may be an additional factor responsible for ovarian cancer . in recent years there has been a growing evidence that exposure to chemicals in the environment poses a serious threat to human and animals reproduction via disrupting effects on endocrine function . despite the fact that these substances are persistent , they may be metabolized into more toxic compounds than the parent molecule in endocrine organs . this endocrine disrupting chemicals ( edcs ) adversely affect health and reproduction even at very low concentrations and may exert their effects on the embryo and fetus . the complexity and diversity of factors belonging to edcs , its direct action on the ovary , and disorders of the reproductive function of women indicate that the impact of environmental pollution as an important determinant factor in fertility should not be minimize . current estimates of cancer risk in humans do not account properly for transplacental and environmental ( including occupational ) exposure to xenoestrogens . it is important to reevaluate the role of xenoestrogens in cancer development using new approaches that better reflect the complexity of carcinogenesis . testing new compounds before they are allowed to use should be expanded to determine their effect on the endocrine system , in order to assess the hormonal activity . in addition , attention should be directed towards dose - response relationships in environmental toxicology . such studies can provide useful information that might have a significant impact on the strategies for risk assessment of toxic substances .

persistent organic pollutants ( pops ) , such as polychlorinated dibenzo - p - dioxins ( pcdds ) and dibenzofurans ( pcdfs ) , polychlorinated biphenyls ( pcbs ) , and polybrominated ethers ( pbdes ) , chloronaftalens ( pcns ) , and bisphenol a ( bpa ) , are stable , lipophilic pollutants that affect fertility and cause serious reproductive problems , including ovotoxic action , lack of ovulation , premature ovarian failure ( pof ) , or polycystic ovarian syndrome ( pcos ) . most of the representatives of pops influence the activation of transcription factors , not only activation of aromatic hydrocarbon receptor ( ahr ) , but also the steroid hormone receptors . this minireview will focus on a variety of pah activities in oocyte , ovary , placenta , and mammary gland . the complexity and diversity of factors belonging to pops and disorders of the reproductive function of women indicate that the impact of environmental pollution as an important determinant factor in fertility should not be minimize .

1. Introduction 2. Actions on the Oocyte 3. Actions on the Ovary 4. Hormone-Dependent Cancer 5. Conclusions

for the past decade , scientists , institutions , governments , and policymakers have warned the general public about serious health hazards associated with chemicals known as endocrine disruptors ( eds ) . eds are exogenous compounds that interfere with the synthesis , secretion , transport , metabolism , and/or action of endogenous hormones that are responsible for normal homeostasis , reproduction , and development . chemicals with hormonal activity can be divided into three main groups : ( i ) synthetic compounds used in industry and agriculture as well as in consumer products , ( ii ) synthetic compounds used in pharmaceutical drugs , and ( iii ) natural compounds present in the food chain ( i.e. within this class , compounds can be further subcategorized into those that are persistent in all elements of the environment , bio - accumulative , transportable over long distances , and capable of adversely affecting life forms that reside within short and long distances from the site of contamination . these compounds include dichlorodiphenyltrichloroethane ( ddt , a pesticide ) , polychlorinated biphenyls ( pcbs ) , polychlorinated naphthalenes ( pcns ) , polychlorinated dibenzodioxins ( pcdds ) , polychlorinated dibenzofurans ( pcdfs ) , and polybrominated diphenyl ethers ( pbdes ) ( figure 1 ) . , decades ) for instance , pesticides and other synthetic compounds used in the 1950s have polluted the air that we breathe , the water that we drink , and the soil that we grow our food in . studies have shown the presence of eds both in adipose tissue and other organs , in almost all humans and many animals . human exposure to environmental toxicants is mediated via food and water chains , breathing , and dermal absorption , with approximately 90% of the exposure coming from food . , ovary , breast , uterus , cervix , bone , muscle , and skin ) rely on sex steroids for normal function , and these organs are especially vulnerable to endocrine disruption by environmental toxicants . much of the information on the impact of eds on human health has come from in vivo studies where animals were exposed to a single compound , usually at an acute ( i.e. . , pesticides and heavy metals ) and solvents ( e.g. , benzene , toluene , and chloroform ) , and air is filled with hundreds of chemicals ( e.g. the ability of these environmental toxicants to affect human health depends on several factors , including interactions between compounds ; absorption , metabolism , accumulation , and excretion of compounds ; and the ability of some compounds bind to cell receptors which can affect hormone action . it is known that such xenobiotic acts in different ways depending on whether their action is observed on the fetus , newborn babies , or adult individuals , depending on the period during whose body has been exposed to these factors . the female reproductive cycle is a complex process comprised of gametogenesis , embryogenesis , menstruation , ovulation , possible pregnancy , endometrial , and mammary gland changes . women are born with all the oocytes they will ever have through their life , and therefore oocytes are more vulnerable to toxic chemicals than germ cells in men who continue to make more germ cells . follicles are especially susceptible to the adverse effects of environmental toxicants , and developing oocytes may be damaged directly or indirectly by action on follicular cells ( i.e. chloroorganic mixtures such as pcbs and ddt , as well as its metabolites , have also been reported to affect puberty , development , and oocyte viability adversely . some of these studies point to unpredictable changes of translational regulation within the oocyte under the influence of pcbs . hormone - mimicking compounds can bind to cell receptors , interfere with hormone action , and affect ovarian function . how eds affect ovarian function is not yet clear , but a disruption in gonadotropin ( i.e. , follicle stimulating hormone ( fsh ) and luteinizing hormone ( lh ) ) secretion and feedback mechanisms involving e2 and progesterone ( p4 ) may be involved . , a decrease in e2 and p4 , but an increase in fsh and lh ) and ovarian failure . fertility in sexually mature women depends largely on the maintenance of healthy follicles , and their steady production ensures that an adequate number of follicles reach the antral stage . the stage of development at which the follicles are destroyed determines the influence of these factors on the fertility of women . complete depletion of the follicle reserve results in irreversible infertility ; partial depletion of the follicle reserve results in moderate effects on periodicity . damage to large follicles can also lead to reversible acyclic disorders that affect hormone production and ovulation . nevertheless , the effects of pops on the hypothalamic - pituitary - ovarian ( hpo ) axis are generally reversible because they do not permanently affect the follicle reserve . tcdd ( 2 , 3 , 7 , 8-teterachlorodibenzo - para - dioxin ) is an isomer of pcdd , and one of the most toxic man - made compounds to pollute our environment . in a study from our laboratory , tcdd inhibited e2 secretion by follicular cells and p4 secretion by luteal cells dose - dependently . in luteal cells , tcdd action was independent of e2 receptor stimulation , instead involving the aryl hydrocarbon receptor ( ahr ) . on a final note , other environmental toxicants ( i.e. , pcdfs , biphenyls , ddt , and methoxychlor ) can also block the surge in lh and fsh secretion during the female reproductive cycle [ 10 , 11 ] . polychlorinated biphenyls ( pcbs ) are the man - made chemicals that may disrupt follicular steroidogenesis either by mimicking natural hormones as agonist or antagonist , altering the pattern of hormone synthesis , modulating hormone receptor affinities or numbers , or by altering enzymes involved in hormone secretion . in our previous study , we showed that the orthosubstituted pcb 153 congener accumulated preferentially in the follicular wall when compared to the nonorthosubstituted pcb 126 congener . 71% , 71.4% , and 30.4% of the total exposure for pcb 153 were in small , medium , and large follicles , respectively ( figure 1 ) . the consequence was a reduction in estradiol secretion by early antral and antral follicles and lack of influence on estradiol secretion by preovulatory follicles . a similar dose - responsive relationship was reported after exposure of follicular cells to a mixture of organic pollutants or a mixture of pbdes . polybrominated dibenzoethers ( pbdes ) are persistent and ubiquitous environmental toxicants found at increasing levels in humans and animals . despite recent bans by the european union , united states , and china on the production of penta- and octa - bde as well as on the diminished use of pbde in japan , 2,2,4,4-tetra - bde ( bde-47 ) , 2,2,4,4,5-penta - bde ( bde-99 ) , and 2,2,4,4,6-penta - bde ( bde-100 ) are the major pbde congeners present in humans and animals . the literary data related to the effects of pbde mixture are limited predominantly to commercial mixture de-71 which contains mainly penta - bdes ( bde-99 and bde-100 ) and tetra - bde ( bde-47 ) . indicated that de-71 ( a mixture of bde-99 , bde-100 , and tetra - bde ) affected the production of thyroid and sex steroids and the development of reproductive organs [ 20 , 21 ] . results from our laboratory have shown an increase in the p4/testosterone ( t ) ratio but a decrease in the t / e2 ratio , in ovarian follicles suggesting premature luteinization of antral follicles . removal of the pbde mixture from cell cultures did not reverse adverse effects . , 17-hydroxysteroid dehydrogenase ( 17-hsd ) , cytochrome p450 , family 17 , subfamily a , polypeptide 1 ( cyp17 ) , and aromatase ( cyp19 ) ) by pbde congeners 47 , 99 , and 100 . last published data showed fast activation of cyp2b1/2 , and late activation of comt ( with a very low basal sult1a activity ) in ovarian follicles by bde-47 indicates a possible action of locally produced hydroxylated metabolites prior to their detoxification . polychlorinated naphthalenes ( pcns ) are members of a large and diverse group of compounds with several industrial applications . , halowax , nibren wax , and seekay wax ) . the toxicological characteristics of pcns are similar to those caused by pcbs , pcdds , and pcdfs [ 27 , 28 ] . presently , there are little data on the toxicity of pcns in experimental animals , and our recently published data was the first showed direct action on ovarian function . we showed an increase in basal testosterone secretion in all doses used , with the highest stimulatory action of the smallest dose , which was accompanied by a parallel decrease in basal estradiol secretion , induced by halowax 1051 suggesting androgenic properties of halowax 1051 . as a mechanism we propose direct stimulatory action on 17-hsd activity and protein expression , enzyme responsible for testosterone synthesis and inhibitory action on cyp19 activity , and enzyme responsible for conversion testosterone to estradiol . examining the effects of the halowax 1051 on phase i ( cyp1a1 ) and phase ii ( sult1a and comt ) enzyme activities and expression in cultured ovarian follicles we showed fast activation of enzymes involved in phase i and concurrent inhibition of enzymes involved in phase ii metabolism confirming our suggestion that the observed effects of halowax 1051 are partially result from the action of metabolites formed locally in ovarian follicles . in a recently published study , we determined in vitro accumulation of hexachlorobenzene ( hcbz ) and pentachlorobenzene ( pecbz ) in porcine ovarian follicles , the effect on steroidogenesis , and the expression of enzymes responsible for steroid synthesis . we showed that sixty percent of the hcbz and almost 100% of the pecbz that was added to the culture medium accumulated in ovarian tissue , and only 1% of each was found in the medium . as a conclusion we mentioned that the greater exposure to an estrogenic action of pecbz than antiestrogenic hcbz would be a consequence of the preferential accumulation of pecbz in the ovarian follicles we showed that hcbz by fast activation of i and ii phase metabolism is probably metabolized to pecbz in placental tissue ( gregoraszczuk et al . demonstrated human follicular fluid obtained from women to contain 1 , 1-dichloro-2 , 2-bis ( p - chlorophenyl)-ethylene ( p , p-dde ) , mirex ( a pesticide ) , hexachloro - ethane 1 , 2 , 4-trichlorobenzene , and numerous pcbs ( i.e. , pcbs 49 , 153 , and 180 ) . several environmental toxicants are er agonists , and they include estrogenic steroids ( natural and synthetic ) , phyto- and mycoestrogens , and xenoestrogens ( e.g. , pesticides , plasticizers , and alkylphenols ) . , toxicokinetic phase ) , or during toxicant binding to receptors and cellular targets ( i.e. moreover , some estrogenic compounds exert their effects , not by binding to er but rather by binding to estrogen plasma transport proteins , resulting in an increase in free endogenous e2 . in our previously published data , to determine which compounds within a pcb - ddt - dde mixture stimulated e2 secretion , we exposed cells to pcbs 118 , 138 , 153 , and 180 , ddt , and dde alone or in different combinations . interestingly , ddt and dde affected e2 secretion , and these results are in agreement with another series of experiments that used mixtures of pbdes . early puberty associates with polycystic ovarian syndrome ( pcos ) , obesity , breast cancer , depression , and a number of social challenges such as experimentation with sex , alcohol , or drugs at a younger age . moreover , earlier menarche and thelarche ages have been reported in girls after exposure to pcbs , pbbs , ddt , and/or phthalate esters [ 46 , 47 ] , and precocious puberty has been observed following exposure to ddt metabolites . the acyclicity of the syndrome is linked with the hyperfunctioning of theca and hypofunctioning of granulosa cells . , neuroendocrine function and ovarian steroidogenesis ) and diseases ( e.g. , insulin resistance , and obesity ) that are regulated by hormonal and metabolic factors . for instance , women with pcos have higher levels of bisphenol a ( bpa , a plastics additive ) and testosterone which is consistent with the decreased clearance of bpa that is often observed . although a cause - and - effect relationship has not been established , the role of environmental toxicants in the pathogenesis of pcos is worthy of further consideration . pof , the cessation of normal ovarian function before the age of 40 , occurs in approximately 1% of women of reproductive age . the underlying causes of pof are largely known in most cases , and any factor that can decrease the ovarian reserve can result in pof . in addition , adult and in utero exposure of mice to bpa resulted in oocyte damage [ 52 , 53 ] , whereas exposure of women to cigarette smoke decreased fertility , in vitro fertilization ( ivf ) success rates , and the ovarian reserve resulted in earlier menopause and increased miscarriage rate . endocrine disruption caused by acute exposure to environmental toxicants such as the ahr agonist tcdd suggests that ahr - mediated apoptosis of oocytes may be involved . the general population is exposed to several hormonally active compounds on a daily basis . , pcahs , pesticides , pcbs , pbdes , ddt , selected drugs , fungicides , phytoestrogens , mycotoxins , bpa , and phthalates ) , and they can possess estrogenic action , affect estrogen levels , and/or bind to ers . the role of pcbs in breast cancer has been investigated intensively . likewise , pang and colleagues reported that exposure of mcf-7 human breast cancer cells to pcbs 81 , 126 , and 39 increased cyp1a1 and cyp1b1 mrna levels , resulting in the formation of e2 metabolites . , dna strand breaks ) in er ( )/mda breast cancer cells but not in er ( + ) /mcf-7 cells , suggesting that the er receptor may play a protective role in breast cancer . demonstrated those pcb congeners such as pcbs 101 , 118 , 138 , 153 , and 180 increased proliferations of mcf-7 cells . our published data demonstrated that from investigated congeners ( 118 , 138 , 153 , and 180 ) , pcb138 and 153 had the highest stimulatory effects on basal mcf-7 cell proliferation as well as the highest inhibitory actions on basal caspase-9 activity . in vivo experiments have also shown pcbs to increase metastasis by triggering the production of reactive oxygen species ( ros ) , thereby activating the rho - associated protein kinase ( rock ) signaling pathway or vascular endothelial growth factor ( vegf ) overexpression that stimulates endothelial hyperpermeability and transendothelial migration of cancer cells . an increase in mcf-7 cell proliferation by de-71 ( a mixture of bde-47 , -99 , -100 , -153 , and -154 ) was also reported by mercado - feliciano and bigsby . pbde-209 also induced mcf-7 cell proliferation by affecting critical steps of the cell cycle . have shown no changes in mcf-7 cell proliferation following exposure to bde-47 , -99 , -100 , and -209 ; however , apoptosis was inhibited by decreasing caspase-9 activity in these cells . when rodents were exposed to bpa early in life , there were changes in mammary gland morphogenesis and tumor susceptibility [ 7274 ] . these cells formed tubule - like structures when cultured in 3d collagen matrix , but spherical masses were noted after bpa treatment , leading the authors to conclude that bpa produces adducts or ros which can inadvertently introduce a variety of dna modifications and cause breast cancer . while most cells undergo neoplastic transformation , including germ cells , granulose , and stromal cells , approximately 90% of tumors are derived from the ovarian surface epithelium ( ose ) . similar to breast cancer , hormonal factors such as estrogen and xenoestrogens have been linked to ovarian cancer [ 80 , 81 ] ; however , the role of environmental toxicants in ovarian cancer requires further study . studies in adult c57bl mice showed that orally administered pbdes-47 , -85 , and -99 to accumulate in the liver , adrenal cortex , and ovary in adult c57bl mice suggest a possible carcinogenic activity , especially in light of research showing that exposure of cho and ovcar-3 cells to pbde-209 initiated s and g2/m phases of the cell cycle , respectively . moreover , ovarian cyst formation was observed in mice treated neonatally with bpa . ovarian cancer may stem from incessant ovulation , which may be linked to the formation of cysts that are frequently found in perimenopausal women . specifically , we showed bpa to promote the cell cycle by upregulating the expression of cyclin d1 , cdk4 , e2f1 , e2f3 , and pcna ( a mediator of g1 to s - phase progression ) and cyclin a ( a mediator of g2-phase progression to mitosis ) , but by downregulating the expression of p21waf1/cip1 , weel-1 , and gadd45 . , fas , fadd , raidd , caspase-8 , -10 , -3 , -6 , and 7 , cad , bax , bak , bok , and apaf-1 ) but an increase in the expression of prosurvival genes ( i.e. in a subsequent study , we showed bpa to trigger phosphorylation of stat3 , erk1/2 , and akt in ovcar-3 cells . cited results of research indicated that bpa acting as a mitogen as well as an antiapoptotic factor may be an additional factor responsible for ovarian cancer . this endocrine disrupting chemicals ( edcs ) adversely affect health and reproduction even at very low concentrations and may exert their effects on the embryo and fetus . the complexity and diversity of factors belonging to edcs , its direct action on the ovary , and disorders of the reproductive function of women indicate that the impact of environmental pollution as an important determinant factor in fertility should not be minimize . it is important to reevaluate the role of xenoestrogens in cancer development using new approaches that better reflect the complexity of carcinogenesis .

the last 50 years have witnessed an impressive evolution in the antibiotic armamentarium at the clinician s disposal . the availability of effective antibiotic treatment has substantially impacted on overall mortality of patients with lower respiratory tract infections ( lrtis ) . international guideline drafting and implementation have been important in highlighting key factors in improving care in community - acquired pneumonia ( cap ) and acute exacerbation of chronic bronchitis ( aecb ) ( woodhead et al 2005 ; mandell et al 2007 ) . this reflects the paucity of appropriately designed large scale clinical trials addressing such an important treatment issue . conventionally , antibiotics are continued until clinical and laboratory indices of infection are normal in patients with lrtis . it is recognized that patients tend to interupt treatment once they feel better , decreasing compliance to treatment after that time point . antibiotic exposure is unavoidably associated with the phenomenon of antibiotic resistance due to a selection pressure on bacterial strains with natural or acquired resistance . ideally , antibiotics should be maintained only up until bacteria are effectively cleared as further protraction of therapy increases unnecessary bacterial exposure to drugs . currently , antimicrobial resistance is a global problem resulting in high hospitalization rates , mortality and costs in lrtis ( sahm 2003 ) . for example , low dose and prolonged duration of -lactam antibiotic treatment may contribute to promoting pharyngeal carriage of drug - resistant streptococcus pneumoniae ( guillemot et al 1998 ) , whereas high - dose short course amoxicillin may be associated with significantly lower penicillin - resistant s. pneumoniae carriage ( schrag et al 2001 ) . patient adherence to prescribed antibiotic regimens is affected by variables including dosing interval , treatment duration , adverse effects , and palatability in pediatric patients . adherence to once - daily regimens has been shown to be far superior to that of twice- ( kardas 2007 ) or thrice - daily dosing schemes ( claxton et al 2001 ) . it is fairly well established that patient compliance with antibiotic therapy begins to drop after 37 days of treatment ( kardas 2002 ) . additional potential benefits of short course antibiotic therapy in lrtis patients include diminished impact on human endogenous flora , decreased total drug exposure with reduced side effects , reduced health care worker time expenditure , and reduced costs . in order to avoid falling short of effectively controlling infection , short - course antibiotic therapy should be based on robust pharmacokinetic and pharmacodynamic considerations . candidate drugs for short - course regimens should be able to achieve adequate tissue penetrations and concentrations at the site of infection for a sufficiently long length of time to ensure bacterial eradication ( nicolau 2001 ) . the development of an effective short course regimen requires a concentration - dependent antimicrobial effect . the rate and extent of bacterial killing increase with increasing drug concentrations . for such agents the pharmacodynamic / pharmacokinetic predictors of efficacy are the area under the concentration - time curve / mic ratio ( auc / mic ) and the maximal serum drug concentration / mic ratio ( cmax / mic ) ( nicolau 2001 ) it acts by interfering with bacterial protein synthesis through binding of the 50s ribosomal subunit of susceptible microorganisms . azithromycin is an ideal candidate for short - course antibiotic regimens for a number of reasons . compared with earlier macrolides , azithromycin shows improved pharmacokinetic / dynamic properties that determine a prolonged half - life and excellent tissue penetration . the drug rapidly moves from the bloodstream into the interstitial compartment resulting in low serum concentrations but high and persistent tissue concentrations ( amsden et al 1997 ) . characteristically , azithromycin concentration in lung tissue may be over a 100-fold greater than in the circulation . considering that respiratory tract infections reside in the interstitial space , tissue accumulation of azithromycin may guarantee high drug concentrations in the active site of infection . a further interesting characteristic is the ability of azithromycin to concentrate within inflammatory cells , particularly neutrophils and monocytes within the bloodstream , and tissue macrophages ( mandell et al 2001 ) . the ongoing inflammatory process in the lung recalls azithromycinladen neutrophils from the circulation which then unload the drug in the presence of bacteria within inflammation sites ( hand et al 2001 ) . inflammatory cells therefore act as a trojan horse delivering local concentrations of azithromycin that are several orders of magnitude greater than that in plasma . neutrophil unloading and the extended half life of azithromycin ( ~60 hours ) sustain therapeutic drug concentrations at the site of infection for prolonged periods of time ( blumer 2005 ) . furthermore , inflammatory cell uptake of azithromycin is non - saturable and concentration dependent , suggesting that administration of higher doses of the drug early in the course of the infection may result in even greater drug concentration at the sites of infection . short - course treatment of cap patients with azithromycin has been shown to be effective , with the 3-day ( 500 mg single daily dose ) regimen and the 5-day ( 500 mg first day , 250 mg following days ) regimens obtaining comparable results ( socan 1998 ) . clinical studies have shown that a 3-day or 5-day treatment with azithromycin is at least as effective as more prolonged regimens of benzylpenicillin or erythromycin ( bohte et al 1995 ) , and clarithromycin ( odoherty et al 1998 ) . azithromycin and comparators were similar in terms of clinical , microbiological , and radiological results in outpatient or hospitalized cases of mild - to moderate cap . similarly , 3 or 5-day azithromycin regimens were clinically and microbiologically as effective as more prolonged treatment with amoxicillin - clavulanic acid ( hoepelman et al 1997 ) , levofloxacin ( amsden et al 2003 ) , clarithromycin ( bradbury et al 1993 ) or 5-day courses of moxifloxacin ( deabate et al 2000 ) or dirithromycin ( castaldo et al 2003 ) in patients with aecb . table 1 shows a summary of clinical data for the quoted studies on azithromycin versus comparators in lrtis . given its prolonged half - life , propensity to accumulate in tissues and inflammatory cells , and post antibiotic effect , attempts have been made to condense conventional treatment into single large dose schemes of azithromycin ( 1.5 g in adults , 30 mg / kg in children ) for the management of lrtis . administration of a single , higher dose of azithromycin has been shown to achieve more rapid bacterial eradication and enhanced survival than the same dose divided over several days in pre - clinical infection models of otitis media , murine pneumonia and septicemia ( girard et al 2005 , babl et al 2002 ) . this suggests that antibiotic front - loading , attaining higher systemic exposure early in the course of infection ( ie , the auc from time zero to 24 hours [ auc024 ] post dosing ) , may result in more rapid and efficient bacterial clearance following the single - dose regimen . this may offer benefits in terms of minimizing emergence of resistance . in an open randomized study , the efficacy and safety of a single 1.5 g oral dose of azithromycin was compared with the standard 3-day regimen in the treatment of 100 patients with community - acquired atypical pneumonia ( schnwald et al 1999 ) . side effects were observed in 2 patients in the single - dose group , and one patient in the 3-day group . due to the relatively small number of patients studied , however , the study does indicate that a single 1.5 g dose of azithromycin may be an alternative to the standard 3-day azithromycin regimen in the treatment of outpatients with atypical pneumonia syndrome . a later study analyzed the serum and white blood cell pharmacokinetic behavior of 1.5 g single dose azithromycin compared to the same dose over 3 days ( amsden et al 2001 ) . the single drug bolus resulted in significantly higher peak serum concentration compared to the 3-day regimen , although the subjects overall drug exposure did not differ significantly . at 10 days from the start of therapy both regimens showed intracellular white blood cell concentrations that were above the mic90 values of the vast majority of community - acquired respiratory pathogens . the highest single oral dose of azithromycin currently approved is an immediate - release 2.0 g sachet formulation of oral powder for suspension in the treatment of chlamydial and gonococcal urethritis and cervicitis ( handsfield et al 1994 ) . the reported 34% incidence of nausea and 14% incidence of diarrhea indicate that this formulation is likely to achieve limited clinical diffusion due to the high level of gastrointestinal intolerance . recently , microspheres have been developed as a means of effectively delivering antibiotics in periodontal ( paquette 2007 ) and ocular diseases ( pijls et al 2006 ) , for minimizing toxicity of antimycobacterial treatment ( rastogi et al 2006 ) , and as an aid in helicobacter pylori eradication ( patel et al 2007 ) . a novel azithromycin microsphere formulation ( recently approved as zmax in the usa ) has been developed to address the challenge of administering a higher oral dose of the drug as a single dose while maintaining tolerability . the characteristics of the microspheres , with a mean diameter ranging from approximately 100300 m , were selected based on a combination of palatability , release profile , and manufacturability . azithromycin is released slowly via diffusion through pores formed in - situ in the microspheres . alkalinizing agents ( sodium phosphate tribasic and magnesium hydroxide ) were incorporated in the formulation . following ingestion , the alkaline nature of the microsphere formulation delays initial release of drug in the stomach . local drug activation of the motilin receptors is thought to mediate much of the macrolide - related gastrointestinal side effects ( takeshita et al 2006 ) . all the drug is released prior to the colon , thus ensuring maximal bioavailability ( approximately 83% ) ( chandra et al 2007 ) . in an open - label , randomized , parallel - group study of 24 healthy adult subjects the pharmacokinetic profiles of azithromycin given as 2.0 g microspheres were characterized in serum and white blood cells and compared with those of a 3-day regimen totaling 1.5 g ( liu et al 2007 ) . compared with the conventional 3-day 1.5 g azithromycin treatment , the single - dose 2.0 g microsphere formulation obtained a 2-fold higher maximum plasma concentration ( cmax ) and a 3-fold higher 24-hour area under the concentration - time curve ( auc024 ) at day 1 . the total systemic exposure in serum over 5 days following the start of dosing ( auc0120 ) was similar for the two regimens . similarly , in white blood cells , threefold higher drug exposure and maximal concentrations were found at day 1 following single dose administration compared with the 3-day regimen . in an observer - blind , parallel group , single - dose study , the tolerability profiles of azithromycin 2.0 g microspheres were compared with the immediate - release azithromycin 2.0 g sachet formulation of oral powder for suspension ( chandra et al 2007 ) . this was a complex study that also evaluated pharmacokinetic profiles of the two compounds and the effect of food ( high fat meal and a standard meal ) and an antiacid on the rate of drug absorption . compared with the sachet oral powder suspension , the microsphere formulation was associated with a slower absorption rate ( 57% decrease in mean cmax with a 2.5 hour delay in time to reach cmax ) . however , because the auc values were comparable , overall drug exposure was similar . the incidence of gastrointestinal side effects ( abdominal pain , nausea , vomiting , and diarrhea ) was significantly lower with azithromycin microspheres compared to the immediate - release formulation . one non - gastrointestinal adverse event common to azithromycin slow - release and immediate release was headache ( 12% and 6% , respectively ) . in both groups over 90% of adverse events were mild in intensity and occurred within the first 2 hours after dosing . both the high fat meal and the standard meal increased the rate of drug absorption ( 2-fold higher cmax values , and 2- to 4-hour shortening of time to maximal concentration ) but were associated with an increased incidence of nausea and vomiting . it was assumed that meal - triggered gastric acid secretions led to faster release of azithromycin from the microspheres ( chandra et al 2007 ) . because of the decreased tolerability of the formulation when taken with food , it was suggested that azithromycin micro - spheres be taken on an empty stomach . pharmacokinetic properties of the azithromycin microspheres were not significantly affected by co - administration of an antiacid . two phase iii multinational , multicenter , double - blind , double - dummy studies analyzed the efficacy and safety of a new microsphere ( ms ) formulation of azithromycin , 2.0 g given as a single dose , compared with either the extended - release formulation of clarithromycin ) ( 1 g od for 7 days ) ( drehobl et al 2005 ) or with levofloxacin ( 500 mg od for 7 days ) ( dignazio et al 2005 ) in the treatment of adults with mild - to - moderate cap . in the clarithromycin extended - release comparator trial , eligible subjects were required to be 16 years of age or older , with cough productive of sputum and a diagnosis of pneumonia as demonstrated by two or more of the following signs or symptoms : auscultatory findings on pulmonary examination of rales and/or evidence of pulmonary consolidation ; dyspnea or tachypnea ; body temperature > 38 c ( oral ) ; or an elevated total peripheral white blood cell count ( > 10,000/mm ) or > 15% immature neutrophils ( bands ) . in addition , subjects had to have a prospectively calculated modified fine risk score of ~70 ( fine classes i and ii ) ( fine et al 1997 ) . patients were recruited at 58 centers in 7 countries among the us , canada , europe , and asia . in the levofloxacin trial , subjects were aged 18 years or older with a clinical diagnosis of mild - to - moderate cap with a fine mortality risk class of i , ii , or iii ( ie , a risk score of ~90 ) . in total , 427 patients were randomized , of whom 423 received study medication . patients were enrolled at 56 centers in 8 countries among north and south america , europe , and asia . in both studies , clinical assessments were conducted at baseline ( day 1 ) , during treatment ( days 35 ) , at the end of treatment ( days 811 ) , post treatment at the test of cure ( toc ) visit ( days 1421 ) and at a long - term follow - up visit ( days 2835 ) . subjects with a clinical response of cure at the toc visit were assessed for relapse at the long - term follow - up visit ( days 2835 ) . bacteriological response was assessed in those subjects from whom a pathogen was identified at baseline . table 2 shows the demographics and baseline characteristics of the patients enrolled in the two studies . no differences in terms of either clinical response or bacteriological response were noted between azithromycin microspheres and comparators at the toc visit ( days 1421 ) ( table 3 ) . in the azithromycin vs clarithromycin study , at the long - term follow - up visit ( days 2835 ) , only 1 subject ( 0.6% ) in the azalide study arm and 5 subjects ( 2.8% ) in the clarithromycin study arm were classified as having relapsed . clinical cure rates in patients with multilobar pneumonia were only slightly lower than those in patients with single lobe pneumonia ( 88.5% vs 94.4% ) . two azithromycin - treated subjects and one clarithromycin - treated subject were hospitalized for worsening of pneumonia . in this study there were 4 deaths , all of which were in the clarithromycin extended release arm . none of the deaths were attributed to study therapy or progression of cap . in the study comparing azithromycin microspheres with levofloxacin , 6 subjects ( 2 azithromycin and 4 levofloxacin ) were hospitalized for worsening pneumonia ; 2 of the levofloxacin - treated and 1 of the azithromycin - treated subjects ultimately died of causes other than pneumonia . in the azithromycin vs clarithromycin study a pathogen was identified in 281/499 ( 56% ) of treated patients , with evidence of mixed infection in 16% . an atypical pathogen was identified in 24% of azithromycin - treated subjects . in the azithromycin vs levofloxacin study a pathogenic microrganism was identified in 219/423 ( 52% ) of treated patients , a mixed infection being present in 11% . data on s. pneumoniae eradication rates and clinical response , according to bacterial susceptibility to macrolides and penicillin , were analyzed in both studies ( tables 4 and 5 ) . no clear relationship between resistance patterns and clinical and bacteriological outcomes was observed . in the two studies combined , 13 azithromycin - resistant s. pneumoniae strains were found , 7 of which were treated with azithromycin , with clinical cure and bacteriological eradication ( presumed or documented ) being obtained in 4/7 . however , the clinical significance of this finding is still debated as resistance has been associated with clinical failure in some ( fogarty et al 2000 ) but not all studies ( aspa et al 2004 ) . particularly , treatment failure is not unusual in cases of bacteremic pneumococcal pneumonia ( lonks 2002 ) , although most clinical failures showed underlying conditions that would have contraindicated the empirical choice of a macrolide as first line treatment . as mentioned above , the administration of a single high dose of azithromycin is likely to result in concentrations of azithromycin in lung tissue and fluids that would be effective against s. pneumoniae with low - level macrolide resistance . for azithromycin microspheres , most adverse events occurred on the day of administration and resolved within 2 days . table 6 shows the adverse reactions recorded in the two studies . in both studies , the azithromycin group showed a greater number of diarrhea and loose stools adverse events . in most cases the effect was limited to the day of therapy or the following day . owing to the single - dose nature of the regimen , all azithromycin - treated patients completed the treatment course , whereas 15 of 254 subjects ( 5.9% ) randomized to the clarithromycin xl arm did not complete the entire 7-day course of active treatment and 10 of the 212 levofloxacin - treated subjects ( 4.7% ) did not complete the full 7-day treatment course . the results of these controlled trials in cap demonstrated that a single 2.0 g dose of azithromycin microspheres is at least as effective as a 7-day treatment with either clarithromycin extended release or levofloxacin , confirming the suitability of the new formulation of azithromycin . proper management of an aecb should result in rapid resolution of the acute episode and decrease in the likelihood of treatment failure and of an early recurrence ( martinez 2005 ) . it is felt that only approximately 50% of exacerbations are sustained by bacteria ( monso et al 1995 ) and the issue is complicated by the fact that approximately 25%40% of copd patients present stable bacterial colonization of the airways outside periods of acute deterioration ( cabello et al 1997 ) . attempts have been made to identify patients more likely to present bacterial aetiology of an exacerbation based on the presence of at least 2 out of 3 of the clinical anthonisen criteria ( increased cough , increased in sputum production , change in sputum color ) ( anthonisen et al 1987 ) . among the three symptoms , sputum purulence has been most strongly associated with bacterial exacerbation ( stockley et al 2000 ; allegra et al 2005 ) . additionally , patient baseline severity must also be considered as antibiotics have a greater beneficial effect in patients with more severe disease ( allegra et al 2001 ) , which translates into a survival advantage in those sufficiently severe to require mechanical ventilation ( nouira et al 2001 ) . regrettably , in most older antibiotic efficacy studies on patients with exacerbations a precise definition of copd was often absent , with enrolment of young , non - obstructed never - smokers , unsatisfactory definitions of exacerbation , high population severity heterogeneity , and lacked stratification for steroid use . a recent double - blind , double - dummy study compared azithromycin microspheres ( 2 g single dose ) with levofloxacin ( 500 mg once daily for 7 days ) in patients with aecb ( zervos et al 2005 ) . patients were clearly defined with stratification for the use of systemic steroids , and strict inclusion and exclusion criteria . a total of 551 subjects were randomized and 438 were treated at 62 centers in 13 countries . most enrolled subjects were advanced - age patients with a past history of smoking , documented airflow obstruction , at least one aecb in the previous year , and presented all three cardinal anthonisen symptoms in the current exacerbation . the clinical cure rate at the test - of - cure ( toc ) visit ( days 1421 ) in the treated population was comparable between the two agents ( 93.6% for azithromycin compared with 92.7% for levofloxacin ) . baseline microbiological testing yielded positive results in approximately 49% of subjects . in these patients , the overall bacteriological eradication rate was comparable between the two treatment regimens ( 91.9% for azithromycin versus 94.4% for levofloxacin ) . additional post hoc analyses of the 272 subjects in this study with spirometric evidence of airway obstruction suggests equal efficacy of azithromycin microspheres compared with levofloxacin across all strata of copd severity using the gold and ats / ers stratification schemes ( pauwels et al 2001 ; celli et al 2004 ) ( table 8) . the majority of enrolled patients presented advanced disease severity . during the course of the trial and the follow - up period , eight of these occurred in the levofloxacin - treated arm while 4 occurred in azithromycin microsphere - treated patients . there was no clear pattern noted , although the majority of hospitalized patients had more severe airway obstruction at baseline . among the patients treated with azithromycin microspheres , all patients were reported as clinically cured . in 1 patient with the highest macrolide mic values ( > 256 g / ml ) , persistence of the baseline pathogen was established at the 10-day toc visit despite clinical resolution of the acute symptoms . optimal duration for antimicrobial treatment in lower respiratory tract infections ( lrtis ) is still undetermined . short - course treatments may be a means for maintaining clinical efficacy while containing antibiotic resistance spread , increasing patient compliance , and limiting drug - related adverse events . as recently pointed out by thomas m. file ( file 2004 ) , the basic rationale behind short - course antibiotic treatment in cap is to hit hard and stop early . candidate drugs must show favorable pharmacokinetic and pharmacodynamic properties allowing high and prolonged antimicrobial concentrations at the site of infection . the novel azithromycin microsphere formulation takes the concept of short - course treatment one step further by allowing single - dose antibiotic administration in lrtis . use of this formulation allows greater peak concentrations of the drug early in the course of infection with similar overall total antimicrobial exposure compared to traditional 3- to 5-day dosing regimens . the results of controlled trials in cap and copd exacerbations demonstrate that a single dose of azithromycin microspheres is at least as effective and well tolerated as a 7-day treatment with either extended release clarithromycin or levofloxacin . an undoubted advantage of single - dose azithromycin administration is the facility in ensuring that patients complete their prescribed course of therapy . failure to complete therapy may be associated with deterioration in the patient s condition , treatment failure , and increased use and cost of healthcare resources such as the requirement for additional drugs and hospital admission . another advantage of single - dose therapy is the potential for use as directly observed therapy ( dot ) . the use of a dot in lrtis is intriguing , particularly in congested clinical situations ( such as emergency departments ) where assuring patient compliance may be troublesome or barriers to filling prescriptions may exist . physicians may be doubtful as to whether a single dose regimen may be reliable and effective for the treatment of their patients with lrtis . similarly , patients are accustomed to taking medications everyday while they are sick , with interruption of treatment often coinciding with symptom resolution . clearly , further sound clinical demonstration of efficacy and recognition of patient subsets most likely to benefit are needed before widespread use of this approach to lrtis can be considered .

few adequately designed clinical trials have addressed optimal treatment duration in lower respiratory tract infections . drugs possessing favourable pharmacokinetic and pharmacodynamic profiles may obtain early bacterial eradication allowing shorter treatment duration . this may be associated with a number of advantages including reduced resistance induction , increased compliance , lesser adverse events , and cost containment . recently , a novel 2.0 g single dose of azithromycin microspheres has been compared with 7-day levofloxacin 500 mg or extended release clarithromycin in over 400 patients with community - acquired pneumonia . clinical cure and bacteriological eradication rates , hospitalizations , and deaths were similar between azithromycin and comparators . azithromycin 2.0 g microspheres proved as effective as 7 days of levofloxacin 500 mg in acute exacerbation of chronic bronchitis patients across all degrees of obstruction severity . in both settings azithromycin microspheres obtained clinical cure in most patients harbouring macrolide - resistant streptococcus pneumoniae strains . the drug was well tolerated in clinical studies and in healthy volunteers with modest and transitory adverse events . an undoubted advantage of single - dose azithromycin administration is the facility in ensuring that patients complete their prescribed course of therapy . a further advantage of single - dose therapy is the potential for use as directly - observed therapy , which may be useful in specific clinical conditions .

Introduction Short-course azithromycin treatment in lower respiratory tract infections Azithromycin microsphere formulation Azithromycin microspheres in community-acquired pneumonia Azithromycin microspheres in acute exacerbations of chronic bronchitis Conclusions

the availability of effective antibiotic treatment has substantially impacted on overall mortality of patients with lower respiratory tract infections ( lrtis ) . international guideline drafting and implementation have been important in highlighting key factors in improving care in community - acquired pneumonia ( cap ) and acute exacerbation of chronic bronchitis ( aecb ) ( woodhead et al 2005 ; mandell et al 2007 ) . this reflects the paucity of appropriately designed large scale clinical trials addressing such an important treatment issue . conventionally , antibiotics are continued until clinical and laboratory indices of infection are normal in patients with lrtis . it is recognized that patients tend to interupt treatment once they feel better , decreasing compliance to treatment after that time point . antibiotic exposure is unavoidably associated with the phenomenon of antibiotic resistance due to a selection pressure on bacterial strains with natural or acquired resistance . currently , antimicrobial resistance is a global problem resulting in high hospitalization rates , mortality and costs in lrtis ( sahm 2003 ) . for example , low dose and prolonged duration of -lactam antibiotic treatment may contribute to promoting pharyngeal carriage of drug - resistant streptococcus pneumoniae ( guillemot et al 1998 ) , whereas high - dose short course amoxicillin may be associated with significantly lower penicillin - resistant s. pneumoniae carriage ( schrag et al 2001 ) . patient adherence to prescribed antibiotic regimens is affected by variables including dosing interval , treatment duration , adverse effects , and palatability in pediatric patients . adherence to once - daily regimens has been shown to be far superior to that of twice- ( kardas 2007 ) or thrice - daily dosing schemes ( claxton et al 2001 ) . it is fairly well established that patient compliance with antibiotic therapy begins to drop after 37 days of treatment ( kardas 2002 ) . additional potential benefits of short course antibiotic therapy in lrtis patients include diminished impact on human endogenous flora , decreased total drug exposure with reduced side effects , reduced health care worker time expenditure , and reduced costs . in order to avoid falling short of effectively controlling infection , short - course antibiotic therapy should be based on robust pharmacokinetic and pharmacodynamic considerations . candidate drugs for short - course regimens should be able to achieve adequate tissue penetrations and concentrations at the site of infection for a sufficiently long length of time to ensure bacterial eradication ( nicolau 2001 ) . azithromycin is an ideal candidate for short - course antibiotic regimens for a number of reasons . the drug rapidly moves from the bloodstream into the interstitial compartment resulting in low serum concentrations but high and persistent tissue concentrations ( amsden et al 1997 ) . considering that respiratory tract infections reside in the interstitial space , tissue accumulation of azithromycin may guarantee high drug concentrations in the active site of infection . a further interesting characteristic is the ability of azithromycin to concentrate within inflammatory cells , particularly neutrophils and monocytes within the bloodstream , and tissue macrophages ( mandell et al 2001 ) . the ongoing inflammatory process in the lung recalls azithromycinladen neutrophils from the circulation which then unload the drug in the presence of bacteria within inflammation sites ( hand et al 2001 ) . inflammatory cells therefore act as a trojan horse delivering local concentrations of azithromycin that are several orders of magnitude greater than that in plasma . neutrophil unloading and the extended half life of azithromycin ( ~60 hours ) sustain therapeutic drug concentrations at the site of infection for prolonged periods of time ( blumer 2005 ) . furthermore , inflammatory cell uptake of azithromycin is non - saturable and concentration dependent , suggesting that administration of higher doses of the drug early in the course of the infection may result in even greater drug concentration at the sites of infection . short - course treatment of cap patients with azithromycin has been shown to be effective , with the 3-day ( 500 mg single daily dose ) regimen and the 5-day ( 500 mg first day , 250 mg following days ) regimens obtaining comparable results ( socan 1998 ) . clinical studies have shown that a 3-day or 5-day treatment with azithromycin is at least as effective as more prolonged regimens of benzylpenicillin or erythromycin ( bohte et al 1995 ) , and clarithromycin ( odoherty et al 1998 ) . azithromycin and comparators were similar in terms of clinical , microbiological , and radiological results in outpatient or hospitalized cases of mild - to moderate cap . similarly , 3 or 5-day azithromycin regimens were clinically and microbiologically as effective as more prolonged treatment with amoxicillin - clavulanic acid ( hoepelman et al 1997 ) , levofloxacin ( amsden et al 2003 ) , clarithromycin ( bradbury et al 1993 ) or 5-day courses of moxifloxacin ( deabate et al 2000 ) or dirithromycin ( castaldo et al 2003 ) in patients with aecb . given its prolonged half - life , propensity to accumulate in tissues and inflammatory cells , and post antibiotic effect , attempts have been made to condense conventional treatment into single large dose schemes of azithromycin ( 1.5 g in adults , 30 mg / kg in children ) for the management of lrtis . administration of a single , higher dose of azithromycin has been shown to achieve more rapid bacterial eradication and enhanced survival than the same dose divided over several days in pre - clinical infection models of otitis media , murine pneumonia and septicemia ( girard et al 2005 , babl et al 2002 ) . this suggests that antibiotic front - loading , attaining higher systemic exposure early in the course of infection ( ie , the auc from time zero to 24 hours [ auc024 ] post dosing ) , may result in more rapid and efficient bacterial clearance following the single - dose regimen . in an open randomized study , the efficacy and safety of a single 1.5 g oral dose of azithromycin was compared with the standard 3-day regimen in the treatment of 100 patients with community - acquired atypical pneumonia ( schnwald et al 1999 ) . side effects were observed in 2 patients in the single - dose group , and one patient in the 3-day group . due to the relatively small number of patients studied , however , the study does indicate that a single 1.5 g dose of azithromycin may be an alternative to the standard 3-day azithromycin regimen in the treatment of outpatients with atypical pneumonia syndrome . a later study analyzed the serum and white blood cell pharmacokinetic behavior of 1.5 g single dose azithromycin compared to the same dose over 3 days ( amsden et al 2001 ) . at 10 days from the start of therapy both regimens showed intracellular white blood cell concentrations that were above the mic90 values of the vast majority of community - acquired respiratory pathogens . the highest single oral dose of azithromycin currently approved is an immediate - release 2.0 g sachet formulation of oral powder for suspension in the treatment of chlamydial and gonococcal urethritis and cervicitis ( handsfield et al 1994 ) . recently , microspheres have been developed as a means of effectively delivering antibiotics in periodontal ( paquette 2007 ) and ocular diseases ( pijls et al 2006 ) , for minimizing toxicity of antimycobacterial treatment ( rastogi et al 2006 ) , and as an aid in helicobacter pylori eradication ( patel et al 2007 ) . a novel azithromycin microsphere formulation ( recently approved as zmax in the usa ) has been developed to address the challenge of administering a higher oral dose of the drug as a single dose while maintaining tolerability . the characteristics of the microspheres , with a mean diameter ranging from approximately 100300 m , were selected based on a combination of palatability , release profile , and manufacturability . local drug activation of the motilin receptors is thought to mediate much of the macrolide - related gastrointestinal side effects ( takeshita et al 2006 ) . all the drug is released prior to the colon , thus ensuring maximal bioavailability ( approximately 83% ) ( chandra et al 2007 ) . in an open - label , randomized , parallel - group study of 24 healthy adult subjects the pharmacokinetic profiles of azithromycin given as 2.0 g microspheres were characterized in serum and white blood cells and compared with those of a 3-day regimen totaling 1.5 g ( liu et al 2007 ) . compared with the conventional 3-day 1.5 g azithromycin treatment , the single - dose 2.0 g microsphere formulation obtained a 2-fold higher maximum plasma concentration ( cmax ) and a 3-fold higher 24-hour area under the concentration - time curve ( auc024 ) at day 1 . similarly , in white blood cells , threefold higher drug exposure and maximal concentrations were found at day 1 following single dose administration compared with the 3-day regimen . in an observer - blind , parallel group , single - dose study , the tolerability profiles of azithromycin 2.0 g microspheres were compared with the immediate - release azithromycin 2.0 g sachet formulation of oral powder for suspension ( chandra et al 2007 ) . compared with the sachet oral powder suspension , the microsphere formulation was associated with a slower absorption rate ( 57% decrease in mean cmax with a 2.5 hour delay in time to reach cmax ) . the incidence of gastrointestinal side effects ( abdominal pain , nausea , vomiting , and diarrhea ) was significantly lower with azithromycin microspheres compared to the immediate - release formulation . in both groups over 90% of adverse events were mild in intensity and occurred within the first 2 hours after dosing . both the high fat meal and the standard meal increased the rate of drug absorption ( 2-fold higher cmax values , and 2- to 4-hour shortening of time to maximal concentration ) but were associated with an increased incidence of nausea and vomiting . it was assumed that meal - triggered gastric acid secretions led to faster release of azithromycin from the microspheres ( chandra et al 2007 ) . pharmacokinetic properties of the azithromycin microspheres were not significantly affected by co - administration of an antiacid . two phase iii multinational , multicenter , double - blind , double - dummy studies analyzed the efficacy and safety of a new microsphere ( ms ) formulation of azithromycin , 2.0 g given as a single dose , compared with either the extended - release formulation of clarithromycin ) ( 1 g od for 7 days ) ( drehobl et al 2005 ) or with levofloxacin ( 500 mg od for 7 days ) ( dignazio et al 2005 ) in the treatment of adults with mild - to - moderate cap . patients were recruited at 58 centers in 7 countries among the us , canada , europe , and asia . in the levofloxacin trial , subjects were aged 18 years or older with a clinical diagnosis of mild - to - moderate cap with a fine mortality risk class of i , ii , or iii ( ie , a risk score of ~90 ) . patients were enrolled at 56 centers in 8 countries among north and south america , europe , and asia . in both studies , clinical assessments were conducted at baseline ( day 1 ) , during treatment ( days 35 ) , at the end of treatment ( days 811 ) , post treatment at the test of cure ( toc ) visit ( days 1421 ) and at a long - term follow - up visit ( days 2835 ) . subjects with a clinical response of cure at the toc visit were assessed for relapse at the long - term follow - up visit ( days 2835 ) . no differences in terms of either clinical response or bacteriological response were noted between azithromycin microspheres and comparators at the toc visit ( days 1421 ) ( table 3 ) . clinical cure rates in patients with multilobar pneumonia were only slightly lower than those in patients with single lobe pneumonia ( 88.5% vs 94.4% ) . in this study there were 4 deaths , all of which were in the clarithromycin extended release arm . none of the deaths were attributed to study therapy or progression of cap . in the study comparing azithromycin microspheres with levofloxacin , 6 subjects ( 2 azithromycin and 4 levofloxacin ) were hospitalized for worsening pneumonia ; 2 of the levofloxacin - treated and 1 of the azithromycin - treated subjects ultimately died of causes other than pneumonia . an atypical pathogen was identified in 24% of azithromycin - treated subjects . in the azithromycin vs levofloxacin study a pathogenic microrganism was identified in 219/423 ( 52% ) of treated patients , a mixed infection being present in 11% . data on s. pneumoniae eradication rates and clinical response , according to bacterial susceptibility to macrolides and penicillin , were analyzed in both studies ( tables 4 and 5 ) . no clear relationship between resistance patterns and clinical and bacteriological outcomes was observed . in the two studies combined , 13 azithromycin - resistant s. pneumoniae strains were found , 7 of which were treated with azithromycin , with clinical cure and bacteriological eradication ( presumed or documented ) being obtained in 4/7 . however , the clinical significance of this finding is still debated as resistance has been associated with clinical failure in some ( fogarty et al 2000 ) but not all studies ( aspa et al 2004 ) . as mentioned above , the administration of a single high dose of azithromycin is likely to result in concentrations of azithromycin in lung tissue and fluids that would be effective against s. pneumoniae with low - level macrolide resistance . for azithromycin microspheres , most adverse events occurred on the day of administration and resolved within 2 days . in both studies , the azithromycin group showed a greater number of diarrhea and loose stools adverse events . in most cases the effect was limited to the day of therapy or the following day . owing to the single - dose nature of the regimen , all azithromycin - treated patients completed the treatment course , whereas 15 of 254 subjects ( 5.9% ) randomized to the clarithromycin xl arm did not complete the entire 7-day course of active treatment and 10 of the 212 levofloxacin - treated subjects ( 4.7% ) did not complete the full 7-day treatment course . the results of these controlled trials in cap demonstrated that a single 2.0 g dose of azithromycin microspheres is at least as effective as a 7-day treatment with either clarithromycin extended release or levofloxacin , confirming the suitability of the new formulation of azithromycin . attempts have been made to identify patients more likely to present bacterial aetiology of an exacerbation based on the presence of at least 2 out of 3 of the clinical anthonisen criteria ( increased cough , increased in sputum production , change in sputum color ) ( anthonisen et al 1987 ) . among the three symptoms , sputum purulence has been most strongly associated with bacterial exacerbation ( stockley et al 2000 ; allegra et al 2005 ) . additionally , patient baseline severity must also be considered as antibiotics have a greater beneficial effect in patients with more severe disease ( allegra et al 2001 ) , which translates into a survival advantage in those sufficiently severe to require mechanical ventilation ( nouira et al 2001 ) . regrettably , in most older antibiotic efficacy studies on patients with exacerbations a precise definition of copd was often absent , with enrolment of young , non - obstructed never - smokers , unsatisfactory definitions of exacerbation , high population severity heterogeneity , and lacked stratification for steroid use . a recent double - blind , double - dummy study compared azithromycin microspheres ( 2 g single dose ) with levofloxacin ( 500 mg once daily for 7 days ) in patients with aecb ( zervos et al 2005 ) . patients were clearly defined with stratification for the use of systemic steroids , and strict inclusion and exclusion criteria . most enrolled subjects were advanced - age patients with a past history of smoking , documented airflow obstruction , at least one aecb in the previous year , and presented all three cardinal anthonisen symptoms in the current exacerbation . the clinical cure rate at the test - of - cure ( toc ) visit ( days 1421 ) in the treated population was comparable between the two agents ( 93.6% for azithromycin compared with 92.7% for levofloxacin ) . in these patients , the overall bacteriological eradication rate was comparable between the two treatment regimens ( 91.9% for azithromycin versus 94.4% for levofloxacin ) . additional post hoc analyses of the 272 subjects in this study with spirometric evidence of airway obstruction suggests equal efficacy of azithromycin microspheres compared with levofloxacin across all strata of copd severity using the gold and ats / ers stratification schemes ( pauwels et al 2001 ; celli et al 2004 ) ( table 8) . during the course of the trial and the follow - up period , eight of these occurred in the levofloxacin - treated arm while 4 occurred in azithromycin microsphere - treated patients . among the patients treated with azithromycin microspheres , all patients were reported as clinically cured . optimal duration for antimicrobial treatment in lower respiratory tract infections ( lrtis ) is still undetermined . short - course treatments may be a means for maintaining clinical efficacy while containing antibiotic resistance spread , increasing patient compliance , and limiting drug - related adverse events . candidate drugs must show favorable pharmacokinetic and pharmacodynamic properties allowing high and prolonged antimicrobial concentrations at the site of infection . the novel azithromycin microsphere formulation takes the concept of short - course treatment one step further by allowing single - dose antibiotic administration in lrtis . use of this formulation allows greater peak concentrations of the drug early in the course of infection with similar overall total antimicrobial exposure compared to traditional 3- to 5-day dosing regimens . the results of controlled trials in cap and copd exacerbations demonstrate that a single dose of azithromycin microspheres is at least as effective and well tolerated as a 7-day treatment with either extended release clarithromycin or levofloxacin . an undoubted advantage of single - dose azithromycin administration is the facility in ensuring that patients complete their prescribed course of therapy . failure to complete therapy may be associated with deterioration in the patient s condition , treatment failure , and increased use and cost of healthcare resources such as the requirement for additional drugs and hospital admission . another advantage of single - dose therapy is the potential for use as directly observed therapy ( dot ) . the use of a dot in lrtis is intriguing , particularly in congested clinical situations ( such as emergency departments ) where assuring patient compliance may be troublesome or barriers to filling prescriptions may exist . physicians may be doubtful as to whether a single dose regimen may be reliable and effective for the treatment of their patients with lrtis .

one of the first challenges we faced was how to deal with the so - called intentional fall , in which a patient intentionally descends to the floor . this could include cases in which a patient drops or throws himself or herself to the floor in the presence of hospital staff as a way of acting out , as well as cases in which the patient falsely claims to have fallen for some reason ( eg , to get attention ) . these events are known to occur on psychiatric units , and ndnqi hospitals also have reported their occurrence on adult general care units ( medical , surgical , medical - surgical ) . the ndnqi wanted to track these events in response to demand from member hospitals and doing so was especially important with the expansion of the falls indicator to psychiatric units . the problem was defining these events in a manner consistent with the ndnqi fall definition , and in such a way as to minimize subjectivity in reporting . we put the term intentional falls in quotes because such events are not falls by most definitions that we are aware of , as these definitions typically include a word such as inadvertently or unintended.58 prior to the expansion , the ndnqi defined a fall as an unplanned descent to the floor with or without injury to the patient.9 this definition was unclear as to whether the descent must be unplanned either from the perspective of the patient or from the perspective of the hospital staff to qualify as a fall . for example , an intentional descent to the floor planned by the patient would be unplanned from the perspective of the hospital staff , so it might or might not be counted as a fall , depending on one 's interpretation of unplanned . an unplanned descent with a sudden , unintentional descent so as to exclude intentional descents altogether . other changes to the definition are discussed in the following text . with intentional descents clearly excluded from the definition of falls it would be incorrect to call these falls , and we ruled out including them in counts of total or injury falls , in part , because this would disrupt years of continuity in the tracking of units ' total and injury fall rates . we decided to use the term intentional fall events and to define these events as occurring when a patient aged 5 years or older falls on purpose or falsely claims to have fallen . it was important to exclude intentional descents by patients younger than 5 years so that pediatric units would not be burdened with reporting every collapse to the floor by a toddler during a tantrum . consistent with the ndnqi fall definition , we decided to count suspected intentional fall events separately from ( rather than as a subcategory of ) all other falls . the ndnqi relies on the judgment of the nursing staff to distinguish between true falls and intentional descents . according to the revised guidelines , when the nursing staff has reason to suspect that a reported fall is an intentional fall event , it should be reported to ndnqi as such . there is a degree of subjectivity in assessing another person 's intentions or truthfulness , and this may limit the reliability of this measure relative to the reliability of more objective measures . however , the exercise of subjective judgment is unavoidable , given the nature of these events . one concern with the introduction of suspected intentional fall events was that hospitals would use this new category to lower their reported total fall rates by miscategorizing true falls as suspected intentional fall events . we understand concerns about gaming the system and underreporting of adverse events , but we would note that the incentives for such behavior for ndnqi member hospitals are limited . hospitals participate voluntarily , and their data are confidential ; the ndnqi does not participate in public reporting . of course , there may be pressure in some hospitals for unit managers or nursing executives to perform well in benchmarking against peers , especially in hospitals that have taken on the substantial financial commitment required to pursue magnet designation . in any case , the risk of intentional underreporting of adverse events is not unique to falls.10 from a falls - tracking perspective , the pediatric population is unique for several reasons . for one , very young children may be learning to stand or walk , and falling is a natural part of this developmental process . to deal with falls of this sort , we created a category for developmental falls , defined as events in which an infant , toddler , or preschooler who is learning to stand , walk , run , or pivot falls as part of the developmental process of acquiring these skills . asking hospitals to report every developmental fall would be pointless from a quality measurement perspective and substantially increase hospitals ' data collection burden . however , there are cases in which a developmental fall would be reflective of quality of care . for example , if a child with hemophilia falls while learning to run and sustains a laceration , this would be a serious adverse event of the sort that should not occur in a hospital . thus , it seems that some , but not all , developmental falls should be reported . children are not typically injured in developmental falls , and an injurious developmental fall may be indicative of a safety issue ( eg , inadequate adult supervision or environmental hazards making the setting unsuitable for a toddler to practice walking ) . pediatric settings are also unique in that children are more likely than adults to climb furniture , jump on beds , and engage in rough - and - tumble play , and children can be dropped while being held or carried by a caregiver ( a topic addressed in the next section ) . in other words , children may fall in more diverse ways than adults . to accommodate a wider variety of falls , we revised the ndnqi fall definition to allow for descents in which the patient does not land on the floor . this was motivated not only by the expansion to pediatric units , where a patient jumping on a bed might land on the bed rail and sustain an injury , but also by the need for a more flexible definition across unit types . unplanned descent to the floor , along with instructions to include as a fall events in which a patient lands on surface where you would not expect to find a patient . however , this did not include the possibility of a patient landing on a nonsurface such as a hospital staff member or , as one hospital reported , a patient losing his or her balance and coming to rest against a wall , sustaining a minor injury in the process . under the revised guidelines , that results in the patient coming to rest on the floor , on or against some other surface ( eg , a counter ) , on another person , or on an object ( eg , a trash can ) . on neonatal units , the safety concerns are not falls during ambulation but events in which a baby rolls or slides off the edge of a bed or some other piece of furniture and events in which the baby is dropped while being carried , held , or transferred from person to person . these events can also occur with small children . rather than create a separate indicator for these events , we incorporated them in the expanded falls indicator . the revised fall definition includes any sudden , unintentional descent , making it broad enough to encompass both falls from furniture and drops . because of their unique nature , we created a special category for baby / child drops , which we defined as follows : a fall in which a newborn , infant , or child being held or carried by a healthcare professional , patient , family member , or visitor falls or slips from that person 's hands , arms , lap , etc . in defining baby / child drops , the revised guidelines explicitly exclude falls from furniture , noting that drops both falls from furniture and baby / child drops are counted in a unit 's total falls count . one of the first challenges we faced was how to deal with the so - called intentional fall , in which a patient intentionally descends to the floor . this could include cases in which a patient drops or throws himself or herself to the floor in the presence of hospital staff as a way of acting out , as well as cases in which the patient falsely claims to have fallen for some reason ( eg , to get attention ) . these events are known to occur on psychiatric units , and ndnqi hospitals also have reported their occurrence on adult general care units ( medical , surgical , medical - surgical ) . the ndnqi wanted to track these events in response to demand from member hospitals and doing so was especially important with the expansion of the falls indicator to psychiatric units . the problem was defining these events in a manner consistent with the ndnqi fall definition , and in such a way as to minimize subjectivity in reporting . we put the term intentional falls in quotes because such events are not falls by most definitions that we are aware of , as these definitions typically include a word such as inadvertently or unintended.58 prior to the expansion , the ndnqi defined a fall as an unplanned descent to the floor with or without injury to the patient.9 this definition was unclear as to whether the descent must be unplanned either from the perspective of the patient or from the perspective of the hospital staff to qualify as a fall . for example , an intentional descent to the floor planned by the patient would be unplanned from the perspective of the hospital staff , so it might or might not be counted as a fall , depending on one 's interpretation of unplanned . an unplanned descent with a sudden , unintentional descent so as to exclude intentional descents altogether . other changes to the definition are discussed in the following text . with intentional descents clearly excluded from the definition of falls it would be incorrect to call these falls , and we ruled out including them in counts of total or injury falls , in part , because this would disrupt years of continuity in the tracking of units ' total and injury fall rates . we decided to use the term intentional fall events and to define these events as occurring when a patient aged 5 years or older falls on purpose or falsely claims to have fallen . it was important to exclude intentional descents by patients younger than 5 years so that pediatric units would not be burdened with reporting every collapse to the floor by a toddler during a tantrum . consistent with the ndnqi fall definition , we decided to count suspected intentional fall events separately from ( rather than as a subcategory of ) all other falls . the ndnqi relies on the judgment of the nursing staff to distinguish between true falls and intentional descents . according to the revised guidelines , when the nursing staff has reason to suspect that a reported fall is an intentional fall event , it should be reported to ndnqi as such . there is a degree of subjectivity in assessing another person 's intentions or truthfulness , and this may limit the reliability of this measure relative to the reliability of more objective measures . however , the exercise of subjective judgment is unavoidable , given the nature of these events . one concern with the introduction of suspected intentional fall events was that hospitals would use this new category to lower their reported total fall rates by miscategorizing true falls as suspected intentional fall events . we understand concerns about gaming the system and underreporting of adverse events , but we would note that the incentives for such behavior for ndnqi member hospitals are limited . hospitals participate voluntarily , and their data are confidential ; the ndnqi does not participate in public reporting . of course , there may be pressure in some hospitals for unit managers or nursing executives to perform well in benchmarking against peers , especially in hospitals that have taken on the substantial financial commitment required to pursue magnet designation . in any case from a falls - tracking perspective , the pediatric population is unique for several reasons . for one , very young children may be learning to stand or walk , and falling is a natural part of this developmental process . to deal with falls of this sort , we created a category for developmental falls , defined as events in which an infant , toddler , or preschooler who is learning to stand , walk , run , or pivot falls as part of the developmental process of acquiring these skills . asking hospitals to report every developmental fall would be pointless from a quality measurement perspective and substantially increase hospitals ' data collection burden . however , there are cases in which a developmental fall would be reflective of quality of care . for example , if a child with hemophilia falls while learning to run and sustains a laceration , this would be a serious adverse event of the sort that should not occur in a hospital . thus , it seems that some , but not all , developmental falls should be reported . children are not typically injured in developmental falls , and an injurious developmental fall may be indicative of a safety issue ( eg , inadequate adult supervision or environmental hazards making the setting unsuitable for a toddler to practice walking ) . pediatric settings are also unique in that children are more likely than adults to climb furniture , jump on beds , and engage in rough - and - tumble play , and children can be dropped while being held or carried by a caregiver ( a topic addressed in the next section ) . in other words , children may fall in more diverse ways than adults . to accommodate a wider variety of falls , we revised the ndnqi fall definition to allow for descents in which the patient does not land on the floor . this was motivated not only by the expansion to pediatric units , where a patient jumping on a bed might land on the bed rail and sustain an injury , but also by the need for a more flexible definition across unit types . prior to the expansion , the definition included any unplanned descent to the floor , along with instructions to include as a fall events in which a patient however , this did not include the possibility of a patient landing on a nonsurface such as a hospital staff member or , as one hospital reported , a patient losing his or her balance and coming to rest against a wall , sustaining a minor injury in the process . under the revised guidelines , that results in the patient coming to rest on the floor , on or against some other surface ( eg , a counter ) , on another person , or on an object ( eg , a trash can ) . on neonatal units , the safety concerns are not falls during ambulation but events in which a baby rolls or slides off the edge of a bed or some other piece of furniture and events in which the baby is dropped while being carried , held , or transferred from person to person . these events can also occur with small children . rather than create a separate indicator for these events , we incorporated them in the expanded falls indicator . the revised fall definition includes any sudden , unintentional descent , making it broad enough to encompass both falls from furniture and drops . because of their unique nature , we created a special category for baby / child drops , which we defined as follows : a fall in which a newborn , infant , or child being held or carried by a healthcare professional , patient , family member , or visitor falls or slips from that person 's hands , arms , lap , etc . in defining baby / child drops , the revised guidelines explicitly exclude falls from furniture , noting that drops both falls from furniture and baby / child drops are counted in a unit 's total falls count . one drawback of tracking total and injurious fall rates as measures of quality of care is that despite the assertion by the centers for medicare & medicaid services that fall - related injuries are reasonably preventable,11 some falls can not be prevented by reasonable measures and do not reflect on the quality of care provided by the hospital staff . for example , a patient admitted for surgery may sustain an injurious fall during an unrelated stroke or myocardial infarction . in these cases , the total and injurious fall rates are imperfect reflections of quality , and , understandably , hospital staff members do not like to be blamed . thus , there would seem to be a need for differentiating between falls that do reflect negligence or some aspect of quality of care and those that do not . morse,12 for example , proposed a scheme in which falls are categorized using 3 categories . unanticipated physiological falls occur due to physical conditions that can not be predicted until the patient falls , whereas anticipated physiological falls occur in patients whose score on the mfs [ morse fall scale ] indicates that they are at risk of falling.12 morse used the term accidental falls to describe falls by patients who can not be identified before the fall and do not score at risk of falling on a predictive instrument .... a full review of fall categorization schemes is beyond the scope of this article , but others have suggested categorizing falls as preventable or nonpreventable.13,14 one pediatric falls collaborative adopted 9 categories of falls , including environmental , developmental , physical / psychological , response to treatment , family attentiveness , modesty , and horseplay.15 these categorization schemes are based on attempts to understand falls in terms of causal factors , and use of any such scheme in practice requires hospital staff to identify the cause , or primary cause , of every fall . we were reluctant to adopt any of these schemes for the ndnqi for a number of reasons . for one , several of the terms involved are problematic . describing some falls as accidental seems to imply that some falls are not accidental , as if some falls are planned and happen on purpose . however , a descent that is intentional or planned is not a fall by most definitions and it follows that every fall is accidental by the usual definition of the word . first , their use could have negative implications and ramifications for hospital staff members , who might protest , if the fall had been preventable i would have prevented it ! second , a decision by a hospital to document a fall as preventable or anticipated for reporting to the ndnqi places the hospital at risk of litigation . although one might argue after the fact that hospital staff should have anticipated a particular patient falling , it seems unlikely that hospital staff members allow many falls to occur that they have truly anticipated beforehand . another reason we were reluctant to adopt any of these schemes is the difficulty of identifying a single cause for every fall and the data reliability issues that would be associated with any attempt to do so . most real - life phenomena involving human behavior have multiple causes and are difficult to fit neatly into categories based on cause , and falls are no exception . certainly , there are cases in which a fall is caused solely by an unexpected physiological event , and in other cases , an unusual feature of the environment such as a wet floor may clearly be the culprit . for example , if a visually impaired patient falls while ambulating to the bathroom at night , should this be attributed to the visual impairment or the dim lighting ? or suppose an incontinent patient can not reach the bathroom in time and slips on his own urine , or a patient with impaired gait slips because her nonskid socks have become turned around on her feet . in each case , both an intrinsic , physiological factor and an extrinsic , environmental factor are at play . complicating attempts to assign a single cause to every inpatient fall is the widespread use of medications , which are particularly complex risk factors for falling.16 a number of drugs have been linked to fall risk in older adults,17,18 but to our knowledge , there is no agreed - on , comprehensive list of medications known to increase fall risk . even if there were such a list , it would likely change continually as researchers publish new findings , making it impossible for the ndnqi to track physiological fall rates across time using a stable physiological fall definition . moreover , the degree to which various drugs increase the risk of falling differs by drug and may be influenced by interactions with other factors such as patient age , making it difficult to weigh the effect of medication in determining the primary cause of a particular fall . for these reason , we doubted that hospitals could reliably categorize falls by cause , and given the high value the ndnqi places on data reliability , we were reluctant to have hospitals report such data . furthermore , even in cases in which a single causal factor can be clearly identified as primary , making this determination may involve hospital staff spending considerable time and effort for example , reviewing the medical chart , conducting a postfall huddle , or checking the research literature to see if a particular drug has been linked to increased fall risk . the ndnqi currently collects as many as 20 data elements for a single patient fall , in addition to the data collected on numerous other indicators , and we are concerned that the growing data collection burden associated with an ever - expanding list of quality indicators may become detrimental to patient care . clearly , quality measurement requires data collection , but the benefits ( if any ) of collecting highly detailed information on every adverse event must be weighed against the costs , including opportunity costs . time spent gathering and reporting the minutiae of every fall ( injurious or not ) , investigating the primary cause of each fall , reporting which type of hospital staff assisted each fall , and so on , is time not spent in patient care . in the end , the only cause - based fall category introduced by the ndnqi was physiological falls , defined as falls attributable to one or more intrinsic , physiological factors , and we made use of this category optional , not required , for hospitals reporting data on falls . according to the updated guidelines , physiological falls can be caused by a sudden physiological event such as hypotension , dysrhythmia , seizure , transient ischemic attack , or stroke , occur due to side effects of known culprit drugs ( eg , central nervous system active drugs and certain cardiovascular drugs ) , or be attributable to some aspect of the patient 's physical condition such as delirium , intoxication , dementia , gait instability , or visual impairment . from a patient safety perspective , efforts to track and benchmark falls by cause seem to us to be somewhat beside the point . ultimately , what matters most is not why a fall occurred , but ( 1 ) that it did occur , posing risk of harm , and ( 2 ) what its consequences were for the patient . morse19 estimated that 78% of falls are of the anticipated physiological sort , and given a long enough list of intrinsic risk factors , most falls can be attributed to some physiological cause ; in many cases , advanced age alone would suffice . attempts to categorize falls by cause may have unintended consequences , focusing the attention of hospital staff on the question of who / what is to blame for a fall and engendering the sense that an entire category of falls those attributable to a physical condition or medication , say is largely unpreventable . the latter problem would be mitigated by distinguishing between anticipated and unanticipated physiological falls , as morse proposed , but we would prefer not categorizing falls by cause at all to a scheme requiring a second layer of categorization based on judgment about what kinds of falls should be anticipated . if what matters is preventing the consequences of falls , especially injury and death , a couple of approaches to falls tracking and benchmarking are worth considering . one could argue that every fall represents an opportunity for an injury and the best way to prevent fall - related injuries is to prevent falls , much like the best way to prevent drunk driving fatalities is to prevent drunk driving . this approach is useful as far as it goes , and the total fall rate may be a suitable measure of quality and safety if defined in the sense of limiting opportunities for harm due to falling . one potential limitation of the total fall rate is that noninjurious falls may be underreported.10 for this reason , the rate of injurious falls , which are presumably harder to ignore and more likely to result in incident reports , may be a more valid measure of quality and safety , although quality and safety here are construed differently that is , not in the sense of limiting opportunities for harm but in the sense of preventing actual , physical harm . however , a drawback of the injurious fall rate is that 2 otherwise identical falls may have different outcomes in terms of injury due solely to differences between the patients sustaining them ; one could argue that both falls pose a risk of injury , both reflect quality of care and patient safety , and therefore both should be included in any fall - related quality measure . both the total fall and injurious fall rates have an additional limitation , namely , that falls assisted by hospital staff are treated the same as those that are not . unassisted falls pose a greater risk of injury20,21 and uniquely reflect quality of care in that they occur when staff members are absent , unaware that the patient needs assistance , or unable to help for some other reason.22 for these reasons , some have argued that injury prevention efforts should be focused on unassisted falls.21,22 an additional reason is that attempts to prevent all falls could discourage appropriate patient mobilization . we think both the rate of unassisted falls and the percentage of falls assisted are useful metrics for quality tracking and benchmarking . data used for quality tracking and benchmarking should be valid , reliable , and collected at a manageable level of granularity . highly detailed data may be useful for addressing a specific quality - related problem on a particular unit in a limited time frame , but the costs of collecting such data on a long - term basis can outweigh the benefits , especially when the reliability of the data declines as the level of detail increases . in expanding the ndnqi falls indicator , we tried to balance the demand for new categories of falls with the ndnqi 's requirement for definitions that allow for collection of reliable data while keeping in mind the growing data collection burden faced by hospital personnel and its ramifications for quality of care .

in 2012 , the national database of nursing quality indicators launched a project to expand its falls indicator for use on pediatric , neonatal , and psychiatric units . we discuss challenges encountered , argue that schemes for categorizing falls by cause or supposed preventability are not suitable for large - scale efforts to track and prevent falls , express concern about the growing burden of collecting increasingly granular quality data , and discuss limitations of total and injurious fall rates as quality measures .

DEFINING FALLS Intentional falls Pediatric falls Baby drops CATEGORIZING FALLS ARGUMENTS AGAINST ADOPTING A CAUSE-BASED CATEGORIZATION SCHEME CHOOSING FALL MEASURES FOR QUALITY TRACKING AND BENCHMARKING CONCLUSION

this could include cases in which a patient drops or throws himself or herself to the floor in the presence of hospital staff as a way of acting out , as well as cases in which the patient falsely claims to have fallen for some reason ( eg , to get attention ) . these events are known to occur on psychiatric units , and ndnqi hospitals also have reported their occurrence on adult general care units ( medical , surgical , medical - surgical ) . the ndnqi wanted to track these events in response to demand from member hospitals and doing so was especially important with the expansion of the falls indicator to psychiatric units . the problem was defining these events in a manner consistent with the ndnqi fall definition , and in such a way as to minimize subjectivity in reporting . we put the term intentional falls in quotes because such events are not falls by most definitions that we are aware of , as these definitions typically include a word such as inadvertently or unintended.58 prior to the expansion , the ndnqi defined a fall as an unplanned descent to the floor with or without injury to the patient.9 this definition was unclear as to whether the descent must be unplanned either from the perspective of the patient or from the perspective of the hospital staff to qualify as a fall . an unplanned descent with a sudden , unintentional descent so as to exclude intentional descents altogether . other changes to the definition are discussed in the following text . with intentional descents clearly excluded from the definition of falls it would be incorrect to call these falls , and we ruled out including them in counts of total or injury falls , in part , because this would disrupt years of continuity in the tracking of units ' total and injury fall rates . the ndnqi relies on the judgment of the nursing staff to distinguish between true falls and intentional descents . according to the revised guidelines , when the nursing staff has reason to suspect that a reported fall is an intentional fall event , it should be reported to ndnqi as such . there is a degree of subjectivity in assessing another person 's intentions or truthfulness , and this may limit the reliability of this measure relative to the reliability of more objective measures . however , the exercise of subjective judgment is unavoidable , given the nature of these events . one concern with the introduction of suspected intentional fall events was that hospitals would use this new category to lower their reported total fall rates by miscategorizing true falls as suspected intentional fall events . hospitals participate voluntarily , and their data are confidential ; the ndnqi does not participate in public reporting . in any case , the risk of intentional underreporting of adverse events is not unique to falls.10 from a falls - tracking perspective , the pediatric population is unique for several reasons . for one , very young children may be learning to stand or walk , and falling is a natural part of this developmental process . to deal with falls of this sort , we created a category for developmental falls , defined as events in which an infant , toddler , or preschooler who is learning to stand , walk , run , or pivot falls as part of the developmental process of acquiring these skills . asking hospitals to report every developmental fall would be pointless from a quality measurement perspective and substantially increase hospitals ' data collection burden . however , there are cases in which a developmental fall would be reflective of quality of care . children are not typically injured in developmental falls , and an injurious developmental fall may be indicative of a safety issue ( eg , inadequate adult supervision or environmental hazards making the setting unsuitable for a toddler to practice walking ) . pediatric settings are also unique in that children are more likely than adults to climb furniture , jump on beds , and engage in rough - and - tumble play , and children can be dropped while being held or carried by a caregiver ( a topic addressed in the next section ) . in other words , children may fall in more diverse ways than adults . to accommodate a wider variety of falls , we revised the ndnqi fall definition to allow for descents in which the patient does not land on the floor . unplanned descent to the floor , along with instructions to include as a fall events in which a patient lands on surface where you would not expect to find a patient . on neonatal units , the safety concerns are not falls during ambulation but events in which a baby rolls or slides off the edge of a bed or some other piece of furniture and events in which the baby is dropped while being carried , held , or transferred from person to person . these events can also occur with small children . rather than create a separate indicator for these events , we incorporated them in the expanded falls indicator . because of their unique nature , we created a special category for baby / child drops , which we defined as follows : a fall in which a newborn , infant , or child being held or carried by a healthcare professional , patient , family member , or visitor falls or slips from that person 's hands , arms , lap , etc . in defining baby / child drops , the revised guidelines explicitly exclude falls from furniture , noting that drops both falls from furniture and baby / child drops are counted in a unit 's total falls count . these events are known to occur on psychiatric units , and ndnqi hospitals also have reported their occurrence on adult general care units ( medical , surgical , medical - surgical ) . the ndnqi wanted to track these events in response to demand from member hospitals and doing so was especially important with the expansion of the falls indicator to psychiatric units . the problem was defining these events in a manner consistent with the ndnqi fall definition , and in such a way as to minimize subjectivity in reporting . we put the term intentional falls in quotes because such events are not falls by most definitions that we are aware of , as these definitions typically include a word such as inadvertently or unintended.58 prior to the expansion , the ndnqi defined a fall as an unplanned descent to the floor with or without injury to the patient.9 this definition was unclear as to whether the descent must be unplanned either from the perspective of the patient or from the perspective of the hospital staff to qualify as a fall . an unplanned descent with a sudden , unintentional descent so as to exclude intentional descents altogether . with intentional descents clearly excluded from the definition of falls it would be incorrect to call these falls , and we ruled out including them in counts of total or injury falls , in part , because this would disrupt years of continuity in the tracking of units ' total and injury fall rates . we decided to use the term intentional fall events and to define these events as occurring when a patient aged 5 years or older falls on purpose or falsely claims to have fallen . it was important to exclude intentional descents by patients younger than 5 years so that pediatric units would not be burdened with reporting every collapse to the floor by a toddler during a tantrum . according to the revised guidelines , when the nursing staff has reason to suspect that a reported fall is an intentional fall event , it should be reported to ndnqi as such . there is a degree of subjectivity in assessing another person 's intentions or truthfulness , and this may limit the reliability of this measure relative to the reliability of more objective measures . however , the exercise of subjective judgment is unavoidable , given the nature of these events . one concern with the introduction of suspected intentional fall events was that hospitals would use this new category to lower their reported total fall rates by miscategorizing true falls as suspected intentional fall events . we understand concerns about gaming the system and underreporting of adverse events , but we would note that the incentives for such behavior for ndnqi member hospitals are limited . hospitals participate voluntarily , and their data are confidential ; the ndnqi does not participate in public reporting . of course , there may be pressure in some hospitals for unit managers or nursing executives to perform well in benchmarking against peers , especially in hospitals that have taken on the substantial financial commitment required to pursue magnet designation . in any case from a falls - tracking perspective , the pediatric population is unique for several reasons . for one , very young children may be learning to stand or walk , and falling is a natural part of this developmental process . to deal with falls of this sort , we created a category for developmental falls , defined as events in which an infant , toddler , or preschooler who is learning to stand , walk , run , or pivot falls as part of the developmental process of acquiring these skills . asking hospitals to report every developmental fall would be pointless from a quality measurement perspective and substantially increase hospitals ' data collection burden . thus , it seems that some , but not all , developmental falls should be reported . children are not typically injured in developmental falls , and an injurious developmental fall may be indicative of a safety issue ( eg , inadequate adult supervision or environmental hazards making the setting unsuitable for a toddler to practice walking ) . pediatric settings are also unique in that children are more likely than adults to climb furniture , jump on beds , and engage in rough - and - tumble play , and children can be dropped while being held or carried by a caregiver ( a topic addressed in the next section ) . in other words , children may fall in more diverse ways than adults . to accommodate a wider variety of falls , we revised the ndnqi fall definition to allow for descents in which the patient does not land on the floor . this was motivated not only by the expansion to pediatric units , where a patient jumping on a bed might land on the bed rail and sustain an injury , but also by the need for a more flexible definition across unit types . prior to the expansion , the definition included any unplanned descent to the floor , along with instructions to include as a fall events in which a patient however , this did not include the possibility of a patient landing on a nonsurface such as a hospital staff member or , as one hospital reported , a patient losing his or her balance and coming to rest against a wall , sustaining a minor injury in the process . under the revised guidelines , that results in the patient coming to rest on the floor , on or against some other surface ( eg , a counter ) , on another person , or on an object ( eg , a trash can ) . on neonatal units , the safety concerns are not falls during ambulation but events in which a baby rolls or slides off the edge of a bed or some other piece of furniture and events in which the baby is dropped while being carried , held , or transferred from person to person . rather than create a separate indicator for these events , we incorporated them in the expanded falls indicator . because of their unique nature , we created a special category for baby / child drops , which we defined as follows : a fall in which a newborn , infant , or child being held or carried by a healthcare professional , patient , family member , or visitor falls or slips from that person 's hands , arms , lap , etc . in defining baby / child drops , the revised guidelines explicitly exclude falls from furniture , noting that drops both falls from furniture and baby / child drops are counted in a unit 's total falls count . one drawback of tracking total and injurious fall rates as measures of quality of care is that despite the assertion by the centers for medicare & medicaid services that fall - related injuries are reasonably preventable,11 some falls can not be prevented by reasonable measures and do not reflect on the quality of care provided by the hospital staff . for example , a patient admitted for surgery may sustain an injurious fall during an unrelated stroke or myocardial infarction . in these cases , the total and injurious fall rates are imperfect reflections of quality , and , understandably , hospital staff members do not like to be blamed . morse,12 for example , proposed a scheme in which falls are categorized using 3 categories . unanticipated physiological falls occur due to physical conditions that can not be predicted until the patient falls , whereas anticipated physiological falls occur in patients whose score on the mfs [ morse fall scale ] indicates that they are at risk of falling.12 morse used the term accidental falls to describe falls by patients who can not be identified before the fall and do not score at risk of falling on a predictive instrument .... a full review of fall categorization schemes is beyond the scope of this article , but others have suggested categorizing falls as preventable or nonpreventable.13,14 one pediatric falls collaborative adopted 9 categories of falls , including environmental , developmental , physical / psychological , response to treatment , family attentiveness , modesty , and horseplay.15 these categorization schemes are based on attempts to understand falls in terms of causal factors , and use of any such scheme in practice requires hospital staff to identify the cause , or primary cause , of every fall . we were reluctant to adopt any of these schemes for the ndnqi for a number of reasons . describing some falls as accidental seems to imply that some falls are not accidental , as if some falls are planned and happen on purpose . however , a descent that is intentional or planned is not a fall by most definitions and it follows that every fall is accidental by the usual definition of the word . first , their use could have negative implications and ramifications for hospital staff members , who might protest , if the fall had been preventable i would have prevented it ! although one might argue after the fact that hospital staff should have anticipated a particular patient falling , it seems unlikely that hospital staff members allow many falls to occur that they have truly anticipated beforehand . another reason we were reluctant to adopt any of these schemes is the difficulty of identifying a single cause for every fall and the data reliability issues that would be associated with any attempt to do so . most real - life phenomena involving human behavior have multiple causes and are difficult to fit neatly into categories based on cause , and falls are no exception . certainly , there are cases in which a fall is caused solely by an unexpected physiological event , and in other cases , an unusual feature of the environment such as a wet floor may clearly be the culprit . for example , if a visually impaired patient falls while ambulating to the bathroom at night , should this be attributed to the visual impairment or the dim lighting ? or suppose an incontinent patient can not reach the bathroom in time and slips on his own urine , or a patient with impaired gait slips because her nonskid socks have become turned around on her feet . complicating attempts to assign a single cause to every inpatient fall is the widespread use of medications , which are particularly complex risk factors for falling.16 a number of drugs have been linked to fall risk in older adults,17,18 but to our knowledge , there is no agreed - on , comprehensive list of medications known to increase fall risk . even if there were such a list , it would likely change continually as researchers publish new findings , making it impossible for the ndnqi to track physiological fall rates across time using a stable physiological fall definition . moreover , the degree to which various drugs increase the risk of falling differs by drug and may be influenced by interactions with other factors such as patient age , making it difficult to weigh the effect of medication in determining the primary cause of a particular fall . for these reason , we doubted that hospitals could reliably categorize falls by cause , and given the high value the ndnqi places on data reliability , we were reluctant to have hospitals report such data . furthermore , even in cases in which a single causal factor can be clearly identified as primary , making this determination may involve hospital staff spending considerable time and effort for example , reviewing the medical chart , conducting a postfall huddle , or checking the research literature to see if a particular drug has been linked to increased fall risk . the ndnqi currently collects as many as 20 data elements for a single patient fall , in addition to the data collected on numerous other indicators , and we are concerned that the growing data collection burden associated with an ever - expanding list of quality indicators may become detrimental to patient care . clearly , quality measurement requires data collection , but the benefits ( if any ) of collecting highly detailed information on every adverse event must be weighed against the costs , including opportunity costs . time spent gathering and reporting the minutiae of every fall ( injurious or not ) , investigating the primary cause of each fall , reporting which type of hospital staff assisted each fall , and so on , is time not spent in patient care . in the end , the only cause - based fall category introduced by the ndnqi was physiological falls , defined as falls attributable to one or more intrinsic , physiological factors , and we made use of this category optional , not required , for hospitals reporting data on falls . from a patient safety perspective , efforts to track and benchmark falls by cause seem to us to be somewhat beside the point . ultimately , what matters most is not why a fall occurred , but ( 1 ) that it did occur , posing risk of harm , and ( 2 ) what its consequences were for the patient . morse19 estimated that 78% of falls are of the anticipated physiological sort , and given a long enough list of intrinsic risk factors , most falls can be attributed to some physiological cause ; in many cases , advanced age alone would suffice . attempts to categorize falls by cause may have unintended consequences , focusing the attention of hospital staff on the question of who / what is to blame for a fall and engendering the sense that an entire category of falls those attributable to a physical condition or medication , say is largely unpreventable . the latter problem would be mitigated by distinguishing between anticipated and unanticipated physiological falls , as morse proposed , but we would prefer not categorizing falls by cause at all to a scheme requiring a second layer of categorization based on judgment about what kinds of falls should be anticipated . if what matters is preventing the consequences of falls , especially injury and death , a couple of approaches to falls tracking and benchmarking are worth considering . one could argue that every fall represents an opportunity for an injury and the best way to prevent fall - related injuries is to prevent falls , much like the best way to prevent drunk driving fatalities is to prevent drunk driving . this approach is useful as far as it goes , and the total fall rate may be a suitable measure of quality and safety if defined in the sense of limiting opportunities for harm due to falling . one potential limitation of the total fall rate is that noninjurious falls may be underreported.10 for this reason , the rate of injurious falls , which are presumably harder to ignore and more likely to result in incident reports , may be a more valid measure of quality and safety , although quality and safety here are construed differently that is , not in the sense of limiting opportunities for harm but in the sense of preventing actual , physical harm . however , a drawback of the injurious fall rate is that 2 otherwise identical falls may have different outcomes in terms of injury due solely to differences between the patients sustaining them ; one could argue that both falls pose a risk of injury , both reflect quality of care and patient safety , and therefore both should be included in any fall - related quality measure . both the total fall and injurious fall rates have an additional limitation , namely , that falls assisted by hospital staff are treated the same as those that are not . we think both the rate of unassisted falls and the percentage of falls assisted are useful metrics for quality tracking and benchmarking . data used for quality tracking and benchmarking should be valid , reliable , and collected at a manageable level of granularity . highly detailed data may be useful for addressing a specific quality - related problem on a particular unit in a limited time frame , but the costs of collecting such data on a long - term basis can outweigh the benefits , especially when the reliability of the data declines as the level of detail increases . in expanding the ndnqi falls indicator , we tried to balance the demand for new categories of falls with the ndnqi 's requirement for definitions that allow for collection of reliable data while keeping in mind the growing data collection burden faced by hospital personnel and its ramifications for quality of care .

shallow portions of major continental strike - slip faults are exposed to brittle deformation conditions . at crustal levels 34 km , kakirites ( heitzmann , 1985 ) dominate such strike - slip faults , the porosity is therefore high , and vertical fluid flow is dominant resulting in fluid rock interaction and formation of fault gouge ( caine et al . , 1996 ; wibberley et al . , 2008 ) . in terms of fluid motion , this crustal section is considered to represent the hydrothermal convection zone ( saishu et al . , 2014 ) . ductile fabrics respectively plastically formed fabrics are generally missing within quartzofeldspathic fault rocks at these shallow crustal levels . in contrast , evaporitic rocks show plastic behavior even at low temperatures and pressures ( rutter , 1986 ) . consequently , the presence of viscously behaving rocks like evaporites ( halite , gypsum ) at such shallow levels may significantly change the fault zone architecture and related porosity and permeability structure . evaporitic layers frequently act as detachment horizons ( e.g. de paola et al . , 2008 and references therein ) . however , evaporitic strike - slip fault zones are rare ( schorn and neubauer , 2014 ) and detailed descriptions of the internal structure of such shear zones are hampered by intense and quick surface weathering of friable mylonitic gypsum . furthermore , structures and fabrics of secondary tectonic origin within evaporitic sequences are often misinterpreted as primary , depositional features , as outlined by schreiber and helman ( 2005 ) . here we present structural observations from the comeglians - paularo strike - slip shear zone in the southern alps , which propagated entirely through the subvertically tilted gypsum - dominated member of the lower bellerophon formation . in such a case the steeply dipping trend of a rheologically weak layer entirely controls propagation and the structural trend of the shear zone , whereas in strike - slip fault systems within gently to moderately dipping sedimentary successions , the lithology virtually does not play a major role . there , fault location in the cover rocks is primarily controlled by the strength of the basement rocks ( mandl , 1987 ; wilcox et al . , 1973 ) . a change in trend of the bellerophon formation also seems to control the position of fault termination . such terminations are geometrically and kinematically complex and a number of distinct brittle structures such as extensive and compressive horsetails are often associated with fault tips ( sylvester , 1988 and references therein ) . we demonstrate how the ductilely behaving shear zone accommodates n s shortening and dextral displacement by two mechanisms , ( 1 ) non - coaxial and coaxial deformation and ( 2 ) a compressive horse - tail at the western fault termination . this paper first gives an overview of the geological and tectonic setting of the comeglians - paularo shear zone . then we describe detailed field observations of the internal fault structures and then assess the microscale deformation structures . in the discussion , we focus on the low - temperature fabrics and assess the role of viscously behaving evaporites within the otherwise brittle shallow crust for the hydrothermal convection zone . the fella - sava fault , and its western extension , the comeglians - paularo shear zone apparently form the northern boundary of a seismogenic zone , which is affected by intense seismicity with dominant n s thrusting . on a large scale , the active dextral comeglians - paularo shear zone / fella - sava fault represents the pliocene - recent southern boundary of the east - directed lateral extruding block . the study area is located in the ( eastern ) southern alps , which are bordered in the north by the periadriatic fault , the major strike - slip fault of the alpine orogen ( handy et al . , 2010 ) . the n - ward moving adriatic microplate and the southern alps at its front act as a rigid indenter . north of the periadriatic fault , part of the shortening is accommodated by lateral extrusion and tectonic escape to the east along e - trending strike - slip faults and n - trending extensional detachments ( fig . 1 ) ( ratschbacher et al . , 1991a , 1991b ) . the dextral periadriatic fault in the south and the sinistral innsbruck - salzburg - amstetten ( isam ) , salzach - enns - mariazell - puchberg ( semp ) and mur - mrz faults in the north ( fig . eastward extrusion starts in the oligocene mainly on the isam and periadriatic faults ( wlfler et al . , 2011 ) . in the early miocene activity migrates to the semp fault , which then defines the northern border of the extrusion block . a successive rejuvenation of fault activity from the northernmost faults ( e. g. isam fault ) to the more in the se located faults ( e. g. mur - mrz fault ) has been suggested by wlfler et al . the eastern sector of the periadriatic fault is transected by the dextral ca . nw se striking mll valley and lavanttal faults , which were active during the miocene and pliocene ( pischinger et al . , 2008 ) . based on the opening of the gorenjska basin since late miocene times a displacement shift to the fella - sava fault is evident ( fodor et al . , 1998 ; the comeglians - paularo shear zone is the westernmost segment of the fella - sava fault ( e.g. venturini et al . , 2009a ) . lateral offsets of 3060 km ( fodor et al . , 1998 ; pinter et al . , 2006 ; vrabec et al . , 2006 ) or up to 6570 km ( placer , 1996 ) along the fella - sava fault have been inferred . the long - term slip rate for the last 20 ma has been reported to range from 1 to 5 mm / a ( jamek rupnik et al . , 2012 ) . ongoing dextral offset of 1.2 mm / a was reported for the central fella - sava fault in slovenia , whereas no movement was observed along its eastern part and along the periadriatic fault within the scope of the same study ( vrabec et al . , 2006 ) . se contraction and large e w oriented shear strain with strain rates of 20 ppb / a ( grenerczy , 2013 ) . the steeply southward dipping fella - sava fault can be traced to the ne corner of the gorenjska basin ( poljak , 2007 ) , where it disappears beneath neogene sediments . an eastward continuation of the fault to celje and to the croatian border has been assumed ( fig . 1 , placer , 1996 ; plenicar , 2009 ) . between the periadriatic and the fella - sava fault systems en echelon sedimentary basins formed along left - stepping oversteps ( vrabec et al . the fella - sava fault separates mainly the ladinian schlern formation in the south from the permian grden formation and part of the bellerophon formation in the north ( selli , 1963 ) . its westernmost segment was introduced by gortani ( 1926 ) as the comeglians - paularo line and later mapped in detail by braga et al . the comeglians - paularo shear zone developed within the gypsiferous member of the bellerophon formation ( venturini et al . , 2009a ) and approximately juxtaposes the older pre - variscan basement with its pennsylvanian cover in the north with permian to mesozoic post - variscan sedimentary strata in the south ( venturini et al . the entire sedimentary succession is steeply tilted to the south ( merlini et al . , 2002 ) . reconstruction of the evolution of the main river system of friuli ( tagliamento river ) indicates a watershed between the fella - sava fault and the friuli foreland up to messinian times ( monegato and stefani , 2010 ; monegato et al . , the supposed river along the fella - sava fault probably drained eastwards into the gorenjska and similar slovenian basins ( dunkl et al . , 2005 ; monegato and stefani , 2010 ) . in zanclean times , the watershed migrated northwards and the e w drainage along the fella - sava fault was captured by the southward dewatering tagliamento river moneg ( monegato and stefani , 2010 ) . the southern alps are tilted southward and are divided into a basement , the carnic alps , which are exposed along the periadriatic fault , and the southalpine cover unit ( fig . both parts represent sedimentary mega - sequences : the carnic alps show the evolution of an early paleozoic passive continental margin of the pre - variscan cycle ( rantitsch , 1997 ) , whereas the southalpine cover unit represents the upper post - variscan cycle ( schnlaub and heinisch , 1993 ) . the basement of the carnic alps contains a nearly continuous geological section ranging from late ordovician to early pennsylvanian , which is overprinted by both the variscan and the alpine orogenies ( e.g. brime et al . , upper ordovician clastic sediments are overlain by thick devonian shelf margin carbonates . during the earliest visan , the extensional regime shifted to a contractional regime and mississippian synorogenic flysch was deposited ( mader et al . , 2007 ) . a pennsylvanian unconformity separates the folded and faulted variscan basement from the post - variscan cover . the deposition of the cover started with intracontinental siliciclastic molasse sediments ( leppig et al . , 2005 ) the relative sea level changed as a result of gondwana glaciation and shelf and later massive limestone were deposited during stages of high sea - level ( krainer , 2007 ) . a hiatus indicates regression at the end of the lower permian during which the carbonate platform was partly destructed ( schnlaub , 1992 ) . the semiarid grden formation , with conglomerates , sandstones and fine - clastic layers subsequently , a west to east progressing transgression began ( cassinis et al . , 2011 ) and the grden formation locally interfingered with the marine bellerophon formation ( venturini , 1990 ) . the upper permian bellerophon formation ( stratigraphic log in fig . 2b ) was originally a gently to the se dipping carbonate ramp located between sabkhas in the west and an open marine regime in the east ( schnlaub , 1992 and references therein ) . the bellerophon formation was deposited in the shallow tropical bellerophon sea , an embayment of the paleo - tethys ( posenato , 2010 ) . the bellerophon formation is divided into two parts : the lower part contains evaporites and cataclastic dolomite at the base ( massari and neri , 1997 ) . the gypsiferous member acted as a major dcollement horizon in the southalpine unit ( schnborn , 1999 ) . the upper part of the bellerophon formation consists of shoal - water deposits of dolomite , often tectonically brecciated , while the uppermost part is made up of bituminous limestone ( massari and neri , 1997 ) deposited during a regression ( scholger et al . , 2000 and references therein ) . in the study area , the permian bellerophon formation is approximately 260 m thick ( discenza and venturini , 2002 ) and is concordantly overlain by the lower triassic werfen formation . during the opening of the alpine tethys in jurassic times , the southalpine cover unit represents part of the passive continental margin of the adriatic plate and sedimentation continued until the eocene ( bertotti et al . , 1993 ; carminati et al . , the southalpine unit shows no or only a very low - grade metamorphic overprint ( e.g. brime et al . , 2008 ; rantitsch , 1997 and references therein ) . in the variscan basement , the metamorphic grade increases from east to west and from south to north ( sassi et al . , 2004 and references therein ) , and a cretaceous age ( eo - alpine ) for the metamorphism the width of the comeglians - paularo shear zone varies from 100 to 500 m and depends on the thickness of the gypsum - bearing member of the bellerophon formation . the high erodibility of gypsum within the shear zone led to the formation of e w striking linear valleys and mountain passes in between ( fig . approximately 11 km west of comeglians the valley changes its strike from e w to ene n s draining confluents of the tagliamento river cross the shear zone perpendicularly . in the area around paluzza and comeglians , the rivers degano and bt are dextrally deflected along the shear zone and lateral offsets of 0.5 km respectively 1 km of the drainages are evident ( fig . in addition to detailed field observations we performed map interpretation in order to characterize the brittle , large - scale structures associated with the comeglians - paularo shear zone . a schematic overview of the whole shear - zone ( fig . 5b d ) of the rio turiea valley in the east ( fig . 5b , after venturini , 1990 ) , the area around comeglians in the west ( fig . 5c , after venturini et al . , 2002 ) and the western termination of the shear zone ( fig 5d , after cantelli et al . , 1960 ) are presented . along the gypsiferous comeglians - paularo shear zone 5 ) dextral nw se striking riedel shears ( b in fig . 5 ) and sinistral nnw 5 ) developed . in the area around the rio turiea valley venturini ( 1990 ) described sw- vergent folds with axes plunging to the se parallel to the riedel shears ( b in fig . 5 ) . in the area near comeglians , a restraining bend ( d in fig . 5c , 2 ) with ene wsw striking thrusts and folds with sse - vergency is consistent with the dextral movement of the main shear zone . 5d ) parallel to the trend of the valley the e w gypsum - bearing strike - slip shear zone passes into a ene wsw striking thrust fault ( g in fig . the thrust fault transported sandstones of the permian grden formation over triassic carbonates and gypsum is lacking along the thrust ( fig . the thrust accommodates , therefore , dextral displacement along the comeglians - paularo shear zone . further structures were formed during a different deformation event : the structures e and f are interpreted as conjugate strike - slip fault system indicating n s shortening . we performed detailed structural studies , including measurements of the penetrative foliation s1 and stretching lineations l1 , fold - axes ( f2 ) and shear bands between paularo and the valley west of comeglians ( fig . outcrop locations and results of orientation data , including composite diagrams , are given in fig . structural investigations along the shear zone revealed that the entire gypsiferous part of the lower bellerophon formation is strongly deformed in a ductile respectively plastic manner representing the shear zone . brittle behavior is restricted to boudinaged dolomitic layers within gypsum and more competent lithologies of the wall rocks . although poorly exposed , a sharp contact between foliated gypsum and brittely deformed bedded dolomites in the hangingwall respectively footwall was observed in a few outcrops ( e.g. , locations 1 , 2 and 9 of fig . these rocks represent the damage zone adjacent to the gypsum - bearing fault core ( according to caine et al . , 1996 ) . the subvertical mylonitic foliation s1 and the stretching lineation l1 are the most prominent micro- and meso - scale structure ( fig . the foliation of gypsum - rich rocks generally dips steeply to the s to sse ( fig . 4 ) . the foliation dips steeply to the ese consistent with its location within the restraining bend . gypsum attains its maximum thickness and is mined commercially in an opencast ( photo ii in fig . 2 ) . l1 dominantly trends wsw ene and plunges mostly eastward with an angle between 10 and 25. only at locations 13 ( fig . the stretching lineation l1 is defined by stretched and/or elongated millimeter - sized gypsum grains and rigid dolomite clasts . the latter indicate a non - coaxial deformation component and show asymmetric pressure shadows clearly indicating dextral sense of shear ( fig . no significant variations of type and strain of fault rocks were observed along - strike of the shear zone . shear bands dip moderately to steeply to the ssw , the associated fault striae plunge gently to the w and show dextral or normal movement ( fig . 4 ) . in the western part of the comeglians - paularo shear zone , subvertical shear bands with subhorizontal lineation ( fig . , 1979a , 1979b ) also indicate dextral movement of the northern block to the sw . in the trail of the shear bands , the dark dolomite formed pinch - and - swell shaped boudins with visible dextral displacement . additionally , 6c ) were found . gently plunging fold axes show a wide scatter with a preferential trend to the e to se ( fig . the folds are isoclinal , containing a cm - spaced axial plane foliation s1 and a stretching lineation l1 . the foliation s1 and the stretching lineation l1 are refolded during the younger event during which open folds ( f2 ) with moderately s - dipping axial plane foliation s2 were formed . the same e all thin sections were cut parallel to the lineation and perpendicular to the foliation and are composed of mylonitic gypsum . 0.050.2 mm ) and matrix supported , with gypsum as matrix ( figs . 7 and 8) . the gypsum grains are mostly subhedral , subrounded , inequigranular and elongated parallel to the stretching lineation . this is consistent with the preservation of micro - shear zones as shown in fig . 8a c ) shows one generation of gypsum whereas in type 2 ( fig . only the older generation is deformed , in type 3 both gypsum generations are deformed and recrystallization is common . macroscopically , the mylonitic foliation and the stretching lineation are visible in all microfabric types . the grain size of gypsum in type 1 varies from 10 to 50 m , in type 2 from 30 to 220 m and in type 3 from 30 to 150 m . in type 1 ( fig . 8c ) the matrix is inequigranular , the grains are elongated and undulatory extinction is visible , the grain boundaries are diffuse and interlobate . 8f ) has an inequigranular matrix and subhedral grains , the grain boundaries are polygonal and triple points are visible . two generations of gypsum are present in type 2 , the older grains are fine - grained with sutured grain boundaries , the younger grains are large , elongated and show undulatory extinction . fig . 8h illustrates a disintegrated grain next to grains of the older generation with polygonal grain boundaries and triple points . 8i ) is foliated , the matrix is fine - grained and inequigranular , the grain boundaries are interlobate and few triple points were found . the majority of the porphyroclasts is symmetric , consisting of carbonate and showing eye - shaped patterns with tails consistent to the principal matrix lineation . in some porphyroclasts ( fig . 9e ) with stair stepping tails indicate dextral shear sense consistent with observations at outcrop - scale . in fringes around many porphyroclasts , two stages of crystal growth are visible ; the fringe at the strain cap ( passchier and trouw , 2005 ) is even - grained and blocky and fibrous at the tails ( fig . the fibers are straight , parallel to the lineation and represent the x - direction of the finite strain . the existence of fibrous structures indicates conditions with a high fluid pressure and solution transfer during deformation ( passchier and trouw , 2005 ) . we interpret this as a result of extension parallel to the foliation s1 and lineation l1 . the new data on the comeglians - paularo shear zone show ductile fabrics along the entire length of the subvertical to steeply ca . the country rocks in the north and south are less affected by strike - slip deformation . there mainly riedel - type brittle fault zones , oversteps and bends can be observed . for simplification , the gypsum - bearing part of the comeglians - paularo shear zone can be considered as the fault core and the adjacent brittely deformed country rocks as damage zones applying the model of caine et al . the above mentioned riedel - type brittle fault zones are considered as separate systems , each with a separate fault core ( without gypsum ) and a separate damage zone . in the following discussion we first focus on the low - temperature ductile deformation of the gypsum rocks and its potential implications on fluid flow . finally we address the role of the fella - sava fault and comeglians - paularo shear zone for earthquake distribution and propagation . the microfabric study of gypsum mylonites reveals extremely small grain sizes and the lack of significant annealing and recrystallization structures . most gypsum grains of microfabrics type 1 and 2 are undulose and amoeboid grain boundaries indicate bulging as the main deformation mechanism . even in the partly recrystallized gypsum grains of microfabrics type 3 amoeboid / interlobate deformation microstructures prevail and grain sizes remained small in contrast to what is observed in many other gypsum - dominated evaporitic deformation zones ( de paola et al . , 2008 ) . the absence of annealing indicates that no thermal overprint following deformation occurred and that no hydrous , hot fluids invaded the comeglians - paularo shear zone . we expected both plastic and cataclastic behavior of gypsum as described by de paola et al . within the comeglians - paularo shear zone with dominant gypsum , low - temperature ductile respectively plastic deformation occurred while in the adjacent damage zone brittle deformation prevailed . in all investigated outcrops , gypsum was found as the sole sulfate mineral , and no bassanite or anhydrite was observed . in experiments , gypsum dehydrates at 100 c to bassanite , at temperatures of 140 c a further alteration to anhydrite takes place ( brantut et al . , 2011 ) . the absence of evidence for dehydration and annealing structures in thin - sections indicates deformation temperatures well below 100 c . we did not find any indication ( e.g. coarse , undeformed gypsum grains , loss of foliation and lineation ) for rehydration processes as observed within many other deformed gypsum - dominated deformation zones ( de paola et al . , 2008 ; de paola et al . this implies dry country rocks and limited fluid flow also in the damage zones adjacent to the shear zone . the presence of gypsum results in low porosity and low permeability at shallow crustal levels . the core of the shear zone acts as a barrier and impedes fluid flow as indicated by the absence of rehydration products . thus it potentially separates two different convection zones of thermal waters within the adjacent damage zones , which dissolve gypsum from shear zone boundaries . sulfur - bearing thermal waters are known at comeglians and at the fella - sava fault in the east ( fig . 4 ) ( e.g. italiano et al . , 2009 ; petrini et al . , 2011 ) . applying the model of caine et al . ( 1996 ) , the shear zone is a barrier for fluid flow , whereas the adjacent damage zones represents a conduit . the missing annealing is in contrast to gypsum - rich shear zones observed elsewhere , e.g. , in the appennines ( de paola et al . , 2008 ; de paola et al . , 2007 ) or northern calcareous alps ( leitner and neubauer , 2011 ; schorn and neubauer , 2011 ) , where higher temperature conditions are assumed and access of hydrous fluids resulted in pervasive recrystallization of gypsum rocks . the parallel strike of adjacent units and the resulting lack of convincing piercing points renders determination of the amount of structural offset along the fella - sava fault difficult . based on structurally offset oligocene and triassic rocks , fodor et al . ( 1998 ) and references therein inferred a displacement of 3040 km for the easternmost part . 20 km as indicated by the northern margin of the gorenjska ( ljubljana ) basin . ( 2006 ) suggest a transfer of parts of the displacement to the pull - apart gorenjska basin ( fig . ( placer , 1999 ) , presenting a transition zone toward the slovenian basin and the bosnian basin in the n and e ( plenicar , 2009 ) . the comeglians - paularo shear zone has been assumed to be the western extension of the fella - sava fault , ( placer , 1999 ; plenicar , 2009 ) . a contact of the comeglians - paularo shear zone with the valsugana thrust has also been discussed ( venturini et al . , 2009a and references therein ) . as a matter of fact displacement along a fault decreases towards its tips ( e.g. barnett et al . , 1987 ) . no movement will be observed at the tip and fault internal deformation will accommodate strain ( e.g. froitzheim et al . , 2006 ) . further to the west of comeglians , the gypsum member of the bellerophon formation changes its strike from e w to ene wsw and is thrusted onto the triassic werfen formation ( g in fig . 5c ) , which accommodated part of the dextral displacement and consider it to be the western tip of the shear zone . if this is correct , dextral splay faults close to the western end around comeglians ( b in fig . 5b ) could be considered as extensive horsetails , which partly accommodated the lateral displacement , too . such shortening has been found by kinematic and paleostress analyses by peresson and decker ( 1997 ) who ascribed a late miocene age to this compressional event . along the comeglians - paularo shear zone we observe accommodation of n s shortening and dextral displacement by a major pure shear and a subordinate simple shear component . the subsimple shear zone and shows evidence for a combination of two stretching faults ( means , 1989 , 1990 ) as illustrated by an interpretative sketch in fig . symmetric boudins , - and -porphyroclasts and additional extension veins within the clasts ( fig . a subordinate simple - shear component is evidenced by asymmetric clasts , shear bands , micro - shear zones , en chelon structures and block rotation with internal antithetic shear . at the surface , the comeglians - paularo shear zone delimits a zone of widespread seismic activity in the south from a zone of insignificant seismicity in the north ( fig . present - day or historical seismicity is restricted to a few minor earthquakes ( e.g. paluzza ml 4.8 , 1956 ; de panfilis , 1959 ; feliziani and marcelli , 1965 , 1966 ; reinecker and lenhardt , 1999 ) . along its eastern extension , the fella - sava fault , the large historical earthquake of 1348 a. d. ( intensity of ca . 10 ) was recently placed on the fault ( tiberi et al . , 2012 ) . in 1895 , ljubljana was destroyed by a major earthquake ( mm 6.1 earthquake ; ribari , 1982 ) , which was rather located within the gorenjska basin than on the fella - sava fault itself . ongoing n s thrusting activity within the gorenjska basin has recently been documented by folded quaternary sediments and the active vodice reverse thrust fault n of ljubljana ( jamek rupnik et al . , 2013 and references therein ) . we speculate that the occurrence of gypsum along the western fault trace ( comeglians - paularo shear zone ) may have absorbed stresses by viscous behavior and thus may have prevented earthquake nucleation . in contrast , the predominance of carbonate - dominated sequences along the eastern segment of the fault may have favored earthquake nucleation . south of the comeglians - paularo shear zone along a parallel fault in the area of tolmezzo few larger earthquakes ( international seismological centre , 2011 ) with dextral focal mechanisms ( bressan et al . , 1998 ; willingshofer and cloetingh , 2003 ) this observation supports the theory of further southward migration of the displacement due to the younging of fault activity from the periadriatic fault to southern parallel faults ( vrabec et al . , 2006 ) . thus the fella - sava fault represents the southernmost and the semp and mur - mrz faults the northern boundaries of the extruding blocks during late miocene / pliocene reactivation and ongoing lateral extrusion ( caporali et al . , 2013 ; grenerczy , 2013 ) . structural observations along the comeglians - paularo shear zone allow the following major conclusions:we corroborate that the comeglians - paularo shear zone represents the westernmost extension of the fella - sava fault . it terminates west of comeglians with a horse - tail type thrust.the rheologically weak , gypsum - dominated lithology determines shear zone propagation and termination . fault rocks are gypsum - mylonites with a consistently developed steep e w striking foliation and a subhorizontal stretching lineation.missing dehydration products of gypsum and the lack of annealing indicate unusually low temperatures < 100 c during mylonitic deformation of the gypsum . resulting low porosity and low permeability may lead to the core of the shear zone acting as a barrier , impeding fluid flow.the structural and fabric elements are generally consistent in style and orientation at all scale and suggest pure shear e w extension during n s shortening and subordinate dextral displacement along the shear zone.the subsimple shear zone is confined by two stretching faults with stretching occurring within the rheologically weak gypsum layer in between.both internal stretching within the gypsum and brittle structures like the horsetail west of comeglians accommodated portions of the displacement at the comeglians - paularo shear zone . the displacement migrated from along the periadriatic fault southward to the fella - sava fault and as earthquake data suggests , even further to the south during recent times.the comeglians - paularo shear zone impedes fluid flow and possibly earthquake propagation . we corroborate that the comeglians - paularo shear zone represents the westernmost extension of the fella - sava fault . fault rocks are gypsum - mylonites with a consistently developed steep e w striking foliation and a subhorizontal stretching lineation . missing dehydration products of gypsum and the lack of annealing indicate unusually low temperatures < 100 c during mylonitic deformation of the gypsum . resulting low porosity and low permeability may lead to the core of the shear zone acting as a barrier , impeding fluid flow . the structural and fabric elements are generally consistent in style and orientation at all scale and suggest pure shear e w extension during n s shortening and subordinate dextral displacement along the shear zone . the subsimple shear zone is confined by two stretching faults with stretching occurring within the rheologically weak gypsum layer in between . both internal stretching within the gypsum and brittle structures like the horsetail west of comeglians accommodated portions of the displacement at the comeglians - paularo shear zone . the displacement migrated from along the periadriatic fault southward to the fella - sava fault and as earthquake data suggests , even further to the south during recent times .

based on structural and fabric analyses at variable scales we investigate the evaporitic gypsum - dominated comeglians - paularo shear zone in the southern alps ( friuli ) . it represents the lateral western termination of the brittle fella - sava fault . missing dehydration products of gypsum and the lack of annealing indicate temperatures below 100 c during development of the shear zone . despite of such low temperatures the shear zone clearly exhibits mylonitic flow , thus evidencing laterally coeval activity of brittle and viscous deformation.the dominant structures within the gypsum rocks of the lower bellerophon formation are a steeply to gently s - dipping foliation , a subhorizontal stretching lineation and pure shear - dominated porphyroclast systems . a subordinate simple shear component with dextral displacement is indicated by scattered -clasts . both meso- and microscale structures are characteristic of a subsimple shear type of deformation with components of both coaxial and non - coaxial strain . shortening in a transpressive regime was accommodated by right - lateral displacement and internal pure shear deformation within the comeglians - paularo shear zone . the shear zone shows evidence for a combination of two stretching faults , where stretching occurred in the rheologically weaker gypsum member and brittle behavior in enveloping lithologies .

Introduction Geological setting Results Discussion Conclusions

however , evaporitic strike - slip fault zones are rare ( schorn and neubauer , 2014 ) and detailed descriptions of the internal structure of such shear zones are hampered by intense and quick surface weathering of friable mylonitic gypsum . here we present structural observations from the comeglians - paularo strike - slip shear zone in the southern alps , which propagated entirely through the subvertically tilted gypsum - dominated member of the lower bellerophon formation . in such a case the steeply dipping trend of a rheologically weak layer entirely controls propagation and the structural trend of the shear zone , whereas in strike - slip fault systems within gently to moderately dipping sedimentary successions , the lithology virtually does not play a major role . there , fault location in the cover rocks is primarily controlled by the strength of the basement rocks ( mandl , 1987 ; wilcox et al . a change in trend of the bellerophon formation also seems to control the position of fault termination . we demonstrate how the ductilely behaving shear zone accommodates n s shortening and dextral displacement by two mechanisms , ( 1 ) non - coaxial and coaxial deformation and ( 2 ) a compressive horse - tail at the western fault termination . this paper first gives an overview of the geological and tectonic setting of the comeglians - paularo shear zone . in the discussion , we focus on the low - temperature fabrics and assess the role of viscously behaving evaporites within the otherwise brittle shallow crust for the hydrothermal convection zone . the fella - sava fault , and its western extension , the comeglians - paularo shear zone apparently form the northern boundary of a seismogenic zone , which is affected by intense seismicity with dominant n s thrusting . on a large scale , the active dextral comeglians - paularo shear zone / fella - sava fault represents the pliocene - recent southern boundary of the east - directed lateral extruding block . the study area is located in the ( eastern ) southern alps , which are bordered in the north by the periadriatic fault , the major strike - slip fault of the alpine orogen ( handy et al . the n - ward moving adriatic microplate and the southern alps at its front act as a rigid indenter . north of the periadriatic fault , part of the shortening is accommodated by lateral extrusion and tectonic escape to the east along e - trending strike - slip faults and n - trending extensional detachments ( fig . the dextral periadriatic fault in the south and the sinistral innsbruck - salzburg - amstetten ( isam ) , salzach - enns - mariazell - puchberg ( semp ) and mur - mrz faults in the north ( fig . in the early miocene activity migrates to the semp fault , which then defines the northern border of the extrusion block . based on the opening of the gorenjska basin since late miocene times a displacement shift to the fella - sava fault is evident ( fodor et al . , 1998 ; the comeglians - paularo shear zone is the westernmost segment of the fella - sava fault ( e.g. , 2006 ) or up to 6570 km ( placer , 1996 ) along the fella - sava fault have been inferred . ongoing dextral offset of 1.2 mm / a was reported for the central fella - sava fault in slovenia , whereas no movement was observed along its eastern part and along the periadriatic fault within the scope of the same study ( vrabec et al . the steeply southward dipping fella - sava fault can be traced to the ne corner of the gorenjska basin ( poljak , 2007 ) , where it disappears beneath neogene sediments . between the periadriatic and the fella - sava fault systems en echelon sedimentary basins formed along left - stepping oversteps ( vrabec et al . the fella - sava fault separates mainly the ladinian schlern formation in the south from the permian grden formation and part of the bellerophon formation in the north ( selli , 1963 ) . its westernmost segment was introduced by gortani ( 1926 ) as the comeglians - paularo line and later mapped in detail by braga et al . the comeglians - paularo shear zone developed within the gypsiferous member of the bellerophon formation ( venturini et al . reconstruction of the evolution of the main river system of friuli ( tagliamento river ) indicates a watershed between the fella - sava fault and the friuli foreland up to messinian times ( monegato and stefani , 2010 ; monegato et al . , the supposed river along the fella - sava fault probably drained eastwards into the gorenjska and similar slovenian basins ( dunkl et al . in zanclean times , the watershed migrated northwards and the e w drainage along the fella - sava fault was captured by the southward dewatering tagliamento river moneg ( monegato and stefani , 2010 ) . the southern alps are tilted southward and are divided into a basement , the carnic alps , which are exposed along the periadriatic fault , and the southalpine cover unit ( fig . both parts represent sedimentary mega - sequences : the carnic alps show the evolution of an early paleozoic passive continental margin of the pre - variscan cycle ( rantitsch , 1997 ) , whereas the southalpine cover unit represents the upper post - variscan cycle ( schnlaub and heinisch , 1993 ) . a hiatus indicates regression at the end of the lower permian during which the carbonate platform was partly destructed ( schnlaub , 1992 ) . , 2011 ) and the grden formation locally interfingered with the marine bellerophon formation ( venturini , 1990 ) . the bellerophon formation was deposited in the shallow tropical bellerophon sea , an embayment of the paleo - tethys ( posenato , 2010 ) . in the study area , the permian bellerophon formation is approximately 260 m thick ( discenza and venturini , 2002 ) and is concordantly overlain by the lower triassic werfen formation . , 2004 and references therein ) , and a cretaceous age ( eo - alpine ) for the metamorphism the width of the comeglians - paularo shear zone varies from 100 to 500 m and depends on the thickness of the gypsum - bearing member of the bellerophon formation . the high erodibility of gypsum within the shear zone led to the formation of e w striking linear valleys and mountain passes in between ( fig . approximately 11 km west of comeglians the valley changes its strike from e w to ene n s draining confluents of the tagliamento river cross the shear zone perpendicularly . in the area around paluzza and comeglians , the rivers degano and bt are dextrally deflected along the shear zone and lateral offsets of 0.5 km respectively 1 km of the drainages are evident ( fig . in addition to detailed field observations we performed map interpretation in order to characterize the brittle , large - scale structures associated with the comeglians - paularo shear zone . a schematic overview of the whole shear - zone ( fig . 5b d ) of the rio turiea valley in the east ( fig . , 2002 ) and the western termination of the shear zone ( fig 5d , after cantelli et al . along the gypsiferous comeglians - paularo shear zone 5 ) dextral nw se striking riedel shears ( b in fig . in the area near comeglians , a restraining bend ( d in fig . 5d ) parallel to the trend of the valley the e w gypsum - bearing strike - slip shear zone passes into a ene wsw striking thrust fault ( g in fig . the thrust accommodates , therefore , dextral displacement along the comeglians - paularo shear zone . we performed detailed structural studies , including measurements of the penetrative foliation s1 and stretching lineations l1 , fold - axes ( f2 ) and shear bands between paularo and the valley west of comeglians ( fig . structural investigations along the shear zone revealed that the entire gypsiferous part of the lower bellerophon formation is strongly deformed in a ductile respectively plastic manner representing the shear zone . brittle behavior is restricted to boudinaged dolomitic layers within gypsum and more competent lithologies of the wall rocks . although poorly exposed , a sharp contact between foliated gypsum and brittely deformed bedded dolomites in the hangingwall respectively footwall was observed in a few outcrops ( e.g. these rocks represent the damage zone adjacent to the gypsum - bearing fault core ( according to caine et al . the foliation of gypsum - rich rocks generally dips steeply to the s to sse ( fig . the foliation dips steeply to the ese consistent with its location within the restraining bend . no significant variations of type and strain of fault rocks were observed along - strike of the shear zone . in the western part of the comeglians - paularo shear zone , subvertical shear bands with subhorizontal lineation ( fig . in the trail of the shear bands , the dark dolomite formed pinch - and - swell shaped boudins with visible dextral displacement . the foliation s1 and the stretching lineation l1 are refolded during the younger event during which open folds ( f2 ) with moderately s - dipping axial plane foliation s2 were formed . the gypsum grains are mostly subhedral , subrounded , inequigranular and elongated parallel to the stretching lineation . macroscopically , the mylonitic foliation and the stretching lineation are visible in all microfabric types . the fibers are straight , parallel to the lineation and represent the x - direction of the finite strain . the new data on the comeglians - paularo shear zone show ductile fabrics along the entire length of the subvertical to steeply ca . for simplification , the gypsum - bearing part of the comeglians - paularo shear zone can be considered as the fault core and the adjacent brittely deformed country rocks as damage zones applying the model of caine et al . in the following discussion we first focus on the low - temperature ductile deformation of the gypsum rocks and its potential implications on fluid flow . finally we address the role of the fella - sava fault and comeglians - paularo shear zone for earthquake distribution and propagation . the microfabric study of gypsum mylonites reveals extremely small grain sizes and the lack of significant annealing and recrystallization structures . even in the partly recrystallized gypsum grains of microfabrics type 3 amoeboid / interlobate deformation microstructures prevail and grain sizes remained small in contrast to what is observed in many other gypsum - dominated evaporitic deformation zones ( de paola et al . the absence of annealing indicates that no thermal overprint following deformation occurred and that no hydrous , hot fluids invaded the comeglians - paularo shear zone . within the comeglians - paularo shear zone with dominant gypsum , low - temperature ductile respectively plastic deformation occurred while in the adjacent damage zone brittle deformation prevailed . the absence of evidence for dehydration and annealing structures in thin - sections indicates deformation temperatures well below 100 c . coarse , undeformed gypsum grains , loss of foliation and lineation ) for rehydration processes as observed within many other deformed gypsum - dominated deformation zones ( de paola et al . this implies dry country rocks and limited fluid flow also in the damage zones adjacent to the shear zone . the core of the shear zone acts as a barrier and impedes fluid flow as indicated by the absence of rehydration products . thus it potentially separates two different convection zones of thermal waters within the adjacent damage zones , which dissolve gypsum from shear zone boundaries . sulfur - bearing thermal waters are known at comeglians and at the fella - sava fault in the east ( fig . ( 1996 ) , the shear zone is a barrier for fluid flow , whereas the adjacent damage zones represents a conduit . , 2007 ) or northern calcareous alps ( leitner and neubauer , 2011 ; schorn and neubauer , 2011 ) , where higher temperature conditions are assumed and access of hydrous fluids resulted in pervasive recrystallization of gypsum rocks . the parallel strike of adjacent units and the resulting lack of convincing piercing points renders determination of the amount of structural offset along the fella - sava fault difficult . 20 km as indicated by the northern margin of the gorenjska ( ljubljana ) basin . ( placer , 1999 ) , presenting a transition zone toward the slovenian basin and the bosnian basin in the n and e ( plenicar , 2009 ) . the comeglians - paularo shear zone has been assumed to be the western extension of the fella - sava fault , ( placer , 1999 ; plenicar , 2009 ) . a contact of the comeglians - paularo shear zone with the valsugana thrust has also been discussed ( venturini et al . further to the west of comeglians , the gypsum member of the bellerophon formation changes its strike from e w to ene wsw and is thrusted onto the triassic werfen formation ( g in fig . 5c ) , which accommodated part of the dextral displacement and consider it to be the western tip of the shear zone . along the comeglians - paularo shear zone we observe accommodation of n s shortening and dextral displacement by a major pure shear and a subordinate simple shear component . the subsimple shear zone and shows evidence for a combination of two stretching faults ( means , 1989 , 1990 ) as illustrated by an interpretative sketch in fig . a subordinate simple - shear component is evidenced by asymmetric clasts , shear bands , micro - shear zones , en chelon structures and block rotation with internal antithetic shear . at the surface , the comeglians - paularo shear zone delimits a zone of widespread seismic activity in the south from a zone of insignificant seismicity in the north ( fig . along its eastern extension , the fella - sava fault , the large historical earthquake of 1348 a. d. ( intensity of ca . in 1895 , ljubljana was destroyed by a major earthquake ( mm 6.1 earthquake ; ribari , 1982 ) , which was rather located within the gorenjska basin than on the fella - sava fault itself . we speculate that the occurrence of gypsum along the western fault trace ( comeglians - paularo shear zone ) may have absorbed stresses by viscous behavior and thus may have prevented earthquake nucleation . in contrast , the predominance of carbonate - dominated sequences along the eastern segment of the fault may have favored earthquake nucleation . south of the comeglians - paularo shear zone along a parallel fault in the area of tolmezzo few larger earthquakes ( international seismological centre , 2011 ) with dextral focal mechanisms ( bressan et al . thus the fella - sava fault represents the southernmost and the semp and mur - mrz faults the northern boundaries of the extruding blocks during late miocene / pliocene reactivation and ongoing lateral extrusion ( caporali et al . structural observations along the comeglians - paularo shear zone allow the following major conclusions:we corroborate that the comeglians - paularo shear zone represents the westernmost extension of the fella - sava fault . it terminates west of comeglians with a horse - tail type thrust.the rheologically weak , gypsum - dominated lithology determines shear zone propagation and termination . fault rocks are gypsum - mylonites with a consistently developed steep e w striking foliation and a subhorizontal stretching lineation.missing dehydration products of gypsum and the lack of annealing indicate unusually low temperatures < 100 c during mylonitic deformation of the gypsum . resulting low porosity and low permeability may lead to the core of the shear zone acting as a barrier , impeding fluid flow.the structural and fabric elements are generally consistent in style and orientation at all scale and suggest pure shear e w extension during n s shortening and subordinate dextral displacement along the shear zone.the subsimple shear zone is confined by two stretching faults with stretching occurring within the rheologically weak gypsum layer in between.both internal stretching within the gypsum and brittle structures like the horsetail west of comeglians accommodated portions of the displacement at the comeglians - paularo shear zone . the displacement migrated from along the periadriatic fault southward to the fella - sava fault and as earthquake data suggests , even further to the south during recent times.the comeglians - paularo shear zone impedes fluid flow and possibly earthquake propagation . we corroborate that the comeglians - paularo shear zone represents the westernmost extension of the fella - sava fault . fault rocks are gypsum - mylonites with a consistently developed steep e w striking foliation and a subhorizontal stretching lineation . missing dehydration products of gypsum and the lack of annealing indicate unusually low temperatures < 100 c during mylonitic deformation of the gypsum . resulting low porosity and low permeability may lead to the core of the shear zone acting as a barrier , impeding fluid flow . the structural and fabric elements are generally consistent in style and orientation at all scale and suggest pure shear e w extension during n s shortening and subordinate dextral displacement along the shear zone . the subsimple shear zone is confined by two stretching faults with stretching occurring within the rheologically weak gypsum layer in between . both internal stretching within the gypsum and brittle structures like the horsetail west of comeglians accommodated portions of the displacement at the comeglians - paularo shear zone . the displacement migrated from along the periadriatic fault southward to the fella - sava fault and as earthquake data suggests , even further to the south during recent times .

atypia of undetermined significance / follicular lesion of undetermined significance ( aus / flus ) is a heterogeneous diagnostic category of the bethesda system for reporting thyroid cytopathology ( tbsrtc ) . it is comprised of cases that can not be definitively diagnosed as benign , suspicious for / consistent with neoplasm , suspicious for malignancy or malignant . there are various interpretive scenarios depicted and illustrated in tbsrtc that may warrant a diagnosis of aus / flus . these include specimens that may show focal architectural or nuclear atypia whose significance can not be further determined ( microfollicle or papillary formation , predominance of oncocytic follicular cells , rare cells with atypical nuclear features of papillary thyroid cancer , focal nuclear atypia in follicular and lymphoid infiltrates ) and cellular aspirates that are limited by obscuring blood or poor fixation . since these aforementioned features are entirely based on the observer , there exists a low inter - observer agreement and poor reproducibility for cases diagnosed as aus / flus . the recommended management for nodules with an initial aus / flus diagnosis is clinical correlation and repeat fine - needle aspiration ( fna ) at an appropriate interval . in the post - bethesda thyroid fna classification era , the risk of malignancy has been reported to a range between 5% and 15% in patients with a diagnosis of aus / flus . in our previous review of thyroid cytopathology reporting schemes , a number of studies reported a higher risk of malignancy for thyroid fna specimens diagnosed as indeterminate beyond the rates recommended by tbsrtc . in the study by jo et al . , a majority of initial aus / flus cases received follow - up surgery rather than the recommended repeat fna . renshaw , in concordance with other studies , noted that thyroid fna cases subcategorized as aus / flus not otherwise specified and aus / flus - rule out follicular neoplasm had a similar malignancy rate to cases diagnosed as follicular neoplasm ( fn)/suspicious for follicular neoplasm ( sfn ) . therefore , even though these two aus / flus subgroups may theoretically have similar malignancy rates to fn / sfn , the case management may entirely be dictated by which tbsrtc category they are placed into ( repeat fna for aus / flus vs. surgical excision for fn / sfn ) . these studies have shown that even though the overall risk of malignancy is relatively low , sub - classifying aus / flus with focal abnormal cytomorphological features may help in better management of patients . in this study , we present our institutional experience with thyroid nodules diagnosed as aus / flus and further stratified into subcategories . in addition , we also assessed the significance of various clinicopathologic factors that may influence aus / flus diagnosis and their outcomes . a retrospective search of our laboratory information system was performed to identify all in - house thyroid fna cases that were performed under ultrasound guidance and diagnosed as aus / flus from 2008 to 2012 . all thyroid fnas were aspirated by a team of endocrinologists or radiologists using a 25 - 27 gauge needle ; generally with 23 passes . a portion of each pass was used to make an air - dried slide , an alcohol fixed slide , with the remaining specimen in the needle hub rinsed in preservcyt solution ( hologic , marlborough , ma , usa ) to prepare a thinprep slide ( hologic ) . a rapid on - site evaluation ( rose ) by staff cytopathologists was performed in nearly all in - house thyroid fnas . aus / flus cases were further sub - classified into one of the following subcategories ( sc ) : sc1 - favor benign , however , a fn could not be excluded due to increased cellularity ; sc2 - specimens with focal nuclear overlapping and crowding ; sc3 - scant specimens with focal nuclear overlapping and crowding ; sc4 - specimens with focal nuclear overlapping and crowding in a background of lymphocytic thyroiditis ; sc5 - few cells with features suspicious for papillary thyroid cancer ( ptc ) ; and sc6 - specimens in which fn can not be excluded ( with miscellaneous morphologic classifiers ) . data points collected for this study included : patient demographics , size of the thyroid nodule sampled , aspirator , pathologist rendering the final diagnosis , and applicable follow - up . the data were collected and characterized by patient demographic information , cytopathology diagnosis with sub - classifiers and follow - up . only cases that were diagnosed as aus / flus were included in the study , however , patients younger than 18 years , nodules aspirated without ultrasound guidance and cases sent to our institution for a second opinion were excluded . the applicable surgical pathology follow - up was matched to the size and location of the index nodule . data processing was performed by employing the microsoft excel software ( microsoft , redmond , wa , usa ) . the characteristics of the case cohort is illustrated in table 1 and included 5079 thyroid fnas performed over the course of the 5-year study period ( 2008 - 2012 ) ; of these , 457 ( 9% ) cases were diagnosed as aus / flus . the mean age of the patients was 57 years at diagnosis with 353 ( 77% ) females and 104 ( 23% ) males . there was a slight increase in the age at diagnosis of aus / flus for males ( 62 years vs. 55 years for females ) ; 209 ( 45.7% ) nodules occurred in the left and 217 ( 47.5% ) in the right lobe . the size of the aspirated nodule was available in 439 of 457 ( 96.1% ) cases ; the average size was 2.54 cm with a slightly larger average size for males ( 2.72 cm vs. 2.48 cm for females ) . twenty - three ( 5% ) nodules measured < 1.0 cm , 297 ( 65% ) between 1.1 and 3.0 cm and 119 ( 26% ) > 3.1 cm . demographics of aus / flus cases all thyroid fnas were performed under ultrasound guidance ; 237 ( 51.9% ) nodules were aspirated by a team of endocrinologists and 220 ( 48.1% ) by a team of radiologists . the average nodule size was 2.42 cm for those aspirated by endocrinologists and 2.67 cm aspirated by radiologists . nodules measuring < 1.0 - 3.0 cm were more frequently aspirated by an endocrinologist than a radiologist ( 178 [ 55.6% ] vs. 142 [ 44.4% ] ) ; whereas , nodules over 3.1 cm were more frequently aspirated by a radiologist than an endocrinologist ( 62 [ 53% ] vs. 55 [ 47% ] ) . the utilization of aus / flus over the course of the 5 years ranged from 77 cases to 103 cases / year by five cytopathologists [ figure 1 ] . the ratio of aus / flus to all thyroid fna diagnoses ranged from 8.1% to 10.8% . there was substantial variation in the utilization of aus / flus among the five cytopathologists ( cp ) [ figure 2 ] . of 457 cases diagnosed as aus / flus , 268 ( 58.6% ) were interpreted by two ( cp1 : 153 [ 33.5% ] and cp2 : 115 [ 25.2% ] ) and the remaining 189 ( 41.4% ) by three cytopathologists ( cp3 : 67 [ 14.7% ] , cp4 : 80 [ 17.5% ] , and cp5 : 42 [ 9.2% ] ) . all aus / flus cases were further sub - classified into one of the aforementioned subcategories ( sc1-sc6 ) at the time of the diagnosis [ table 2 ] . of the 457 cases , 16 ( 3.5% ) were sub - classified as sc1 , 187 ( 40.9% ) as sc2 , 157 ( 34.5% ) as sc3 , 64 ( 14% ) as sc4 , 22 ( 4.8% ) as sc5 , and 11 ( 2.4% ) as sc6 . the average nodule size was 2.43 , 2.41 , 2.79 , 2.23 , 2.72 , and 2.75 cm for sc1-sc6 , respectively . the average age for cases sub - classified as sc1-sc6 was : 48 , 57 , 59 , 55 , 48 , and 58 years , respectively . overall number of atypia of undetermined significance / follicular lesion of undetermined significance ( aus / flus ) cases by year number of atypia of undetermined significance / follicular lesion of undetermined significance cases by cytopathologist / year . cp = cytopathologist characterization of surgical and repeat fna follow - up by aus / flus subcategory of the 457 aus / flus cases , repeat fna and/or surgical follow - up was available in 363 ( 79.4% ) cases [ table 2 ] . a repeat fna was performed in 285 of 457 cases ; 215 ( 75.4% ) were performed by an endocrinologist and 70 ( 24.6% ) by a radiologist . most cases had one repeat fna ( 266 cases [ 93.3% ] ) ; multiple repeat fnas were performed in only 19 ( 6.7% ) cases . the majority of repeat fnas ( 76.5% , 218 cases ) were performed in aus / flus cases sub - classified as sc2 and sc3 . there were 182 ( 39.8% ) cases with cytologic follow - up only ; 167 ( 91.8% ) received one repeat fna , 10 ( 5.5% ) received two and 5 ( 2.7% ) with three [ table 3 ] . overall , 18 ( 9.9% ) remained as aus / flus , while 158 ( 86.8% ) were re - classified with the majority being benign ( 142 cases ) and 6 ( 3.3% ) were unsatisfactory . histologic follow - up was available in 181 ( 39.6% ) cases ; 78 ( 43.1% ) after the initial aus / flus diagnosis , 99 ( 54.7% ) after the first repeat fna and 4 ( 2.2% ) after the second repeat fna [ table 4 ] . of the 78 cases that went directly to surgery , 58 ( 74.4% ) were benign ( 15 hyperplastic / goiter , 41 follicular adenoma / fn , 1 lymphocytic thyroiditis and 1 hyalinizing trabecular tumor ) and 20 ( 25.6% ) were malignant ( 19 papillary thyroid carcinomas and 1 oncocytic follicular carcinoma / hurthle cell carcinoma ) . of the 103 cases that received one or two follow - up repeat fnas , 63 ( 61.2% ) were diagnosed as benign ( 45 follicular adenoma / fn , 17 hyperplastic / goiter , and 1 cystic nodule ) and 40 ( 38.8% ) as malignant ( 37 papillary thyroid carcinomas and 3 follicular carcinoma ) on histologic follow - up . final diagnosis upon repeat fna follow - up of initial aus / flus . ( n=182 ) final diagnosis upon surgical follow - up of initial aus / flus . ( n=181 ) there were 60 malignant cases confirmed by surgical excision for an overall malignancy rate of 33.1% [ table 5 ] . the malignancy rates for each aus / flus subcategory ( sc ) were : sc1 - 25% , sc2 - 32% , sc3 - 25% , sc4 - 54% , sc5 - 50% , and sc6 - 17% . the size of the malignant nodule ranged from < 1.0 cm to > 4.1 cm ; 5 ( 8.3% ) nodules measured < 1.0 cm , 20 ( 33.3% ) between 1.1 and 2.0 cm , 17 ( 28.3% ) between 2.1 and 3.0 cm , 10 ( 16.7% ) between 3.1 and 4.0 cm and 8 ( 13.3% ) > 4.1 cm . almost half of the malignant cases were initially interpreted as aus / flus by one cytopathologist , cp1 : 25 cases ( 41.7% ) ; followed by cp2 : 13 cases ( 21.7% ) , cp4 : 12 cases ( 20% ) , cp3 : 5 cases ( 8.3% ) , and cp5 : 5 cases ( 8.3% ) . characterization of surgical pathology follow - up by aus / flus subcategory papillary thyroid carcinoma accounted for 93.3% ( 56 cases ) of the malignant cases with 81.7% ( 49 cases ) characterized as the follicular variant of papillary thyroid carcinoma . there were three cases of follicular carcinoma and one case of oncocytic follicular / hurthle cell carcinoma . of the 121 benign cases , 71.1% ( 86 cases ) were follicular adenomas / fns , 26.4% ( 32 cases ) were nodular goiter / hyperplastic nodules and 2.5% ( 3 cases ) were other benign / neoplastic nodules ( hyalinizing trabecular tumor , lymphocytic thyroiditis and benign cystic nodule ) . incidental microcarcinoma was identified in 79 ( 43.6% ) cases , the majority ( 60.8% , 48 cases ) occurring in cases with benign index nodules . the utilization of aus / flus over the course of the 5 years ranged from 77 cases to 103 cases / year by five cytopathologists [ figure 1 ] . the ratio of aus / flus to all thyroid fna diagnoses ranged from 8.1% to 10.8% . there was substantial variation in the utilization of aus / flus among the five cytopathologists ( cp ) [ figure 2 ] . of 457 cases diagnosed as aus / flus , 268 ( 58.6% ) were interpreted by two ( cp1 : 153 [ 33.5% ] and cp2 : 115 [ 25.2% ] ) and the remaining 189 ( 41.4% ) by three cytopathologists ( cp3 : 67 [ 14.7% ] , cp4 : 80 [ 17.5% ] , and cp5 : 42 [ 9.2% ] ) . all aus / flus cases were further sub - classified into one of the aforementioned subcategories ( sc1-sc6 ) at the time of the diagnosis [ table 2 ] . of the 457 cases , 16 ( 3.5% ) were sub - classified as sc1 , 187 ( 40.9% ) as sc2 , 157 ( 34.5% ) as sc3 , 64 ( 14% ) as sc4 , 22 ( 4.8% ) as sc5 , and 11 ( 2.4% ) as sc6 . the average nodule size was 2.43 , 2.41 , 2.79 , 2.23 , 2.72 , and 2.75 cm for sc1-sc6 , respectively . the average age for cases sub - classified as sc1-sc6 was : 48 , 57 , 59 , 55 , 48 , and 58 years , respectively . overall number of atypia of undetermined significance / follicular lesion of undetermined significance ( aus / flus ) cases by year number of atypia of undetermined significance / follicular lesion of undetermined significance cases by cytopathologist / year . cp = cytopathologist characterization of surgical and repeat fna follow - up by aus / flus subcategory of the 457 aus / flus cases , repeat fna and/or surgical follow - up was available in 363 ( 79.4% ) cases [ table 2 ] . a repeat fna was performed in 285 of 457 cases ; 215 ( 75.4% ) were performed by an endocrinologist and 70 ( 24.6% ) by a radiologist . most cases had one repeat fna ( 266 cases [ 93.3% ] ) ; multiple repeat fnas were performed in only 19 ( 6.7% ) cases . the majority of repeat fnas ( 76.5% , 218 cases ) were performed in aus / flus cases sub - classified as sc2 and sc3 . there were 182 ( 39.8% ) cases with cytologic follow - up only ; 167 ( 91.8% ) received one repeat fna , 10 ( 5.5% ) received two and 5 ( 2.7% ) with three [ table 3 ] . overall , 18 ( 9.9% ) remained as aus / flus , while 158 ( 86.8% ) were re - classified with the majority being benign ( 142 cases ) and 6 ( 3.3% ) were unsatisfactory . histologic follow - up was available in 181 ( 39.6% ) cases ; 78 ( 43.1% ) after the initial aus / flus diagnosis , 99 ( 54.7% ) after the first repeat fna and 4 ( 2.2% ) after the second repeat fna [ table 4 ] . of the 78 cases that went directly to surgery , 58 ( 74.4% ) were benign ( 15 hyperplastic / goiter , 41 follicular adenoma / fn , 1 lymphocytic thyroiditis and 1 hyalinizing trabecular tumor ) and 20 ( 25.6% ) were malignant ( 19 papillary thyroid carcinomas and 1 oncocytic follicular carcinoma / hurthle cell carcinoma ) . of the 103 cases that received one or two follow - up repeat fnas , 63 ( 61.2% ) were diagnosed as benign ( 45 follicular adenoma / fn , 17 hyperplastic / goiter , and 1 cystic nodule ) and 40 ( 38.8% ) as malignant ( 37 papillary thyroid carcinomas and 3 follicular carcinoma ) on histologic follow - up . ( n=182 ) final diagnosis upon surgical follow - up of initial aus / flus . ( n=181 ) there were 60 malignant cases confirmed by surgical excision for an overall malignancy rate of 33.1% [ table 5 ] . the malignancy rates for each aus / flus subcategory ( sc ) were : sc1 - 25% , sc2 - 32% , sc3 - 25% , sc4 - 54% , sc5 - 50% , and sc6 - 17% . the size of the malignant nodule ranged from < 1.0 cm to > 4.1 cm ; 5 ( 8.3% ) nodules measured < 1.0 cm , 20 ( 33.3% ) between 1.1 and 2.0 cm , 17 ( 28.3% ) between 2.1 and 3.0 cm , 10 ( 16.7% ) between 3.1 and 4.0 cm and 8 ( 13.3% ) > 4.1 cm . almost half of the malignant cases were initially interpreted as aus / flus by one cytopathologist , cp1 : 25 cases ( 41.7% ) ; followed by cp2 : 13 cases ( 21.7% ) , cp4 : 12 cases ( 20% ) , cp3 : 5 cases ( 8.3% ) , and cp5 : 5 cases ( 8.3% ) . characterization of surgical pathology follow - up by aus / flus subcategory papillary thyroid carcinoma accounted for 93.3% ( 56 cases ) of the malignant cases with 81.7% ( 49 cases ) characterized as the follicular variant of papillary thyroid carcinoma . there were three cases of follicular carcinoma and one case of oncocytic follicular / hurthle cell carcinoma . of the 121 benign cases , 71.1% ( 86 cases ) were follicular adenomas / fns , 26.4% ( 32 cases ) were nodular goiter / hyperplastic nodules and 2.5% ( 3 cases ) were other benign / neoplastic nodules ( hyalinizing trabecular tumor , lymphocytic thyroiditis and benign cystic nodule ) . incidental microcarcinoma was identified in 79 ( 43.6% ) cases , the majority ( 60.8% , 48 cases ) occurring in cases with benign index nodules . the bethesda thyroid fna classification was developed with intent to provide a reporting system for thyroid fna results that was uniform between laboratories and effectively corresponded cytologic interpretations for proper patient follow - up and management . the aus / flus category of this scheme was intended to categorize the subset of cases that could not be definitively classified as benign , suspicious or malignant . it was well understood that this category would represent a heterogeneous set of cases with anticipated usage of 3 - 6% and overall risk of malignancy of 5 - 15% . as more laboratories adopted tbsrtc and shared their experiences , variable usage and malignancy rates surpassing tbsrtc guidelines have been reported in the literature . in the current study , there was a steady increase in the number of thyroid fna cases ( 950 - 1174 cases ) over the 5-year study period ( 2008 - 2012 ) , yet the usage of aus / flus remained steady with an average of 91 cases / year . at our institution , aus / flus cases were further sub - classified based on the cytomorphologic findings and suspicion for or against a neoplastic process . the surgical pathology follow - up showed different malignant outcomes for each of the subcategories . the highest malignancy rates ( 53.8% and 50% ) were encountered in aus / flus cases in which sub - classifier notes focal nuclear overlapping and crowding in a background of chronic lymphocytic thyroiditis and few cells with nuclear features suspicious for papillary thyroid carcinoma were utilized ( sc4 and sc5 ) . our findings are similar to what has been reported by other authors . a study by vanderlaan et al . reported a malignancy rate close to 30% in a subset of cases which exhibit cytomorphologic features suggestive but not diagnostic of papillary thyroid carcinoma as compared to 16% in cases with only architectural atypia . a similar study by renshaw , which pre - dated tbsrtc , also found that cases diagnosed as interestingly , a high rate of malignancy ( 53.8% ) was also noted in aus / flus cases with focal nuclear overlapping and crowding in a background of chronic lymphocytic thyroiditis ( sc4 ) . it is well - known that chronic lymphocytic thyroiditis may obscure subtle features of ptc ( peri - nuclear halos , nuclear grooves , nuclear enlargement and crowding ) . on the other hand , it is also a common practice not to render a suspicion of papillary carcinoma diagnosis based on subtle nuclear atypia such as nuclear chromatin clearing in fna specimens with chronic lymphocytic thyroiditis to avoid over diagnosis of reactive changes . in 344 ( 75.3% ) cases , focal nuclear overlapping and crowding was utilized based on adequate ( sc2 ) or limited cellularity ( sc3 ) with malignancy rates of 32.3% and 25% , respectively . together , these are higher than those reported in the literature for aus / flus cases with architectural atypia . our findings are in agreement with other studies that conclude sub - classifying aus / flus can impact patient follow - up regimens and provide guidance toward a repeat fna or surgery , especially in multi - disciplinary clinical settings . according to tbsrtc , the recommended management for thyroid nodules with an initial aus / flus diagnosis is clinical correlation and repeat fna . this is in contrast to guidelines from the american association of clinical endocrinologists / associazione medici endocrinologi / european thyroid association task force which favors surgery over observation and repeat fna . it has been shown by many studies that a definite diagnosis can be rendered in more than half of cases diagnosed as recent studies have reported variable rates for repeat fna ranging from 7.9% to 46.4% in aus / flus cases . in our study , 285 cases ( 62.4% ) of aus / flus cases underwent repeat fna ; 182 cases received repeat fna with no further surgical follow - up . a definitive diagnosis was rendered in 158 ( 86.8% ) cases ; the majority ( 142 cases ) was benign , 13 were fn / sfn and 3 were suspicious for malignancy . there were 18 cases that remained as aus / flus after the first repeat fna and 6 were unsatisfactory . surgery is indicated for cases with successive aus / flus results and has a higher rate of malignancy . our results support that a repeat fna offers a better selection of patients for further management . for cases with surgical follow - up , surgery was performed in 103 ( 56.9% ) cases with a repeat fna as compared to 78 ( 43.1% ) cases without repeat fna . the malignancy rate was 38.8% for cases with repeat fna versus 25.6% for cases that went directly to surgery without a repeat fna . although beyond the scope of this study , repeat fna may not be indicated in some cases based on suspicious clinical history or ultrasound findings . some authors have suggested eliminating the aus / flus category on the basis that the positive predictive value for most aus / flus cases is similar to the fn category and a repeat fna would not aid in patient management . in our study , even though the overall malignancy rate ( 33.1% ) is similar to that reported for fn / sfn ( 15 - 30% ) , cases where surgery was performed after the initial aus / flus diagnosis were more likely to be benign ( 74.4% ) as compared to those with repeat fna ( 61.2% ) . thus , a repeat fna , for patients without further clinical suspicions , will spare patients from unnecessary surgery and its complications . therefore , we are of the belief that these comparable malignancy rates are a product of better clinical management and selection of patients diagnosed as aus / flus for surgery after a repeat fna . in the current study , 181 thyroid nodules classified as aus / flus that underwent surgical excision were equally divided as being aspirated under ultrasound - guidance by a team of radiologists and endocrinologists . interestingly , the malignancy rate for nodules aspirated by radiologists ( 40.7% ) was almost twice that of endocrinologists ( 25.6% ) . we believe that these results are indicative of operator experience , which may affect obtaining a representative sample from suspicious areas of the nodule ( hypoechoic , solid and micro - calcifications ) for cytologic interpretation . we have shown that the diagnosis of aus / flus is dependent on factors that are occurring at both pre - analytical and analytical phases of fna evaluation of the thyroid nodule . these include experience of the operator obtaining the sample , variable diagnostic thresholds , and experience of the cytopathologist . based on our study , even though the malignancy rate of aus / flus cases is similar to those reported for cases diagnosed as fn / sfn , we are of the belief that these comparable malignancy rates are a product of better clinical management and selection of patients diagnosed as aus / flus for surgery after a repeat fna . all authors of this article declare that we qualify for authorship as defined by the icjme . each author has participated sufficiently in the work and takes public responsibility for appropriate portions of the content of the article . lqw , val and zwb are responsible for planning and revising the article critically for important intellectual content . zwb is responsible for conceiving and coordinating the whole work , execution and analysis of the work and final approval of the version to be published . all authors take the responsibility of maintaining relevant documentation of records and other data used in this study as per the institutional policy . to ensure the integrity and highest quality of cytojournal publications , the review process of this manuscript was conducted under a double - blind model.(authors are blinded for reviewers and vice versa ) through automatic online system .

background : the overall malignancy rate for the thyroid fine - needle aspiration ( fna ) diagnosed as atypia of undetermined significance / follicular lesion of undetermined significance ( aus / flus ) ranges from 5% to 30% . in this study , we present our institutional experience with thyroid nodules diagnosed as aus / flus and further stratified into subcategories . in addition , we also assessed the significance of various clinicopathologic factors that may influence aus / flus diagnoses and their outcomes.design:a search of our laboratory information system was performed to identify all in - house thyroid fna cases diagnosed as aus / flus from 2008 to 2012 . the data were collected and characterized by patient demographic information , cytopathology diagnosis with sub - classifiers and follow-up.results:the case cohort included 457 cases diagnosed as aus / flus . these were further sub - classified into one of six subcategories depending on the cytomorphologic findings and suspicion for or against a neoplastic process . of the 457 cases , repeat fna and/or surgical follow - up was available in 363 cases . there were 182 ( 39.8% ) cases with cytologic follow - up only ; 18 ( 9.9% ) remained as aus / flus , while 158 ( 86.8% ) were re - classified with the majority being benign ( 142 cases ) . histologic follow - up was available in 181 ( 39.6% ) cases . there were 60 malignant cases confirmed by surgical excision , with an overall malignancy rate of 33.1% . the malignancy rate was 38.8% for cases with a repeat fna versus 25.6% for cases that went directly to surgery without a repeat fna . papillary thyroid carcinoma accounted for 93.3% ( 56 cases ) of the malignant cases.conclusion:based on our study , even though the malignancy rate of aus / flus cases is similar to those reported for cases diagnosed as follicular neoplasm / suspicious for follicular neoplasm , we are of the belief that these comparable malignancy rates are a product of better clinical management and selection of patients diagnosed as aus / flus for surgery after a repeat fna .

INTRODUCTION MATERIALS AND METHODS RESULTS Atypia of undetermined significance/follicular lesion of undetermined significance diagnosis and sub-classification Surgical and repeat fine-needle aspiration follow-up DISCUSSION CONCLUSION COMPETING INTERESTS STATEMENT BY ALL AUTHORS AUTHORSHIP STATEMENT BY ALL AUTHORS ETHICS STATEMENT BY ALL AUTHORS EDITORIAL/PEER-REVIEW STATEMENT

atypia of undetermined significance / follicular lesion of undetermined significance ( aus / flus ) is a heterogeneous diagnostic category of the bethesda system for reporting thyroid cytopathology ( tbsrtc ) . since these aforementioned features are entirely based on the observer , there exists a low inter - observer agreement and poor reproducibility for cases diagnosed as aus / flus . the recommended management for nodules with an initial aus / flus diagnosis is clinical correlation and repeat fine - needle aspiration ( fna ) at an appropriate interval . in the post - bethesda thyroid fna classification era , the risk of malignancy has been reported to a range between 5% and 15% in patients with a diagnosis of aus / flus . , a majority of initial aus / flus cases received follow - up surgery rather than the recommended repeat fna . renshaw , in concordance with other studies , noted that thyroid fna cases subcategorized as aus / flus not otherwise specified and aus / flus - rule out follicular neoplasm had a similar malignancy rate to cases diagnosed as follicular neoplasm ( fn)/suspicious for follicular neoplasm ( sfn ) . therefore , even though these two aus / flus subgroups may theoretically have similar malignancy rates to fn / sfn , the case management may entirely be dictated by which tbsrtc category they are placed into ( repeat fna for aus / flus vs. surgical excision for fn / sfn ) . these studies have shown that even though the overall risk of malignancy is relatively low , sub - classifying aus / flus with focal abnormal cytomorphological features may help in better management of patients . in this study , we present our institutional experience with thyroid nodules diagnosed as aus / flus and further stratified into subcategories . in addition , we also assessed the significance of various clinicopathologic factors that may influence aus / flus diagnosis and their outcomes . a retrospective search of our laboratory information system was performed to identify all in - house thyroid fna cases that were performed under ultrasound guidance and diagnosed as aus / flus from 2008 to 2012 . a rapid on - site evaluation ( rose ) by staff cytopathologists was performed in nearly all in - house thyroid fnas . aus / flus cases were further sub - classified into one of the following subcategories ( sc ) : sc1 - favor benign , however , a fn could not be excluded due to increased cellularity ; sc2 - specimens with focal nuclear overlapping and crowding ; sc3 - scant specimens with focal nuclear overlapping and crowding ; sc4 - specimens with focal nuclear overlapping and crowding in a background of lymphocytic thyroiditis ; sc5 - few cells with features suspicious for papillary thyroid cancer ( ptc ) ; and sc6 - specimens in which fn can not be excluded ( with miscellaneous morphologic classifiers ) . data points collected for this study included : patient demographics , size of the thyroid nodule sampled , aspirator , pathologist rendering the final diagnosis , and applicable follow - up . the data were collected and characterized by patient demographic information , cytopathology diagnosis with sub - classifiers and follow - up . only cases that were diagnosed as aus / flus were included in the study , however , patients younger than 18 years , nodules aspirated without ultrasound guidance and cases sent to our institution for a second opinion were excluded . the characteristics of the case cohort is illustrated in table 1 and included 5079 thyroid fnas performed over the course of the 5-year study period ( 2008 - 2012 ) ; of these , 457 ( 9% ) cases were diagnosed as aus / flus . the size of the aspirated nodule was available in 439 of 457 ( 96.1% ) cases ; the average size was 2.54 cm with a slightly larger average size for males ( 2.72 cm vs. 2.48 cm for females ) . demographics of aus / flus cases all thyroid fnas were performed under ultrasound guidance ; 237 ( 51.9% ) nodules were aspirated by a team of endocrinologists and 220 ( 48.1% ) by a team of radiologists . of 457 cases diagnosed as aus / flus , 268 ( 58.6% ) were interpreted by two ( cp1 : 153 [ 33.5% ] and cp2 : 115 [ 25.2% ] ) and the remaining 189 ( 41.4% ) by three cytopathologists ( cp3 : 67 [ 14.7% ] , cp4 : 80 [ 17.5% ] , and cp5 : 42 [ 9.2% ] ) . all aus / flus cases were further sub - classified into one of the aforementioned subcategories ( sc1-sc6 ) at the time of the diagnosis [ table 2 ] . of the 457 cases , 16 ( 3.5% ) were sub - classified as sc1 , 187 ( 40.9% ) as sc2 , 157 ( 34.5% ) as sc3 , 64 ( 14% ) as sc4 , 22 ( 4.8% ) as sc5 , and 11 ( 2.4% ) as sc6 . overall number of atypia of undetermined significance / follicular lesion of undetermined significance ( aus / flus ) cases by year number of atypia of undetermined significance / follicular lesion of undetermined significance cases by cytopathologist / year . cp = cytopathologist characterization of surgical and repeat fna follow - up by aus / flus subcategory of the 457 aus / flus cases , repeat fna and/or surgical follow - up was available in 363 ( 79.4% ) cases [ table 2 ] . a repeat fna was performed in 285 of 457 cases ; 215 ( 75.4% ) were performed by an endocrinologist and 70 ( 24.6% ) by a radiologist . the majority of repeat fnas ( 76.5% , 218 cases ) were performed in aus / flus cases sub - classified as sc2 and sc3 . there were 182 ( 39.8% ) cases with cytologic follow - up only ; 167 ( 91.8% ) received one repeat fna , 10 ( 5.5% ) received two and 5 ( 2.7% ) with three [ table 3 ] . overall , 18 ( 9.9% ) remained as aus / flus , while 158 ( 86.8% ) were re - classified with the majority being benign ( 142 cases ) and 6 ( 3.3% ) were unsatisfactory . histologic follow - up was available in 181 ( 39.6% ) cases ; 78 ( 43.1% ) after the initial aus / flus diagnosis , 99 ( 54.7% ) after the first repeat fna and 4 ( 2.2% ) after the second repeat fna [ table 4 ] . of the 78 cases that went directly to surgery , 58 ( 74.4% ) were benign ( 15 hyperplastic / goiter , 41 follicular adenoma / fn , 1 lymphocytic thyroiditis and 1 hyalinizing trabecular tumor ) and 20 ( 25.6% ) were malignant ( 19 papillary thyroid carcinomas and 1 oncocytic follicular carcinoma / hurthle cell carcinoma ) . of the 103 cases that received one or two follow - up repeat fnas , 63 ( 61.2% ) were diagnosed as benign ( 45 follicular adenoma / fn , 17 hyperplastic / goiter , and 1 cystic nodule ) and 40 ( 38.8% ) as malignant ( 37 papillary thyroid carcinomas and 3 follicular carcinoma ) on histologic follow - up . final diagnosis upon repeat fna follow - up of initial aus / flus . ( n=182 ) final diagnosis upon surgical follow - up of initial aus / flus . ( n=181 ) there were 60 malignant cases confirmed by surgical excision for an overall malignancy rate of 33.1% [ table 5 ] . the malignancy rates for each aus / flus subcategory ( sc ) were : sc1 - 25% , sc2 - 32% , sc3 - 25% , sc4 - 54% , sc5 - 50% , and sc6 - 17% . almost half of the malignant cases were initially interpreted as aus / flus by one cytopathologist , cp1 : 25 cases ( 41.7% ) ; followed by cp2 : 13 cases ( 21.7% ) , cp4 : 12 cases ( 20% ) , cp3 : 5 cases ( 8.3% ) , and cp5 : 5 cases ( 8.3% ) . characterization of surgical pathology follow - up by aus / flus subcategory papillary thyroid carcinoma accounted for 93.3% ( 56 cases ) of the malignant cases with 81.7% ( 49 cases ) characterized as the follicular variant of papillary thyroid carcinoma . of the 121 benign cases , 71.1% ( 86 cases ) were follicular adenomas / fns , 26.4% ( 32 cases ) were nodular goiter / hyperplastic nodules and 2.5% ( 3 cases ) were other benign / neoplastic nodules ( hyalinizing trabecular tumor , lymphocytic thyroiditis and benign cystic nodule ) . incidental microcarcinoma was identified in 79 ( 43.6% ) cases , the majority ( 60.8% , 48 cases ) occurring in cases with benign index nodules . of 457 cases diagnosed as aus / flus , 268 ( 58.6% ) were interpreted by two ( cp1 : 153 [ 33.5% ] and cp2 : 115 [ 25.2% ] ) and the remaining 189 ( 41.4% ) by three cytopathologists ( cp3 : 67 [ 14.7% ] , cp4 : 80 [ 17.5% ] , and cp5 : 42 [ 9.2% ] ) . all aus / flus cases were further sub - classified into one of the aforementioned subcategories ( sc1-sc6 ) at the time of the diagnosis [ table 2 ] . of the 457 cases , 16 ( 3.5% ) were sub - classified as sc1 , 187 ( 40.9% ) as sc2 , 157 ( 34.5% ) as sc3 , 64 ( 14% ) as sc4 , 22 ( 4.8% ) as sc5 , and 11 ( 2.4% ) as sc6 . overall number of atypia of undetermined significance / follicular lesion of undetermined significance ( aus / flus ) cases by year number of atypia of undetermined significance / follicular lesion of undetermined significance cases by cytopathologist / year . cp = cytopathologist characterization of surgical and repeat fna follow - up by aus / flus subcategory of the 457 aus / flus cases , repeat fna and/or surgical follow - up was available in 363 ( 79.4% ) cases [ table 2 ] . a repeat fna was performed in 285 of 457 cases ; 215 ( 75.4% ) were performed by an endocrinologist and 70 ( 24.6% ) by a radiologist . the majority of repeat fnas ( 76.5% , 218 cases ) were performed in aus / flus cases sub - classified as sc2 and sc3 . there were 182 ( 39.8% ) cases with cytologic follow - up only ; 167 ( 91.8% ) received one repeat fna , 10 ( 5.5% ) received two and 5 ( 2.7% ) with three [ table 3 ] . overall , 18 ( 9.9% ) remained as aus / flus , while 158 ( 86.8% ) were re - classified with the majority being benign ( 142 cases ) and 6 ( 3.3% ) were unsatisfactory . histologic follow - up was available in 181 ( 39.6% ) cases ; 78 ( 43.1% ) after the initial aus / flus diagnosis , 99 ( 54.7% ) after the first repeat fna and 4 ( 2.2% ) after the second repeat fna [ table 4 ] . of the 78 cases that went directly to surgery , 58 ( 74.4% ) were benign ( 15 hyperplastic / goiter , 41 follicular adenoma / fn , 1 lymphocytic thyroiditis and 1 hyalinizing trabecular tumor ) and 20 ( 25.6% ) were malignant ( 19 papillary thyroid carcinomas and 1 oncocytic follicular carcinoma / hurthle cell carcinoma ) . of the 103 cases that received one or two follow - up repeat fnas , 63 ( 61.2% ) were diagnosed as benign ( 45 follicular adenoma / fn , 17 hyperplastic / goiter , and 1 cystic nodule ) and 40 ( 38.8% ) as malignant ( 37 papillary thyroid carcinomas and 3 follicular carcinoma ) on histologic follow - up . ( n=182 ) final diagnosis upon surgical follow - up of initial aus / flus . ( n=181 ) there were 60 malignant cases confirmed by surgical excision for an overall malignancy rate of 33.1% [ table 5 ] . the malignancy rates for each aus / flus subcategory ( sc ) were : sc1 - 25% , sc2 - 32% , sc3 - 25% , sc4 - 54% , sc5 - 50% , and sc6 - 17% . almost half of the malignant cases were initially interpreted as aus / flus by one cytopathologist , cp1 : 25 cases ( 41.7% ) ; followed by cp2 : 13 cases ( 21.7% ) , cp4 : 12 cases ( 20% ) , cp3 : 5 cases ( 8.3% ) , and cp5 : 5 cases ( 8.3% ) . characterization of surgical pathology follow - up by aus / flus subcategory papillary thyroid carcinoma accounted for 93.3% ( 56 cases ) of the malignant cases with 81.7% ( 49 cases ) characterized as the follicular variant of papillary thyroid carcinoma . of the 121 benign cases , 71.1% ( 86 cases ) were follicular adenomas / fns , 26.4% ( 32 cases ) were nodular goiter / hyperplastic nodules and 2.5% ( 3 cases ) were other benign / neoplastic nodules ( hyalinizing trabecular tumor , lymphocytic thyroiditis and benign cystic nodule ) . incidental microcarcinoma was identified in 79 ( 43.6% ) cases , the majority ( 60.8% , 48 cases ) occurring in cases with benign index nodules . in the current study , there was a steady increase in the number of thyroid fna cases ( 950 - 1174 cases ) over the 5-year study period ( 2008 - 2012 ) , yet the usage of aus / flus remained steady with an average of 91 cases / year . at our institution , aus / flus cases were further sub - classified based on the cytomorphologic findings and suspicion for or against a neoplastic process . the highest malignancy rates ( 53.8% and 50% ) were encountered in aus / flus cases in which sub - classifier notes focal nuclear overlapping and crowding in a background of chronic lymphocytic thyroiditis and few cells with nuclear features suspicious for papillary thyroid carcinoma were utilized ( sc4 and sc5 ) . reported a malignancy rate close to 30% in a subset of cases which exhibit cytomorphologic features suggestive but not diagnostic of papillary thyroid carcinoma as compared to 16% in cases with only architectural atypia . a similar study by renshaw , which pre - dated tbsrtc , also found that cases diagnosed as interestingly , a high rate of malignancy ( 53.8% ) was also noted in aus / flus cases with focal nuclear overlapping and crowding in a background of chronic lymphocytic thyroiditis ( sc4 ) . our findings are in agreement with other studies that conclude sub - classifying aus / flus can impact patient follow - up regimens and provide guidance toward a repeat fna or surgery , especially in multi - disciplinary clinical settings . according to tbsrtc , the recommended management for thyroid nodules with an initial aus / flus diagnosis is clinical correlation and repeat fna . it has been shown by many studies that a definite diagnosis can be rendered in more than half of cases diagnosed as recent studies have reported variable rates for repeat fna ranging from 7.9% to 46.4% in aus / flus cases . in our study , 285 cases ( 62.4% ) of aus / flus cases underwent repeat fna ; 182 cases received repeat fna with no further surgical follow - up . a definitive diagnosis was rendered in 158 ( 86.8% ) cases ; the majority ( 142 cases ) was benign , 13 were fn / sfn and 3 were suspicious for malignancy . there were 18 cases that remained as aus / flus after the first repeat fna and 6 were unsatisfactory . surgery is indicated for cases with successive aus / flus results and has a higher rate of malignancy . for cases with surgical follow - up , surgery was performed in 103 ( 56.9% ) cases with a repeat fna as compared to 78 ( 43.1% ) cases without repeat fna . the malignancy rate was 38.8% for cases with repeat fna versus 25.6% for cases that went directly to surgery without a repeat fna . some authors have suggested eliminating the aus / flus category on the basis that the positive predictive value for most aus / flus cases is similar to the fn category and a repeat fna would not aid in patient management . in our study , even though the overall malignancy rate ( 33.1% ) is similar to that reported for fn / sfn ( 15 - 30% ) , cases where surgery was performed after the initial aus / flus diagnosis were more likely to be benign ( 74.4% ) as compared to those with repeat fna ( 61.2% ) . therefore , we are of the belief that these comparable malignancy rates are a product of better clinical management and selection of patients diagnosed as aus / flus for surgery after a repeat fna . in the current study , 181 thyroid nodules classified as aus / flus that underwent surgical excision were equally divided as being aspirated under ultrasound - guidance by a team of radiologists and endocrinologists . interestingly , the malignancy rate for nodules aspirated by radiologists ( 40.7% ) was almost twice that of endocrinologists ( 25.6% ) . we have shown that the diagnosis of aus / flus is dependent on factors that are occurring at both pre - analytical and analytical phases of fna evaluation of the thyroid nodule . based on our study , even though the malignancy rate of aus / flus cases is similar to those reported for cases diagnosed as fn / sfn , we are of the belief that these comparable malignancy rates are a product of better clinical management and selection of patients diagnosed as aus / flus for surgery after a repeat fna .

the molecular signature of chronic myeloid leukemia ( cml ) and philadelphia - positive ( ph ) acute lymphoblastic leukemia ( all ) is the bcr - abl hybrid gene , originating from a reciprocal t(9;22 ) chromosomal translocation on the 22q- derivative , commonly referred to as the philadelphia chromosome . the resulting fusion protein the disease begins with an indolent chronic phase ( cp ) marked by the expansion of myeloid cells with normal differentiation , and then inexorably proceeds to advanced phases , i.e. , accelerated phase ( ap ) and the terminal blastic phase ( bp ) . imatinib ( gleevec , glivec ; sti571 ) , a relatively selective tyrosine kinase inhibitor that blocks the catalytic activity of bcr - abl , is now the first - line treatment for all newly diagnosed cml patients . despite excellent clinical results , there is still a need to improve therapy for patients with cml and ph all . more than 80% of newly diagnosed cml patients treated with imatinib in cp achieve a complete cytogenetic remission , as typified by the absence of the philadelphia chromosome at the examination of 20 bone marrow meta - phases . however , residual bcr - abl transcripts persist in the majority of these patients , as assessed by sensitive assays such as nested reverse transcription - polymerase chain reaction , and represent the potential pool from which disease recurrence may originate . while responses in cml in cp patients have been shown to last more than five years , most responding patients with ap- and bp - cml , as well as those with ph all , relapse early despite continued therapy . we and other authors have reported that approximately half cml patients have evidence of point mutations within the abl kinase domain at the time of resistance to imatinib . mutations target critical contact points between imatinib and bcr - abl or , more often , induce a conformation to which imatinib is unable to bind . in the remaining patients , the reasons for imatinib resistance have to be traced to bcr - abl gene amplification or overexpression , clonal cytogenetic evolution , or altered levels of transport molecules responsible for imatinib influx and efflux ( abc transporters , hoct1 ) . abl mutations are at present the most extensively investigated and best characterized mechanism of resistance to imatinib . so far , at least 90 different point mutations have been isolated from relapsed cml patients who are resistant to imatinib . the clinical and pathogenetic impact of mutations varies according to their different degree of residual sensitivity to imatinib . indeed , while certain bcr - abl mutations retain in vitro sensitivity to imatinib at physiologically relevant concentrations and therefore may not be clinically meaningful , others require increased doses of imatinib , and some confer a highly resistant phenotype ( table 1 ) . table 1comparison between imatinib , dasatinib and nilotinib ic50 values obtained in ba / f3 cellular proliferation assays . adapted from cellular proliferationabl variantimatinibnilotinibdasatinibic50 ( nm)fold - changeic50 ( nm)fold - changeic50 ( nm)fold - changewild - type26011310.81m244v2,00083831.32g250e1,35054841.82q252h1,32557053.44y253f3,47513125101.42y253h>6,400>25450351.32e255k5,20020200155.67e255v>6,400>25430331114f311l48022321.32t315i>6,400>25>2000>154>200>250f317l1,05045047.49m351t8803151.21.11.4f359v1,8257175132.23l387m1,000449423h396p85034130.60.8h396r1,75074131.32 an amino acid substitution occurring at the so - called gatekeeper residue , i.e. threonine 315 , has attracted particular interest since it confers a high level of resistance not only to imatinib therapy but also to all of the newly developed tyrosine kinase inhibitors entered in clinical trials . co - crystal structure analysis indicates that , on binding , the hydroxyl group of threonine 315 forms a crucial hydrogen bond with imatinib . moreover , the side chain of threonine also sterically controls the binding of the inhibitor to hydrophobic regions adjacent to the atp - binding site . in 1015% of imatinib - resistant patients , especially those in more advanced phases of disease , the t315i abrogates imatinib binding because it disrupts the above mentioned hydrogen bond and introduces a bulkier isoleucine side chain into the gatekeeper position . however , this explanation is not the most up - to - date . in fact , as recently demonstrated , the t315i resistance to imatinib mainly results from the breakdown of interactions between imatinib and both e286 and m290 . as a result , biochemical and cellular ic50 values of imatinib for the t315i - bcr - abl have been shown to be > 6400 times higher than those of wild - type bcr - abl ( table 1 ) . some authors have suggested that the t315i is associated with highly aggressive disease phenotype and poor outcome if no timely therapeutic reassessment is made . however , the effects of the t315i mutation on kinase activity in vitro and transforming efficiency of bcr - abl in vitro and in vivo have been very recently investigated , suggesting that in the absence of imatinib , there is neither increased kinase activity nor any growth advantage for cells carrying t315i - bcr - abl as compared to wild - type bcr - abl . to counteract the problem of resistance due to point mutations , several second - generation inhibitors have been synthesized and tested in pre - clinical assays : nilotinib ( amn107 ) , dasatinib ( bms-354825 ) , bosutinib , vx-680 , ap23464 , bafetinib , pd166326 , pd180970 and pd173955 , and on012380 . two of them are currently being evaluated in phase ii clinical trials the dual - specificity src / abl inhibitor dasatinib and the imatinib derivative nilotinib . it has been shown to be 300 times more potent than imatinib in bcr - abl inhibition assays . excellent results in terms of hematologic and cytogenetic response in cml and ph all patients resistant to imatinib have been reported after dasatinib administration . pre - clinical studies have demonstrated that dasatinib is active against at least fourteen imatinib - resistant bcr - abl mutants ( m244v , g250e , q252h / r , y253f / h , e255k / v , f317l , m351 t , e355 g , f359v , h396r , f486s ) . the only imatinib - resistant bcr - abl isoform that was clearly insensitive to dasatinib was the t315i mutant , which retained kinase activity even in the presence of micromolar concentrations of the compound ( table 1 ) . accordingly , imatinib - resistant patients harboring the t315i mutation have been shown not to benefit from dasatinib in the recent phase i trial . nilotinib is a close relative of imatinib with more than 20-fold improved affinity for wild - type bcr - abl . it is highly efficacious in patients with imatinib - resistant ph cml . in vitro experiment with cell lines transformed with mutated forms of bcr - abl showed ic50 proliferation inhibition for most mutations with the exception of the t315i , which remains refractory to nilotinib ( table 1 ) . accordingly , clinical responses have been observed in patients with various imatinib - resistant bcr - abl mutations but not in patients positive for the t315i in the recent phase i trial . despite the pressing need for a clinically effective t315i - bcr - abl inhibitor , a potential pitfall might be the tendency to screen initially for abl kinase inhibition rather than for t315i - specific inhibition . for example , on012380 , a putative substrate - competitive inhibitor , exhibited low nanomolar activity against imatinib - resistant bcr - abl mutants , including the t315i , in biochemical and cellular assays . between these new promising drugs , vx-680 and pha-739358 , two aurora kinase a , b and c inhibitors , have a leading place . the aurora kinases are a family of serine / threonine kinases involved in many cellular functions , including progression through mitosis , by regulating spindle formation , chromosome segregation and cytokinesis . the overexpression of aurora kinases has been reported in many human solid tumors , leading to defects in centrosome function , aberrant spindle assembly , misalignment of chromosomes , abnormal cytokinesis and genetic instability , determining the activation of oncogenic pathways . many authors reported an aberrant expression of the aurora a and b kinases also in leukemia cells , suggesting a potential role of these molecular targets in the treatment of cml and all . aurora kinase function is mediated by the phosphorylation of several substrates that have important roles in cell division , such as proteins survivin , cenp - a and serine 10 on histone h3 . they have a c terminal domain that is responsible for regulation of the protein levels via proteasomal degradation ; a highly conserved catalytic domain ; and a short n - terminal domain that varies in length between the kinases and contributes to the differing locations of the kinases within cells . ( figure 1 ) . figure 1schematic representation of domain organization of aurora kinases . aurora kinases have three domains : the n - terminal and c - terminal domains which contain most of the aurora 's regulatory motifs and the catalytic domain in the central region . the alignment of auroras a and b allows the identification of one distantly conserved ken motif , spanning 1118 residues . the ken motif acts as a cdh dependant anaphase - promoting complex recognition signal . aurora kinases have three domains : the n - terminal and c - terminal domains which contain most of the aurora 's regulatory motifs and the catalytic domain in the central region . the alignment of auroras a and b allows the identification of one distantly conserved ken motif , spanning 1118 residues . the ken motif acts as a cdh dependant anaphase - promoting complex recognition signal . the aurora a isotype ( also known as aurora , aurora-2 , aik , air-1 , airk1 , ayk1 , btak , eg2 , mmiak1 and stk15 ) is widely expressed in proliferating normal tissues , with expression being cell - cycle - dependent and peaking at the g2/m point of the cell cycle . during mitosis , the kinase is virtually confined to the spindle poles , where it is needed for centrosome separation and maturation . an overexpression of aurora a causes an increase in centrosome numbers and aneuploidy , leading to the transformation of mammalian cells and also causes resistance to apoptosis induced by taxol in human cancer cell lines . moreover , this kinase is a key regulatory component of the p53 pathway as its overexpression leads to increased p53 degradation , which facilitates oncogenic transformation . drugable target in several tumors including pancreatic , hepatocellular , breast , nonendometriod , and ovarian carcinomas , gliomas and aggressive non - hodgkin 's lymphoma . aurora b ( also known as aurora-1 , aim-1 , aik2 , air-2 , airk-2 , ark2 , ial-1 and stk12 ) activity is required for bipolar chromosome orientation and condensation . aurora b kinases are chromosomal passenger proteins , which are found in cells in a complex with inner centromere protein ( incenp ) and survivin . the overexpression of an aurora b kinase - dead mutant ( k r ) causes multiple defects in the mitotic machinery , including the loss of kinetochore attachment to microtubules and the exit from mitosis without anaphase or cytokinesis . increased aurora b expression correlates with increased grade in glioma and colon cancer and with anaplasia in thyroid carcinoma . aurora c ( also known as aik3 ) expression plays a role in spermatogenesis at the time when cells assemble the two meiotic spindles and also cooperates with aurora b to regulate mitotic chromosome dynamics in mammalian cells . aurora c overexpression has been detected in tumor cell lines in vitro and in biopsy samples from colorectal carcinoma . several compounds have been pre - clinically screened for their inhibitory activity against aurora kinases ( vx680 , mln8054 , azd1152 , r766 , r763 , pha-739358 , at9283 ) and many of them are being tested in clinical phase i / ii trials ( table 2 ) . mk-0457 ( vx-680 ) is a pan - aurora kinase inhibitor with demonstrated in vitro activity against wild - type and mutated bcr - abl , including the t315i form , as well as flt3 and jak-2 . fascinatingly , carter et al . have found that the aurora kinase inhibitor vx-680 , already in phase i trials , and the p38 inhibitor birb-796 , in clinical trials for inflammatory disease , inhibit the imatinib- and dasatinib - resistant t315i - bcr - abl with high affinity ( tables 3 and 4 ) . in fact , contrasting results related to this compound have been published . in particular , birb-796 binds with good affinity to t315i - bcr - abl ( kd = 40 nm ) , but has significantly weaker affinity for wild - type and other imatinib - resistant forms of abl , with kd values > 1 m . in contrast , as reported by other authors , the compound fails to inhibit the proliferation of cells expressing t315i , suggesting a lack of clinical benefit for patients harboring such a mutation . table 2novel compound aurora - kinase inhibitors in clinical trial development.tk inhibitorcompanyphasetarget(s)mk-0457mercki - iiaurora , flt-3 , jak-2pha-739358nervianoiiaurorakw-2449kyowaiauroradecipheradeciferaiablas703569merck seronoi - iiaurora , abl , jak-2azd1152astra - zenecai - iiaurora table 3comparison between the binding affinity of imatinib and of the aurora kinase inhibitors birb-796 and mk-0457 for wild - type and drug - resistant abl variants . adapted from abl variantimatinibbirb-796mk-0457wild - type22,00020q252h204,00010y253f402,00020e255k100>10,00050m351t102,0008f359v208,00020t315i6,000405h396p60>10,0007 table 4comparison between the binding affinity of imatinib and of the aurora kinase inhibitors pha-739358 and mk-0457 for wild - type and drug - resistant abl variants . adapted from .wte255v ( p loop)t315im351timatinib0.2300.610>20.0000.100pha-7393580.0210.0140.0050.015mk-04570.0830.2050.0850.045 in a recent phase i - ii study , mk-0457 was shown to be active in patients with t315i phenotype - refractory cml or ph - positive all , with no significant extramedullary toxicity . because of a potential heart safety issue revealed in one patient who experienced qtc prolongation , the enrolment on phase ii protocol was halted in november 2007 . furthermore , an innovative phase i clinical study of sequential and concomitant treatment with dasatinib and mk-0457 has been conducted , based on the suggestion that such a combinatory approach would suppress the emergence of t315i and other resistant clones , improving upon the response rate for dasatinib and the durability of response . to date , 3 patients with wild - type chronic myeloid leukemia ( cml ) or ph - positive acute lymphoblastic leukemia ( all ) have been enrolled , and this innovative therapeutic combination showed a relevant hematologic activity and a good safety profile . pha-739358 is a small molecule that selectively inhibits the atp site of aurora - a ( ic50=13 nm ) and aurora - b ( ic50=79 nm ) kinases . starting from the rationale that aurora kinases play an important role in mitosis and that the interruption of their function has significant potential in the treatment of cancer , the drug , formulated for intravenous infusion , is being developed for therapeutic use in solid tumors and in patients with philadelphia positive leukemias . interestingly , pha-739358 , when tested against a panel of more than 30 kinases , has shown a strong cross - reactivity with c - abl ( ic50=25 nm ) . its inhibitory activity on abl in cells was confirmed in k562 leukemia cells which bear the philadelphia chromosome related translocation bcr - abl . furthermore pha-739358 inhibits phosphorylation of tyr412 , which is located in the kinase activation loop of abl and is also active against the t315i mutant of abl , which is resistant to other atp competitive inhibitors in the clinic , such as gleevec , and second generation tk inhibitors . a multicentric phase i / ii study , aimed to test pha-739358 in patients with chronic , accelerated or blast phase cml relapsing on gleevec or c - abl therapy and preferably with t315i mutation in bcr - abl kinase is ongoing . the compound vx-680 , developed by vertex pharmaceuticals as an inhibitor of the aurora kinases , is a y - shaped molecule , with a n - methyl - piperazine group forming the base or leg of the y , a pyrimidine group at the fork , and a methylpyrazole group at one arm and a substituted phenyl group at the other arm ( figure 2 ) . a recent study showed that vx-680 forms a hydrogen bond with the strictly conserved asp381 of the asp - phe - gly ( dfg ) motif in the abl kinase domain and maintains it in an orientation close to one that is normally seen in active kinases , although the activation loop of abl is not phosphorylated in this structure ( figure 3 ) . furthermore , vx-680 does not deeply penetrate into the kinase domain as imatinib does and it is anchored to it by four hydrogen bonds . three of these are formed between two carbonyl groups ( glu316 and met318 ) and an amide nitrogen ( met318 ) in the hinge region of the kinaseand three nitrogen atoms , one in the linker between the pyrimidine group and the methylpyrazole group , and the other two in the methylpyrazole group . these bonds are a common feature of kinase inhibitors and are independent of the sequence of the kinase . likewise , the fourth hydrogen bond , made by vx-680 to the side chain of asp381 of the dfg motif , is to a strictly invariant catalytic residue . using these four anchors , the inhibitor makes contact with 14 side chains within the kinase domain , eight of which are identical between abl and aurora . one of the non - conservative substitutions is at the gatekeeper position , where thr315 in bcr - abl is replaced by leu210 in aurora a kinase ( figure 4 ) . the side chains of isoleucine ( at position 315 of bcr - abl ) and leucine ( at position 210 of aurora a kinase ) can be accommodated readily between the two sides of the y of vx-680 . for this reason , vx-680 , in contrast to imatinib , is able to inhibit the kinase activity of both wild - type bcr - abl and t315i - bcr - abl . to understand the structural basis of the capability of pha-739358 to bind and inhibit the t315i mutant , the crystal structure of the inhibitor - protein complex was determined ( figure 5 ) . the protein is in the typical conformation of active kinases , with the activation loop in the extended dfg in conformation . indeed , asp381 points into the active site and interacts with mg ion that occupies a position similar to the one usually seen in the structures of kinases in complex with atp . the glycine loop adopts an extended conformation , in contrast to the other publicly available abl structures where the loop is more distorted , which could be due to the specific binding mode of our inhibitor . the purified t315i abl kinase domain used for crystallization experiments is predominantly phosphorylated on the activation loop at tyr393 , whereas tyr253 , tyr257 , and tyr264 are phosphorylated at lower levels . these interactions probably stabilize the active conformation of the activation loop , which is , however , very similar to the structures reported for dasatinib in complex with the wt abl kinase domain and of mk-0457 in complex with the abl mutant h396p . the mutation of the threonine to the more bulky isoleucine does not seem to cause any widespread conformational changes but creates a steric hindrance that would interfere with the binding of inhibitors , such as imatinib , nilotinib , and dasatinib , which make use of the hydrophobic pocket . the binding mode of pha-739358 is very similar to that reported for the complex of the same compound with aurora a ( figure 5b and d ) , although the conformation of the proteins around the atp - binding site shows some differences because in the aurora a structure the dfg motif is more similar to the out conformation . however , all of the essential contacts between pha-739358 and abl t315i involve highly conserved elements . the molecule makes three hydrogen bonds with the protein backbone of the hinge region : the two nitrogen atoms of the pyrrolopyrazole core interact with the carbonyl oxygen of glu316 and with the amide nitrogen of met318 , whereas the nitrogen of the amide group hydrogen bonds to the carbonyl oxygen of met318 . in addition , the side chain nitrogen of the conserved lys271 is within hydrogen bonding distance of the oxygen of the carbonyl group and the oxygen of the methoxy group . as in the aurora structure , the benzyl group packs against leu370 ( leu263 in aurora ) , whereas the n - methyl - piperazine points toward the solvent accessible area of the kinase pocket . the gatekeeper residue in the aurora kinases is leu210 , a large and hydrophobic residue very similar to isoleucine , and we have observed that pha-739358 binds in the atp - binding pocket of aurora a without any steric hindrance with the gatekeeper residue . indeed , the co - crystal structure reported here reveals that the compound is bound to the abl t315i kinase domain in a way that accommodates the substitution of isoleucine for threonine . figure 6 shows the structure of the abl t315i complex with pha-739358 superimposed on those of the abl wt with imatinib and abl h396p with mk-0457 . in the t315i mutant , the isoleucine side chain causes a steric clash with imatinib and the hydrogen bond between imatinib and the side chain oxygen of threonine is lost ( figure 6a and b ) . on the contrary , both pha-739358 and mk-0457 bind in such a way to avoid the gatekeeper residue ( figure 6c ) and this provides an explanation for the ability of both compounds to accommodate the isoleucine substitution . furthermore , the pyrrolopyrazole scaffold of pha-739358 is situated within van der waals distance of the side chain of ile315 mimicking the interaction between the inhibitor and leu210 in aurora a. it is possible that this favorable hydrophobic packing interaction may explain why pha-739358 is more active against the mutant than the wt protein . pha-739358 could represent a valuable novel agent to target the t315i bcr - abl mutation , and pre - clinical and clinical data are coming through to support this concept . figure 2chemical structure of vx-680 aurora kinase inhibitor . [ reprinted and adapted with permission from http://kinasepro.wordpress.com ] . chemical structure of vx-680 aurora kinase inhibitor . [ reprinted and adapted with permission from http://kinasepro.wordpress.com ] . figure 3structure of abl domain kinase bound to imatinib ( left ) and to vx-680 ( right ) . [ reprinted and adapted with permission of aacr from : young ma , et al . structure of the kinase domain of an imatinib - resistant abl mutant in complex with the aurora kinase inhibitor vx-680 . structure of abl domain kinase bound to imatinib ( left ) and to vx-680 ( right ) . [ reprinted and adapted with permission of aacr from : young ma , et al . structure of the kinase domain of an imatinib - resistant abl mutant in complex with the aurora kinase inhibitor vx-680 . figure 4binding mode of vx-680 to aurora a and t315i - bcr - abl . both leucine and isoleucine side chains can be accommodated between the two arms of the y of vx-680 . [ reprinted and adapted with permission of aacr from : young ma , et al . structure of the kinase domain of an imatinib - resistant abl mutant in complex with the aurora kinase inhibitor vx-680 . both leucine and isoleucine side chains can be accommodated between the two arms of the y of vx-680 . [ structure of the kinase domain of an imatinib - resistant abl mutant in complex with the aurora kinase inhibitor vx-680 . ( a ) ribbon representation of the structure of t315i abl mutant with pha-739358 . the mutated gatekeeper residue ile315 and the activation loop with the phosphorylated residue tyr393 ( b ) close - up view of the binding site of pha-739358 showing the final 2 fo - fc electron density map , contoured at 1 j , associated with the ligand . details of the binding of pha-739358 to abl ( green carbon atoms ) and to aurora a ( yellow carbon atoms ) showing the residues of the hinge region and of the dfg motif of both proteins . [ reprinted and adapted with permission of aacr from : modugno m , et al . crystal structure of the t315i abl mutant in complex with the aurora kinases inhibitor pha-739358 . structure of abl - t315i - pha 739358 complex . ( a ) ribbon representation of the structure of t315i abl mutant with pha-739358 . the mutated gatekeeper residue ile315 and the activation loop with the phosphorylated residue tyr393 are highlighted in green . ( b ) close - up view of the binding site of pha-739358 showing the final 2 fo - fc electron density map , contoured at 1 j , associated with the ligand . details of the binding of pha-739358 to abl ( green carbon atoms ) and to aurora a ( yellow carbon atoms ) showing the residues of the hinge region and of the dfg motif of both proteins . [ reprinted and adapted with permission of aacr from : modugno m , et al . crystal structure of the t315i abl mutant in complex with the aurora kinases inhibitor pha-739358 . cancer res 2007 sep1;67(17):798790 ] . ( a ) and ( b ) pha-739358 complex ( green carbon atoms ) superimposed with the structure of imatinib ( magenta carbon atoms ) . the gatekeeper residues ile315 of pha-739358 complex ( a ) and thr315 of the imatinib complex ( b ) are shown with van der waals spheres . in the t315i mutant , the isoleucine side chain causes a steric clash with imatinib ; in addition , the hydrogen bond between imatinib and the side chain oxygen of threonine is lost . ( c ) structure of abl - pha-739358 complex ( green carbon atoms ) superimposed on the structure of mk-0457 . [ reprinted and adapted with permission of aacr from : modugno m , et al . crystal structure of the t315i abl mutant in complex with the aurora kinases inhibitor pha-739358 . ( a ) and ( b ) pha-739358 complex ( green carbon atoms ) superimposed with the structure of imatinib ( magenta carbon atoms ) . the gatekeeper residues ile315 of pha-739358 complex ( a ) and thr315 of the imatinib complex ( b ) are shown with van der waals spheres . in the t315i mutant , the isoleucine side chain causes a steric clash with imatinib ; in addition , the hydrogen bond between imatinib and the side chain oxygen of threonine is lost . ( c ) structure of abl - pha-739358 complex ( green carbon atoms ) superimposed on the structure of mk-0457 . [ reprinted and adapted with permission of aacr from : modugno m , et al . crystal structure of the t315i abl mutant in complex with the aurora kinases inhibitor pha-739358 . the t315i is responsible for approximately 15% of the cases of relapse in cml and ph all patients on imatinib therapy . the clinical relevance of this mutant is likely to increase considerably as to date it seems to represent the main mechanism of resistance to dasatinib and nilotinib , the second - generation inhibitors already being developed clinically . structural analyses indicate that the substitution of threonine with isoleucine at residue 315 eliminates a crucial hydrogen - bonding interaction and introduces a steric clash which abrogates binding and effective inhibition of bcr - abl by imatinib as well as by several novel inhibitors . a possible approach to the development of second - line strategies overcoming resistance induced by the t315i mutation is to design inhibitors binding regions of bcr - abl other than the atp binding pocket . an intriguing alternative is to explore the possibility of whether molecules that have been developed as inhibitors for other protein kinases and are already undergoing clinical trials might include the t315i - bcr - abl mutant among their off - targets . although off - target activity may lead to undesirable side effects , it has to be recognized that focusing on compounds that are already being tested in clinical practice may speed up the development of successful therapeutic strategies . recent studies have shown that mk-0457 ( vx-680 ) and pha-739358 , two small - molecule aurora kinase inhibitors , have in vitro activity against the t315i - bcr - abl . moreover , preliminary data showed promising clinical efficacy in patients affected by philadelphia positive leukemias , relapsing or resistant to first and second generation tk inhibitors . such a remarkable efficacy raises the question of whether aurora kinases may also harbor some pathogenetic significance in cml and/or ph all or may be selectively deregulated by the t315i - bcr - abl , and whether auroras may be a suitable secondary target for inhibition .

at present , there are no compounds in clinical development in the field of chronic myeloid leukemia ( cml ) or philadelphia - positive ( ph+ ) acute lymphoblastic leukemia ( all ) that have been documented to harbor significant activity against the imatinib - resistant t315i mutation . recent reports on the pre - clinical activity of some emerging tyrosine kinase inhibitors such as on012380 , vx-680 and pha-739358 promise possible clinical efficacy against this specific bcr - abl mutant form . here , we focus on the role of aurora kinase inhibitor vx-680 and pha-739358 in blocking the leukemogenic pathways driven by wild - type and t315i - bcr - abl in cml or ph+ all by reviewing recent research evidence . we also discuss the possibility of employing aurora kinase inhibitors as a promising new therapeutic approach in the treatment of cml and ph+ all patients resistant to first and second generation tk inhibitors .

Introduction The T315I mutation is highly resistant to imatinib The two second-generation inhibitors in clinical development, dasatinib and nilotinib, are ineffective against the T315I mutant Aurora kinases as targets for cancer Novel aurora kinase inhibitors effective against the T315I-Bcr-Abl Binding mode of VX-680 and PHA-739358 to Abl Conclusions

the molecular signature of chronic myeloid leukemia ( cml ) and philadelphia - positive ( ph ) acute lymphoblastic leukemia ( all ) is the bcr - abl hybrid gene , originating from a reciprocal t(9;22 ) chromosomal translocation on the 22q- derivative , commonly referred to as the philadelphia chromosome . imatinib ( gleevec , glivec ; sti571 ) , a relatively selective tyrosine kinase inhibitor that blocks the catalytic activity of bcr - abl , is now the first - line treatment for all newly diagnosed cml patients . despite excellent clinical results , there is still a need to improve therapy for patients with cml and ph all . however , residual bcr - abl transcripts persist in the majority of these patients , as assessed by sensitive assays such as nested reverse transcription - polymerase chain reaction , and represent the potential pool from which disease recurrence may originate . while responses in cml in cp patients have been shown to last more than five years , most responding patients with ap- and bp - cml , as well as those with ph all , relapse early despite continued therapy . mutations target critical contact points between imatinib and bcr - abl or , more often , induce a conformation to which imatinib is unable to bind . in the remaining patients , the reasons for imatinib resistance have to be traced to bcr - abl gene amplification or overexpression , clonal cytogenetic evolution , or altered levels of transport molecules responsible for imatinib influx and efflux ( abc transporters , hoct1 ) . abl mutations are at present the most extensively investigated and best characterized mechanism of resistance to imatinib . so far , at least 90 different point mutations have been isolated from relapsed cml patients who are resistant to imatinib . indeed , while certain bcr - abl mutations retain in vitro sensitivity to imatinib at physiologically relevant concentrations and therefore may not be clinically meaningful , others require increased doses of imatinib , and some confer a highly resistant phenotype ( table 1 ) . threonine 315 , has attracted particular interest since it confers a high level of resistance not only to imatinib therapy but also to all of the newly developed tyrosine kinase inhibitors entered in clinical trials . in 1015% of imatinib - resistant patients , especially those in more advanced phases of disease , the t315i abrogates imatinib binding because it disrupts the above mentioned hydrogen bond and introduces a bulkier isoleucine side chain into the gatekeeper position . as a result , biochemical and cellular ic50 values of imatinib for the t315i - bcr - abl have been shown to be > 6400 times higher than those of wild - type bcr - abl ( table 1 ) . however , the effects of the t315i mutation on kinase activity in vitro and transforming efficiency of bcr - abl in vitro and in vivo have been very recently investigated , suggesting that in the absence of imatinib , there is neither increased kinase activity nor any growth advantage for cells carrying t315i - bcr - abl as compared to wild - type bcr - abl . to counteract the problem of resistance due to point mutations , several second - generation inhibitors have been synthesized and tested in pre - clinical assays : nilotinib ( amn107 ) , dasatinib ( bms-354825 ) , bosutinib , vx-680 , ap23464 , bafetinib , pd166326 , pd180970 and pd173955 , and on012380 . it has been shown to be 300 times more potent than imatinib in bcr - abl inhibition assays . excellent results in terms of hematologic and cytogenetic response in cml and ph all patients resistant to imatinib have been reported after dasatinib administration . pre - clinical studies have demonstrated that dasatinib is active against at least fourteen imatinib - resistant bcr - abl mutants ( m244v , g250e , q252h / r , y253f / h , e255k / v , f317l , m351 t , e355 g , f359v , h396r , f486s ) . the only imatinib - resistant bcr - abl isoform that was clearly insensitive to dasatinib was the t315i mutant , which retained kinase activity even in the presence of micromolar concentrations of the compound ( table 1 ) . accordingly , imatinib - resistant patients harboring the t315i mutation have been shown not to benefit from dasatinib in the recent phase i trial . nilotinib is a close relative of imatinib with more than 20-fold improved affinity for wild - type bcr - abl . it is highly efficacious in patients with imatinib - resistant ph cml . in vitro experiment with cell lines transformed with mutated forms of bcr - abl showed ic50 proliferation inhibition for most mutations with the exception of the t315i , which remains refractory to nilotinib ( table 1 ) . accordingly , clinical responses have been observed in patients with various imatinib - resistant bcr - abl mutations but not in patients positive for the t315i in the recent phase i trial . despite the pressing need for a clinically effective t315i - bcr - abl inhibitor , a potential pitfall might be the tendency to screen initially for abl kinase inhibition rather than for t315i - specific inhibition . for example , on012380 , a putative substrate - competitive inhibitor , exhibited low nanomolar activity against imatinib - resistant bcr - abl mutants , including the t315i , in biochemical and cellular assays . between these new promising drugs , vx-680 and pha-739358 , two aurora kinase a , b and c inhibitors , have a leading place . many authors reported an aberrant expression of the aurora a and b kinases also in leukemia cells , suggesting a potential role of these molecular targets in the treatment of cml and all . aurora kinase function is mediated by the phosphorylation of several substrates that have important roles in cell division , such as proteins survivin , cenp - a and serine 10 on histone h3 . figure 1schematic representation of domain organization of aurora kinases . the ken motif acts as a cdh dependant anaphase - promoting complex recognition signal . aurora kinases have three domains : the n - terminal and c - terminal domains which contain most of the aurora 's regulatory motifs and the catalytic domain in the central region . the ken motif acts as a cdh dependant anaphase - promoting complex recognition signal . several compounds have been pre - clinically screened for their inhibitory activity against aurora kinases ( vx680 , mln8054 , azd1152 , r766 , r763 , pha-739358 , at9283 ) and many of them are being tested in clinical phase i / ii trials ( table 2 ) . mk-0457 ( vx-680 ) is a pan - aurora kinase inhibitor with demonstrated in vitro activity against wild - type and mutated bcr - abl , including the t315i form , as well as flt3 and jak-2 . have found that the aurora kinase inhibitor vx-680 , already in phase i trials , and the p38 inhibitor birb-796 , in clinical trials for inflammatory disease , inhibit the imatinib- and dasatinib - resistant t315i - bcr - abl with high affinity ( tables 3 and 4 ) . in particular , birb-796 binds with good affinity to t315i - bcr - abl ( kd = 40 nm ) , but has significantly weaker affinity for wild - type and other imatinib - resistant forms of abl , with kd values > 1 m . table 2novel compound aurora - kinase inhibitors in clinical trial development.tk inhibitorcompanyphasetarget(s)mk-0457mercki - iiaurora , flt-3 , jak-2pha-739358nervianoiiaurorakw-2449kyowaiauroradecipheradeciferaiablas703569merck seronoi - iiaurora , abl , jak-2azd1152astra - zenecai - iiaurora table 3comparison between the binding affinity of imatinib and of the aurora kinase inhibitors birb-796 and mk-0457 for wild - type and drug - resistant abl variants . adapted from abl variantimatinibbirb-796mk-0457wild - type22,00020q252h204,00010y253f402,00020e255k100>10,00050m351t102,0008f359v208,00020t315i6,000405h396p60>10,0007 table 4comparison between the binding affinity of imatinib and of the aurora kinase inhibitors pha-739358 and mk-0457 for wild - type and drug - resistant abl variants . adapted from .wte255v ( p loop)t315im351timatinib0.2300.610>20.0000.100pha-7393580.0210.0140.0050.015mk-04570.0830.2050.0850.045 in a recent phase i - ii study , mk-0457 was shown to be active in patients with t315i phenotype - refractory cml or ph - positive all , with no significant extramedullary toxicity . furthermore , an innovative phase i clinical study of sequential and concomitant treatment with dasatinib and mk-0457 has been conducted , based on the suggestion that such a combinatory approach would suppress the emergence of t315i and other resistant clones , improving upon the response rate for dasatinib and the durability of response . to date , 3 patients with wild - type chronic myeloid leukemia ( cml ) or ph - positive acute lymphoblastic leukemia ( all ) have been enrolled , and this innovative therapeutic combination showed a relevant hematologic activity and a good safety profile . pha-739358 is a small molecule that selectively inhibits the atp site of aurora - a ( ic50=13 nm ) and aurora - b ( ic50=79 nm ) kinases . starting from the rationale that aurora kinases play an important role in mitosis and that the interruption of their function has significant potential in the treatment of cancer , the drug , formulated for intravenous infusion , is being developed for therapeutic use in solid tumors and in patients with philadelphia positive leukemias . interestingly , pha-739358 , when tested against a panel of more than 30 kinases , has shown a strong cross - reactivity with c - abl ( ic50=25 nm ) . its inhibitory activity on abl in cells was confirmed in k562 leukemia cells which bear the philadelphia chromosome related translocation bcr - abl . furthermore pha-739358 inhibits phosphorylation of tyr412 , which is located in the kinase activation loop of abl and is also active against the t315i mutant of abl , which is resistant to other atp competitive inhibitors in the clinic , such as gleevec , and second generation tk inhibitors . a multicentric phase i / ii study , aimed to test pha-739358 in patients with chronic , accelerated or blast phase cml relapsing on gleevec or c - abl therapy and preferably with t315i mutation in bcr - abl kinase is ongoing . a recent study showed that vx-680 forms a hydrogen bond with the strictly conserved asp381 of the asp - phe - gly ( dfg ) motif in the abl kinase domain and maintains it in an orientation close to one that is normally seen in active kinases , although the activation loop of abl is not phosphorylated in this structure ( figure 3 ) . three of these are formed between two carbonyl groups ( glu316 and met318 ) and an amide nitrogen ( met318 ) in the hinge region of the kinaseand three nitrogen atoms , one in the linker between the pyrimidine group and the methylpyrazole group , and the other two in the methylpyrazole group . these bonds are a common feature of kinase inhibitors and are independent of the sequence of the kinase . one of the non - conservative substitutions is at the gatekeeper position , where thr315 in bcr - abl is replaced by leu210 in aurora a kinase ( figure 4 ) . the side chains of isoleucine ( at position 315 of bcr - abl ) and leucine ( at position 210 of aurora a kinase ) can be accommodated readily between the two sides of the y of vx-680 . for this reason , vx-680 , in contrast to imatinib , is able to inhibit the kinase activity of both wild - type bcr - abl and t315i - bcr - abl . indeed , asp381 points into the active site and interacts with mg ion that occupies a position similar to the one usually seen in the structures of kinases in complex with atp . the purified t315i abl kinase domain used for crystallization experiments is predominantly phosphorylated on the activation loop at tyr393 , whereas tyr253 , tyr257 , and tyr264 are phosphorylated at lower levels . these interactions probably stabilize the active conformation of the activation loop , which is , however , very similar to the structures reported for dasatinib in complex with the wt abl kinase domain and of mk-0457 in complex with the abl mutant h396p . the mutation of the threonine to the more bulky isoleucine does not seem to cause any widespread conformational changes but creates a steric hindrance that would interfere with the binding of inhibitors , such as imatinib , nilotinib , and dasatinib , which make use of the hydrophobic pocket . as in the aurora structure , the benzyl group packs against leu370 ( leu263 in aurora ) , whereas the n - methyl - piperazine points toward the solvent accessible area of the kinase pocket . the gatekeeper residue in the aurora kinases is leu210 , a large and hydrophobic residue very similar to isoleucine , and we have observed that pha-739358 binds in the atp - binding pocket of aurora a without any steric hindrance with the gatekeeper residue . in the t315i mutant , the isoleucine side chain causes a steric clash with imatinib and the hydrogen bond between imatinib and the side chain oxygen of threonine is lost ( figure 6a and b ) . furthermore , the pyrrolopyrazole scaffold of pha-739358 is situated within van der waals distance of the side chain of ile315 mimicking the interaction between the inhibitor and leu210 in aurora a. it is possible that this favorable hydrophobic packing interaction may explain why pha-739358 is more active against the mutant than the wt protein . pha-739358 could represent a valuable novel agent to target the t315i bcr - abl mutation , and pre - clinical and clinical data are coming through to support this concept . figure 2chemical structure of vx-680 aurora kinase inhibitor . chemical structure of vx-680 aurora kinase inhibitor . structure of the kinase domain of an imatinib - resistant abl mutant in complex with the aurora kinase inhibitor vx-680 . structure of the kinase domain of an imatinib - resistant abl mutant in complex with the aurora kinase inhibitor vx-680 . figure 4binding mode of vx-680 to aurora a and t315i - bcr - abl . structure of the kinase domain of an imatinib - resistant abl mutant in complex with the aurora kinase inhibitor vx-680 . [ structure of the kinase domain of an imatinib - resistant abl mutant in complex with the aurora kinase inhibitor vx-680 . ( a ) ribbon representation of the structure of t315i abl mutant with pha-739358 . crystal structure of the t315i abl mutant in complex with the aurora kinases inhibitor pha-739358 . structure of abl - t315i - pha 739358 complex . ( a ) ribbon representation of the structure of t315i abl mutant with pha-739358 . crystal structure of the t315i abl mutant in complex with the aurora kinases inhibitor pha-739358 . in the t315i mutant , the isoleucine side chain causes a steric clash with imatinib ; in addition , the hydrogen bond between imatinib and the side chain oxygen of threonine is lost . ( c ) structure of abl - pha-739358 complex ( green carbon atoms ) superimposed on the structure of mk-0457 . crystal structure of the t315i abl mutant in complex with the aurora kinases inhibitor pha-739358 . the gatekeeper residues ile315 of pha-739358 complex ( a ) and thr315 of the imatinib complex ( b ) are shown with van der waals spheres . in the t315i mutant , the isoleucine side chain causes a steric clash with imatinib ; in addition , the hydrogen bond between imatinib and the side chain oxygen of threonine is lost . ( c ) structure of abl - pha-739358 complex ( green carbon atoms ) superimposed on the structure of mk-0457 . the t315i is responsible for approximately 15% of the cases of relapse in cml and ph all patients on imatinib therapy . structural analyses indicate that the substitution of threonine with isoleucine at residue 315 eliminates a crucial hydrogen - bonding interaction and introduces a steric clash which abrogates binding and effective inhibition of bcr - abl by imatinib as well as by several novel inhibitors . a possible approach to the development of second - line strategies overcoming resistance induced by the t315i mutation is to design inhibitors binding regions of bcr - abl other than the atp binding pocket . an intriguing alternative is to explore the possibility of whether molecules that have been developed as inhibitors for other protein kinases and are already undergoing clinical trials might include the t315i - bcr - abl mutant among their off - targets . recent studies have shown that mk-0457 ( vx-680 ) and pha-739358 , two small - molecule aurora kinase inhibitors , have in vitro activity against the t315i - bcr - abl . moreover , preliminary data showed promising clinical efficacy in patients affected by philadelphia positive leukemias , relapsing or resistant to first and second generation tk inhibitors . such a remarkable efficacy raises the question of whether aurora kinases may also harbor some pathogenetic significance in cml and/or ph all or may be selectively deregulated by the t315i - bcr - abl , and whether auroras may be a suitable secondary target for inhibition .

cancer - related inflammation affects many aspects of malignancy , including proliferation and survival of malignant cells , angiogenesis , and therapeutic response . in a previous study that utilizes a mouse model , we showed that an inflammatory response is elicited in the early stages of the postsurgical wound healing process , leading to an increase in the number of inflammatory cells in the peritoneum . this further increases pro - matrix metalloproteinase-9 ( pro - mmp-9 ) , which plays a key role in the growth and progression of peritoneal metastases . based on these experimental results , we hypothesize that the local metastatic microenvironment will be changed during the early inflammatory phase in the process of wound healing . clinically , there have been efforts to demonstrate a correlation between operative systemic inflammation and cancer . some biomarkers representing the degree of systemic inflammation , such as the glasgow prognostic score , neutrophil lymphocyte ratio ( nlr ) , and platelet lymphocyte ratio ( plr ) , have been shown to have prognostic value in many kinds of cancer patients [ 5 - 8 ] . however , these results only focus on the effects of preoperative cancer or inflammation , regardless of etiology of the inflammation . until now , there is only a few research regarding postoperative systemic inflammation on cancer prognosis . in this study , we examine not only the effect of the preoperative inflammatory state on cancer , but also the effects of early postoperative systemic inflammation on cancer prognosis , based on the previous animal study results . six hundred thirty - nine consecutive patients underwent surgery for colorectal cancer at yeouido st . mary 's hospital , the catholic university of korea from january 2006 to december 2009 . patients were excluded if they had colorectal cancer other than adenocarcinoma and carcinoma in situ . however , patients who had preoperative radiation therapy and those who were not able to undergo curative resection were included . routine laboratory measurements , including white blood cell ( wbc ) count , neutrophil count , lymphocyte count , monocyte count , and platelet count , were performed preoperatively , daily until day four postoperatively , and subsequently every two days . we did not consider preoperative infection and inflammatory condition and postoperative infection and inflammatory condition . patients were divided into three groups based on the days spent on the leukocyte count to drop below 10,000/mm after surgery ( dsnlc ; group i , 0 - 1 days ; group ii , 2 - 3 days ; group iii , 4 days ) . for a comparative analysis with the results of the other study , grouping of the variables , such as the wbc count , neutrophil count , lymphocyte count , monocyte count , and platelet count , " right side colon " was defined as both the right colon and the transverse colon . " left side colon " was defined as both the left colon and the recto - sigmoid colon above the peritoneal reflection . staging evaluation was carried out according to the guidelines of american joint committee on cancer , sixth edition . this study was approved by the institutional review board of the college of medicine ( sc11tisi0080 ) . the relationship between the number of days required for the leukocyte count to drop below 10,000/mm after surgery ( group i , 0 - 1 day ; group ii , 2 - 3 days ; group iii , 4 days ) and clinicopathologic factors was assessed using the chi - squared and fisher 's exact tests . the overall duration of survival was calculated from the date of surgery until the date of death . the duration of cancer - free survival was calculated from the date of surgery until the date of detection of disease recurrence , defined using clinical , radiographic or pathological findings . overall and cancer - free survival rates were calculated using the kaplan - meier method , and differences among curves were tested using the log - rank test . variables that were significantly related to the survival rate in a univariate analysis were subsequently included in a multivariate analysis employing the cox multiple regression model . this method finds the ' best ' variables ( p<0.05 ) for which the effects on prognosis are not only significant , but also independent when correlated with the other factors included in the model . all statistical tests were two - tailed and the significance level was set at 0.05 . six hundred thirty - nine consecutive patients underwent surgery for colorectal cancer at yeouido st . mary 's hospital , the catholic university of korea from january 2006 to december 2009 . patients were excluded if they had colorectal cancer other than adenocarcinoma and carcinoma in situ . however , patients who had preoperative radiation therapy and those who were not able to undergo curative resection were included . routine laboratory measurements , including white blood cell ( wbc ) count , neutrophil count , lymphocyte count , monocyte count , and platelet count , were performed preoperatively , daily until day four postoperatively , and subsequently every two days . we did not consider preoperative infection and inflammatory condition and postoperative infection and inflammatory condition . patients were divided into three groups based on the days spent on the leukocyte count to drop below 10,000/mm after surgery ( dsnlc ; group i , 0 - 1 days ; group ii , 2 - 3 days ; group iii , 4 days ) . for a comparative analysis with the results of the other study , grouping of the variables , such as the wbc count , neutrophil count , lymphocyte count , monocyte count , and platelet count , " right side colon " was defined as both the right colon and the transverse colon . " left side colon " was defined as both the left colon and the recto - sigmoid colon above the peritoneal reflection . staging evaluation was carried out according to the guidelines of american joint committee on cancer , sixth edition . this study was approved by the institutional review board of the college of medicine ( sc11tisi0080 ) . the relationship between the number of days required for the leukocyte count to drop below 10,000/mm after surgery ( group i , 0 - 1 day ; group ii , 2 - 3 days ; group iii , 4 days ) and clinicopathologic factors was assessed using the chi - squared and fisher 's exact tests . the overall duration of survival was calculated from the date of surgery until the date of death . the duration of cancer - free survival was calculated from the date of surgery until the date of detection of disease recurrence , defined using clinical , radiographic or pathological findings . overall and cancer - free survival rates were calculated using the kaplan - meier method , and differences among curves were tested using the log - rank test . variables that were significantly related to the survival rate in a univariate analysis were subsequently included in a multivariate analysis employing the cox multiple regression model . this method finds the ' best ' variables ( p<0.05 ) for which the effects on prognosis are not only significant , but also independent when correlated with the other factors included in the model . all statistical tests were two - tailed and the significance level was set at 0.05 . the male - to - female ratio of patients was 1.48:1 , and the mean age was 62.311.5 years . three hundred fifty patients ( 58.2% ) were under 65 years old , and 250 patients ( 41.7% ) were over 65 years old . the follow - up period ranged from 1 to 72 months ( mean , 27.418.2 months ) . tumors were localized to the right side colon in 156 patients ( 26.0% ) , the left side colon in 250 patients ( 41.7% ) , the rectum in 184 patients ( 30.7% ) and multiple areas in 10 patients ( 1.7% ) . there were no differences between the groups with regards to complication of cancer , which was defined by perforating or obstructing colorectal lesions . among the rectal cancer patients laparoscopic treatment was performed in 69 patients ( 11.5% ) , and conventional treatment was performed in 531 patients ( 88.5% ) . five hundred thirty - one patients ( 88.5% ) underwent r0 resection and 69 patients ( 11.5% ) underwent r1 resection . we observed stage i disease in 140 patients ( 23.3% ) , stage ii disease in 178 patients ( 29.7% ) , stage iii0 disease in 208 patients ( 34.7% ) , and stage iv disease in 52 patients ( 8.7% ) . there were no differences in the characteristics of patients between the three groups , other than neoadjuvant rt and surgical approach methods ( p=0.003 and p=0.005 , respectively ) ( table 1 ) . in total , 548 patients ( 91.3% ) had preoperative wbc counts under 10,000/mm and 52 patients ( 8.7% ) had increased preoperative wbc counts ( over 10,000/mm ) . postoperative wbc counts exceeded 10,000/mm in 417 patients ( 69.5% ) , including 74 cases ( 45.8% ) in group i , 198 cases ( 33.0% ) in group ii , and 127 cases ( 21.2% ) in group iii . preoperative wbc counts correlated significantly with disease stage ( p=0.047 ) . in particular , increased preoperative wbc counts were noted in patients at stages ii and iii ( data not shown ) . however , neither postoperative wbc counts nor the number of days required for the leukocyte count to fall below 10,000/mm correlated with disease stage ( p=0.394 and p=0.402 , respectively ) . patients who received preoperative radiotherapy and patients who had laparoscopic surgery had statistically significant shorter wbc normalization periods compared to patients without preoperative radiotherapy and with conventional surgery . the results did not show a significant difference in the postoperative wbc normalization period for the combined resection of other organs ( p=0.329 ) . the duration of postoperative hospital stay , according to the wbc normalization period , was as follows : group i , 127.1 days ( median , 11.0 days ) ; group ii , 12.37.3 days ( median , 10.0 days ) ; group iii , 14.87.9 days ( median , 12.0 days ) . however , there were no significant differences , between groups i and ii . there are significant correlations between the groups with regards to preoperative neutrophil count ( p<0.001 ) , lymphocyte count ( p=0.001 ) , monocyte count ( p<0.001 ) , and platelet count ( p=0.001 ) , but no correlation between preoperative nlr ( p=0.47 ) ( table 1 ) . in univariate survival analyses , tumor , node , metastasis ( tnm ) stage and monocyte count were significantly associated with cancer - free survival ( p<0.01 for both parameters ) . in addition , cancer - free survival outcomes were significantly worse in patients who required more than four days for the normalization of wbc count ( hazard ratio [ hr ] , 1.652 ; 95% confidence interval [ ci ] , 1.105 to 2.468 ; p=0.014 ) . a tnm stage greater than ii and nlr is significantly associated with the duration of overall survival ( p<0.050 and p=0.045 , respectively ) , but no correlation was observed between dsnlc and duration of overall survival ( table 2 ) . cancer - free and overall survival curves are shown according to the stage , time for leukocyte count to decline below 10,000/mm and monocyte count in figs . 1 , 2 , and 3 , respectively . no correlation was revealed between the elevated preoperative and postoperative wbc counts ( 1010 ) and cancer - free or overall survival . in a multivariate analysis of these significant variables ( except curative resection ) , tnm stage ( stage ii : hr , 3.545 ; 95% ci , 1.571 to 7.997 ; p=0.002 ; stage iii : hr , 6.115 ; 95% ci , 2.818 to 13.447 ; p<0.001 ; stage iv : hr , 15.928 ; 95% ci , 6.974 to 36.378 ; p<0.001 ) , an interval longer than four days for normalization of wbc counts ( hr , 1.509 ; 95% ci , 0.999 to 2.278 ; p=0.050 ) , and monocyte count ( hr , 2.133 ; 95% ci , 1.262 to 3.605 ; p=0.005 ) were independently associated with cancer - free survival ( table 3 ) . the male - to - female ratio of patients was 1.48:1 , and the mean age was 62.311.5 years . three hundred fifty patients ( 58.2% ) were under 65 years old , and 250 patients ( 41.7% ) were over 65 years old . the follow - up period ranged from 1 to 72 months ( mean , 27.418.2 months ) . tumors were localized to the right side colon in 156 patients ( 26.0% ) , the left side colon in 250 patients ( 41.7% ) , the rectum in 184 patients ( 30.7% ) and multiple areas in 10 patients ( 1.7% ) . there were no differences between the groups with regards to complication of cancer , which was defined by perforating or obstructing colorectal lesions . among the rectal cancer patients laparoscopic treatment was performed in 69 patients ( 11.5% ) , and conventional treatment was performed in 531 patients ( 88.5% ) . five hundred thirty - one patients ( 88.5% ) underwent r0 resection and 69 patients ( 11.5% ) underwent r1 resection . we observed stage i disease in 140 patients ( 23.3% ) , stage ii disease in 178 patients ( 29.7% ) , stage iii0 disease in 208 patients ( 34.7% ) , and stage iv disease in 52 patients ( 8.7% ) . there were no differences in the characteristics of patients between the three groups , other than neoadjuvant rt and surgical approach methods ( p=0.003 and p=0.005 , respectively ) ( table 1 ) . in total , 548 patients ( 91.3% ) had preoperative wbc counts under 10,000/mm and 52 patients ( 8.7% ) had increased preoperative wbc counts ( over 10,000/mm ) . postoperative wbc counts exceeded 10,000/mm in 417 patients ( 69.5% ) , including 74 cases ( 45.8% ) in group i , 198 cases ( 33.0% ) in group ii , and 127 cases ( 21.2% ) in group iii . preoperative wbc counts correlated significantly with disease stage ( p=0.047 ) . in particular , increased preoperative wbc counts were noted in patients at stages ii and iii ( data not shown ) . however , neither postoperative wbc counts nor the number of days required for the leukocyte count to fall below 10,000/mm correlated with disease stage ( p=0.394 and p=0.402 , respectively ) . patients who received preoperative radiotherapy and patients who had laparoscopic surgery had statistically significant shorter wbc normalization periods compared to patients without preoperative radiotherapy and with conventional surgery . the results did not show a significant difference in the postoperative wbc normalization period for the combined resection of other organs ( p=0.329 ) . the duration of postoperative hospital stay , according to the wbc normalization period , was as follows : group i , 127.1 days ( median , 11.0 days ) ; group ii , 12.37.3 days ( median , 10.0 days ) ; group iii , 14.87.9 days ( median , 12.0 days ) . however , there were no significant differences , between groups i and ii . there are significant correlations between the groups with regards to preoperative neutrophil count ( p<0.001 ) , lymphocyte count ( p=0.001 ) , monocyte count ( p<0.001 ) , and platelet count ( p=0.001 ) , but no correlation between preoperative nlr ( p=0.47 ) ( table 1 ) . in univariate survival analyses , tumor , node , metastasis ( tnm ) stage and monocyte count in addition , cancer - free survival outcomes were significantly worse in patients who required more than four days for the normalization of wbc count ( hazard ratio [ hr ] , 1.652 ; 95% confidence interval [ ci ] , 1.105 to 2.468 ; p=0.014 ) . a tnm stage greater than ii and nlr is significantly associated with the duration of overall survival ( p<0.050 and p=0.045 , respectively ) , but no correlation was observed between dsnlc and duration of overall survival ( table 2 ) . cancer - free and overall survival curves are shown according to the stage , time for leukocyte count to decline below 10,000/mm and monocyte count in figs . 1 , 2 , and 3 , respectively . no correlation was revealed between the elevated preoperative and postoperative wbc counts ( 1010 ) and cancer - free or overall survival . in a multivariate analysis of these significant variables ( except curative resection ) , tnm stage ( stage ii : hr , 3.545 ; 95% ci , 1.571 to 7.997 ; p=0.002 ; stage iii : hr , 6.115 ; 95% ci , 2.818 to 13.447 ; p<0.001 ; stage iv : hr , 15.928 ; 95% ci , 6.974 to 36.378 ; p<0.001 ) , an interval longer than four days for normalization of wbc counts ( hr , 1.509 ; 95% ci , 0.999 to 2.278 ; p=0.050 ) , and monocyte count ( hr , 2.133 ; 95% ci , 1.262 to 3.605 ; p=0.005 ) were independently associated with cancer - free survival ( table 3 ) . in univariate survival analyses , tumor , node , metastasis ( tnm ) stage and monocyte count were significantly associated with cancer - free survival ( p<0.01 for both parameters ) . in addition , cancer - free survival outcomes were significantly worse in patients who required more than four days for the normalization of wbc count ( hazard ratio [ hr ] , 1.652 ; 95% confidence interval [ ci ] , 1.105 to 2.468 ; p=0.014 ) . a tnm stage greater than ii and nlr is significantly associated with the duration of overall survival ( p<0.050 and p=0.045 , respectively ) , but no correlation was observed between dsnlc and duration of overall survival ( table 2 ) . cancer - free and overall survival curves are shown according to the stage , time for leukocyte count to decline below 10,000/mm and monocyte count in figs . 1 , 2 , and 3 , respectively . no correlation was revealed between the elevated preoperative and postoperative wbc counts ( 1010 ) and cancer - free or overall survival . in a multivariate analysis of these significant variables ( except curative resection ) , tnm stage ( stage ii : hr , 3.545 ; 95% ci , 1.571 to 7.997 ; p=0.002 ; stage iii : hr , 6.115 ; 95% ci , 2.818 to 13.447 ; p<0.001 ; stage iv : hr , 15.928 ; 95% ci , 6.974 to 36.378 ; p<0.001 ) , an interval longer than four days for normalization of wbc counts ( hr , 1.509 ; 95% ci , 0.999 to 2.278 ; p=0.050 ) , and monocyte count ( hr , 2.133 ; 95% ci , 1.262 to 3.605 ; p=0.005 ) were independently associated with cancer - free survival ( table 3 ) . first , inflammatory diseases increase the risk of developing many types of cancer and inflammatory cells . second , chemokines and cytokines are present in the microenvironment of all tumors in experimental animal models and in humans from the earliest stages of development . as mentioned above , our experimental mouse model creates an inflammatory reaction in the peritoneum and confers an increased metastatic burden at the wound site compared with the undamaged normal peritoneal tissues . furthermore , this model demonstrates an abundance of pro - inflammatory cells at the wound site , which likely induces mesothelial cells in the peritoneum . consequently , an increase in the mesothelial cells and other inflammatory cells constitute a pro - metastatic microenvironment in the normal peritoneum . in addition , in the early stages of wound healing , wound metastasis occurs . for wounds with continued inflammation , we confirmed that wound metastasis occurs even after seven days of wound healing . the glasgow prognostic score ( gps ) , which includes only serum c - reactive protein and serum albumin , has a prognostic value independent of stage in patients with advanced or primary operable cancer . in addition , the nlr and plr have been shown to have an independent prognostic value in a variety of cancers [ 5 - 8 ] . these studies demonstrate that the prognostic values of nlr or plr are independent of tumor stage , conventional scoring systems and treatment modalities . furthermore , studies conducted by leitch et al . show that monocyte count and gps are independently associated with cancer - specific survival . in this study , monocyte count was significantly associated with cancer - free survival in a univariate survival analysis . postoperative wbc count ( 1010 ) did not reveal any correlation with cancer - free survival or overall survival . most studies mentioned above examined the effects of the preoperative inflammatory condition . however , the research presented in this report does not focus on one point of time , but rather on a certain period of an inflammatory condition that will have a great effect on the metastatic microenvironment . this idea was brought on by the fact that mmp-9 increases in the first three days postoperatively during wound healing and that the early postoperative changes affect peritoneal metastasis , as observed in the author 's prior animal model experiment study [ 4 - 12 ] . thus , in the current study , regardless of the preoperative and postoperative inflammatory status , the groups were classified by the normalization of wbc count . the period in which the metastatic microenvironment is exposed to inflammation was defined as the period until the wbc count is normalized . the results did not show significant differences between the groups , the cancer location , the extent of curative resection , or stage . these results demonstrate that the differences in surgical methods according to stage did not affect the postoperative wbc count or dsnlc . in a univariate analysis , the presence of increased wbcs in the preoperative stages did not affect the duration of disease - free survival or overall survival . on the other hand , sustained inflammation over four days did affect disease - free survival and overall survival . we can analyze that this sustained inflammation can also have a systematic effect , like the result of three day inflammatory cell increase of wound healing process having an effect on peritoneal metastasis . moreover , dsnlc was independently associated with cancer - free survival in a multivariate survival analysis . other studies have shown that an elevated gps may reflect an altered innate immune response , as gps was also associated with increased numbers of neutrophils and monocytes . this may suggest that activation of innate immunity , rather than down - regulation of acquired immunity , is the most important factor in determining a poor outcome in patients with colorectal cancer . one study also revealed that an early increase in wbcs during surgery correlates with an increased innate immune response . in addition , an increase in wbcs is a better prognostic parameter than an increase in monocytes or nlr . in this study , an early operative increase in wbc correlates with increased innate immunity , and this increase is predictive of a poorer outcome for colorectal cancer more so than monocyte or nlr increase , which only shows an increase in innate immunity at one point of time . preoperative wbc count shows a correlation with an increase of cancer stage , with the exception of stage iv . iv , since neoadjuvant chemotherapy can be performed , an increase in wbcs may be lower than in stages ii and iii . in addition , patients receiving preoperative radiation show statistically significant shorter periods for normalization of wbc counts compared to patients not receiving radiation therapy . generally , in radiation therapy , numerous inflammatory cells are recruited into the irradiation field . gross inflammatory scores and microscopic inflammatory scores are significantly higher at the colonic anastomotic line after preoperative irradiation . however , in colorectal cancer , the systemic effect of radiotherapy may not be substantial , and instead , the accompanying chemotherapy may be the reason for a shorter period of normalization of wbc count . in addition , there is a shorter wbc count normalization period following laparoscopic surgery ; this is an advantage of minimally invasive surgery . en bloc resection with radical lymphadenectomy is performed by laparoscopic surgery in our center in the same manner of open surgery . since there is no difference in operative extent between surgical methods , the shorter wbc count normalization period and decreased persistence of inflammation is attributable to laparoscopic surgery . however , there is no difference in the duration of disease - free survival between laparoscopic and open surgery . postoperative increase in wbc count has many contributory factors , such as cancer cell itself , surgical complications , radiation therapy , chemotherapy and surgical methods . whatever the causative factor , the increase in postoperative wbcs affects the metastatic microenvironment and eventually contributes to cancer recurrences . wbc count decrease was observed in cases that had preoperative radiation or chemotherapy . for laparoscopic surgery , more research is necessary as there are relatively few cases ; thus other factors , such as selection bias , can influence the results . complicated cancer was defined by perforating or obstructing colorectal lesions shows a correlation with an increase of preoperative wbc count ( p=0.004 ) , but there were no differences between the groups ( p=0.694 , data not shown ) . this fact shows that complicated colorectal cancer was not correlative with the normalization of wbc count unless the lesion persists . in addition , the wbc count normalization period after surgery did not correlate with the presence of combined resection , but rather correlated with the duration of postoperative hospital stay . in this study , three factors ( surgical approach method , preoperative radiation therapy and postoperative hospital stay ) were found to be associated with the difference in time of the period of normalization of wbc count . postoperative complications , factors in the preoperative period or factors that can affect the cancer itself may also be considered . among these additional factors , unfortunately , postoperative complications were not recorded prospectively . postoperative complications were omitted as a factor , leading to inaccuracy in the retrospective study , and this represents a shortcoming of this study . taking into account the postoperative hospital stay , it can be inferred that postoperative complications are likely to have a correlation . in summary , postoperative early inflammatory phase and preoperative monocyte count correlate with the poor prognosis of colon cancer even after the calibration of stage . thus , we can conclude that preoperativeand postoperative inflammatory response and period unfavorably affect the metastatic microenvironment .

purposecancer - related inflammation affects many aspects of malignancy . we confirm the effects of early postoperative systemic inflammation on cancer prognosis.materials and methodssix hundred consecutive patients underwent surgery for colorectal cancer from 2006 to 2009 . measurements of white blood cells , neutrophils , lymphocytes , monocytes , and platelet counts were performed preoperatively , daily until the fourth postoperative day , and subsequently every two days . patients were divided into three groups based on the days spent on the leukocyte count to drop below 10,000/mm3 after surgery.resultspreoperative white blood cell ( wbc ) counts correlated with stage of disease . in univariate survival analyses , tumor , node , metastasis ( tnm ) stage , and monocyte count were associated with cancer - free survival . in addition , cancer - free survival outcomes were worse in patients who required more than four days for the normalization of wbc count . a tnm stage greater than ii and the neutrophil lymphocyte ratio were associated with the duration of overall survival . in a multivariate analysis of these significant variables , tnm stage , an interval longer than four days for normalization of wbc counts and monocyte count independently associated with cancer - free survival.conclusionpostoperative early inflammatory phase and preoperative monocyte count correlate with poor colon cancer prognosis . we can conclude that preoperative and postoperative inflammatory response and period unfavorably affect the metastatic microenvironment .

Introduction Materials and Methods 1. Patient settings 2. Statistical analysis Results 1. Patient characteristics 2. Clinicopathologic features related to systemic inflammatory response 1) Survival in relation to clinicopatholigicfactors and systemic inflammatory response Discussion Conclusion

cancer - related inflammation affects many aspects of malignancy , including proliferation and survival of malignant cells , angiogenesis , and therapeutic response . in a previous study that utilizes a mouse model , we showed that an inflammatory response is elicited in the early stages of the postsurgical wound healing process , leading to an increase in the number of inflammatory cells in the peritoneum . based on these experimental results , we hypothesize that the local metastatic microenvironment will be changed during the early inflammatory phase in the process of wound healing . some biomarkers representing the degree of systemic inflammation , such as the glasgow prognostic score , neutrophil lymphocyte ratio ( nlr ) , and platelet lymphocyte ratio ( plr ) , have been shown to have prognostic value in many kinds of cancer patients [ 5 - 8 ] . however , these results only focus on the effects of preoperative cancer or inflammation , regardless of etiology of the inflammation . until now , there is only a few research regarding postoperative systemic inflammation on cancer prognosis . in this study , we examine not only the effect of the preoperative inflammatory state on cancer , but also the effects of early postoperative systemic inflammation on cancer prognosis , based on the previous animal study results . six hundred thirty - nine consecutive patients underwent surgery for colorectal cancer at yeouido st . patients were excluded if they had colorectal cancer other than adenocarcinoma and carcinoma in situ . routine laboratory measurements , including white blood cell ( wbc ) count , neutrophil count , lymphocyte count , monocyte count , and platelet count , were performed preoperatively , daily until day four postoperatively , and subsequently every two days . patients were divided into three groups based on the days spent on the leukocyte count to drop below 10,000/mm after surgery ( dsnlc ; group i , 0 - 1 days ; group ii , 2 - 3 days ; group iii , 4 days ) . for a comparative analysis with the results of the other study , grouping of the variables , such as the wbc count , neutrophil count , lymphocyte count , monocyte count , and platelet count , " right side colon " was defined as both the right colon and the transverse colon . " the relationship between the number of days required for the leukocyte count to drop below 10,000/mm after surgery ( group i , 0 - 1 day ; group ii , 2 - 3 days ; group iii , 4 days ) and clinicopathologic factors was assessed using the chi - squared and fisher 's exact tests . the overall duration of survival was calculated from the date of surgery until the date of death . the duration of cancer - free survival was calculated from the date of surgery until the date of detection of disease recurrence , defined using clinical , radiographic or pathological findings . overall and cancer - free survival rates were calculated using the kaplan - meier method , and differences among curves were tested using the log - rank test . variables that were significantly related to the survival rate in a univariate analysis were subsequently included in a multivariate analysis employing the cox multiple regression model . this method finds the ' best ' variables ( p<0.05 ) for which the effects on prognosis are not only significant , but also independent when correlated with the other factors included in the model . all statistical tests were two - tailed and the significance level was set at 0.05 . six hundred thirty - nine consecutive patients underwent surgery for colorectal cancer at yeouido st . patients were excluded if they had colorectal cancer other than adenocarcinoma and carcinoma in situ . routine laboratory measurements , including white blood cell ( wbc ) count , neutrophil count , lymphocyte count , monocyte count , and platelet count , were performed preoperatively , daily until day four postoperatively , and subsequently every two days . patients were divided into three groups based on the days spent on the leukocyte count to drop below 10,000/mm after surgery ( dsnlc ; group i , 0 - 1 days ; group ii , 2 - 3 days ; group iii , 4 days ) . for a comparative analysis with the results of the other study , grouping of the variables , such as the wbc count , neutrophil count , lymphocyte count , monocyte count , and platelet count , " right side colon " was defined as both the right colon and the transverse colon . " staging evaluation was carried out according to the guidelines of american joint committee on cancer , sixth edition . the relationship between the number of days required for the leukocyte count to drop below 10,000/mm after surgery ( group i , 0 - 1 day ; group ii , 2 - 3 days ; group iii , 4 days ) and clinicopathologic factors was assessed using the chi - squared and fisher 's exact tests . the overall duration of survival was calculated from the date of surgery until the date of death . the duration of cancer - free survival was calculated from the date of surgery until the date of detection of disease recurrence , defined using clinical , radiographic or pathological findings . overall and cancer - free survival rates were calculated using the kaplan - meier method , and differences among curves were tested using the log - rank test . variables that were significantly related to the survival rate in a univariate analysis were subsequently included in a multivariate analysis employing the cox multiple regression model . this method finds the ' best ' variables ( p<0.05 ) for which the effects on prognosis are not only significant , but also independent when correlated with the other factors included in the model . the male - to - female ratio of patients was 1.48:1 , and the mean age was 62.311.5 years . we observed stage i disease in 140 patients ( 23.3% ) , stage ii disease in 178 patients ( 29.7% ) , stage iii0 disease in 208 patients ( 34.7% ) , and stage iv disease in 52 patients ( 8.7% ) . postoperative wbc counts exceeded 10,000/mm in 417 patients ( 69.5% ) , including 74 cases ( 45.8% ) in group i , 198 cases ( 33.0% ) in group ii , and 127 cases ( 21.2% ) in group iii . preoperative wbc counts correlated significantly with disease stage ( p=0.047 ) . in particular , increased preoperative wbc counts were noted in patients at stages ii and iii ( data not shown ) . however , neither postoperative wbc counts nor the number of days required for the leukocyte count to fall below 10,000/mm correlated with disease stage ( p=0.394 and p=0.402 , respectively ) . the duration of postoperative hospital stay , according to the wbc normalization period , was as follows : group i , 127.1 days ( median , 11.0 days ) ; group ii , 12.37.3 days ( median , 10.0 days ) ; group iii , 14.87.9 days ( median , 12.0 days ) . there are significant correlations between the groups with regards to preoperative neutrophil count ( p<0.001 ) , lymphocyte count ( p=0.001 ) , monocyte count ( p<0.001 ) , and platelet count ( p=0.001 ) , but no correlation between preoperative nlr ( p=0.47 ) ( table 1 ) . in univariate survival analyses , tumor , node , metastasis ( tnm ) stage and monocyte count were significantly associated with cancer - free survival ( p<0.01 for both parameters ) . in addition , cancer - free survival outcomes were significantly worse in patients who required more than four days for the normalization of wbc count ( hazard ratio [ hr ] , 1.652 ; 95% confidence interval [ ci ] , 1.105 to 2.468 ; p=0.014 ) . a tnm stage greater than ii and nlr is significantly associated with the duration of overall survival ( p<0.050 and p=0.045 , respectively ) , but no correlation was observed between dsnlc and duration of overall survival ( table 2 ) . cancer - free and overall survival curves are shown according to the stage , time for leukocyte count to decline below 10,000/mm and monocyte count in figs . no correlation was revealed between the elevated preoperative and postoperative wbc counts ( 1010 ) and cancer - free or overall survival . in a multivariate analysis of these significant variables ( except curative resection ) , tnm stage ( stage ii : hr , 3.545 ; 95% ci , 1.571 to 7.997 ; p=0.002 ; stage iii : hr , 6.115 ; 95% ci , 2.818 to 13.447 ; p<0.001 ; stage iv : hr , 15.928 ; 95% ci , 6.974 to 36.378 ; p<0.001 ) , an interval longer than four days for normalization of wbc counts ( hr , 1.509 ; 95% ci , 0.999 to 2.278 ; p=0.050 ) , and monocyte count ( hr , 2.133 ; 95% ci , 1.262 to 3.605 ; p=0.005 ) were independently associated with cancer - free survival ( table 3 ) . the male - to - female ratio of patients was 1.48:1 , and the mean age was 62.311.5 years . we observed stage i disease in 140 patients ( 23.3% ) , stage ii disease in 178 patients ( 29.7% ) , stage iii0 disease in 208 patients ( 34.7% ) , and stage iv disease in 52 patients ( 8.7% ) . postoperative wbc counts exceeded 10,000/mm in 417 patients ( 69.5% ) , including 74 cases ( 45.8% ) in group i , 198 cases ( 33.0% ) in group ii , and 127 cases ( 21.2% ) in group iii . preoperative wbc counts correlated significantly with disease stage ( p=0.047 ) . in particular , increased preoperative wbc counts were noted in patients at stages ii and iii ( data not shown ) . however , neither postoperative wbc counts nor the number of days required for the leukocyte count to fall below 10,000/mm correlated with disease stage ( p=0.394 and p=0.402 , respectively ) . the results did not show a significant difference in the postoperative wbc normalization period for the combined resection of other organs ( p=0.329 ) . the duration of postoperative hospital stay , according to the wbc normalization period , was as follows : group i , 127.1 days ( median , 11.0 days ) ; group ii , 12.37.3 days ( median , 10.0 days ) ; group iii , 14.87.9 days ( median , 12.0 days ) . there are significant correlations between the groups with regards to preoperative neutrophil count ( p<0.001 ) , lymphocyte count ( p=0.001 ) , monocyte count ( p<0.001 ) , and platelet count ( p=0.001 ) , but no correlation between preoperative nlr ( p=0.47 ) ( table 1 ) . in univariate survival analyses , tumor , node , metastasis ( tnm ) stage and monocyte count in addition , cancer - free survival outcomes were significantly worse in patients who required more than four days for the normalization of wbc count ( hazard ratio [ hr ] , 1.652 ; 95% confidence interval [ ci ] , 1.105 to 2.468 ; p=0.014 ) . a tnm stage greater than ii and nlr is significantly associated with the duration of overall survival ( p<0.050 and p=0.045 , respectively ) , but no correlation was observed between dsnlc and duration of overall survival ( table 2 ) . cancer - free and overall survival curves are shown according to the stage , time for leukocyte count to decline below 10,000/mm and monocyte count in figs . no correlation was revealed between the elevated preoperative and postoperative wbc counts ( 1010 ) and cancer - free or overall survival . in a multivariate analysis of these significant variables ( except curative resection ) , tnm stage ( stage ii : hr , 3.545 ; 95% ci , 1.571 to 7.997 ; p=0.002 ; stage iii : hr , 6.115 ; 95% ci , 2.818 to 13.447 ; p<0.001 ; stage iv : hr , 15.928 ; 95% ci , 6.974 to 36.378 ; p<0.001 ) , an interval longer than four days for normalization of wbc counts ( hr , 1.509 ; 95% ci , 0.999 to 2.278 ; p=0.050 ) , and monocyte count ( hr , 2.133 ; 95% ci , 1.262 to 3.605 ; p=0.005 ) were independently associated with cancer - free survival ( table 3 ) . in univariate survival analyses , tumor , node , metastasis ( tnm ) stage and monocyte count were significantly associated with cancer - free survival ( p<0.01 for both parameters ) . in addition , cancer - free survival outcomes were significantly worse in patients who required more than four days for the normalization of wbc count ( hazard ratio [ hr ] , 1.652 ; 95% confidence interval [ ci ] , 1.105 to 2.468 ; p=0.014 ) . a tnm stage greater than ii and nlr is significantly associated with the duration of overall survival ( p<0.050 and p=0.045 , respectively ) , but no correlation was observed between dsnlc and duration of overall survival ( table 2 ) . cancer - free and overall survival curves are shown according to the stage , time for leukocyte count to decline below 10,000/mm and monocyte count in figs . no correlation was revealed between the elevated preoperative and postoperative wbc counts ( 1010 ) and cancer - free or overall survival . in a multivariate analysis of these significant variables ( except curative resection ) , tnm stage ( stage ii : hr , 3.545 ; 95% ci , 1.571 to 7.997 ; p=0.002 ; stage iii : hr , 6.115 ; 95% ci , 2.818 to 13.447 ; p<0.001 ; stage iv : hr , 15.928 ; 95% ci , 6.974 to 36.378 ; p<0.001 ) , an interval longer than four days for normalization of wbc counts ( hr , 1.509 ; 95% ci , 0.999 to 2.278 ; p=0.050 ) , and monocyte count ( hr , 2.133 ; 95% ci , 1.262 to 3.605 ; p=0.005 ) were independently associated with cancer - free survival ( table 3 ) . as mentioned above , our experimental mouse model creates an inflammatory reaction in the peritoneum and confers an increased metastatic burden at the wound site compared with the undamaged normal peritoneal tissues . consequently , an increase in the mesothelial cells and other inflammatory cells constitute a pro - metastatic microenvironment in the normal peritoneum . in addition , in the early stages of wound healing , wound metastasis occurs . in addition , the nlr and plr have been shown to have an independent prognostic value in a variety of cancers [ 5 - 8 ] . these studies demonstrate that the prognostic values of nlr or plr are independent of tumor stage , conventional scoring systems and treatment modalities . show that monocyte count and gps are independently associated with cancer - specific survival . in this study , monocyte count was significantly associated with cancer - free survival in a univariate survival analysis . postoperative wbc count ( 1010 ) did not reveal any correlation with cancer - free survival or overall survival . most studies mentioned above examined the effects of the preoperative inflammatory condition . however , the research presented in this report does not focus on one point of time , but rather on a certain period of an inflammatory condition that will have a great effect on the metastatic microenvironment . thus , in the current study , regardless of the preoperative and postoperative inflammatory status , the groups were classified by the normalization of wbc count . the period in which the metastatic microenvironment is exposed to inflammation was defined as the period until the wbc count is normalized . these results demonstrate that the differences in surgical methods according to stage did not affect the postoperative wbc count or dsnlc . in a univariate analysis , the presence of increased wbcs in the preoperative stages did not affect the duration of disease - free survival or overall survival . on the other hand , sustained inflammation over four days did affect disease - free survival and overall survival . moreover , dsnlc was independently associated with cancer - free survival in a multivariate survival analysis . this may suggest that activation of innate immunity , rather than down - regulation of acquired immunity , is the most important factor in determining a poor outcome in patients with colorectal cancer . in addition , an increase in wbcs is a better prognostic parameter than an increase in monocytes or nlr . in this study , an early operative increase in wbc correlates with increased innate immunity , and this increase is predictive of a poorer outcome for colorectal cancer more so than monocyte or nlr increase , which only shows an increase in innate immunity at one point of time . preoperative wbc count shows a correlation with an increase of cancer stage , with the exception of stage iv . iv , since neoadjuvant chemotherapy can be performed , an increase in wbcs may be lower than in stages ii and iii . in addition , patients receiving preoperative radiation show statistically significant shorter periods for normalization of wbc counts compared to patients not receiving radiation therapy . however , in colorectal cancer , the systemic effect of radiotherapy may not be substantial , and instead , the accompanying chemotherapy may be the reason for a shorter period of normalization of wbc count . in addition , there is a shorter wbc count normalization period following laparoscopic surgery ; this is an advantage of minimally invasive surgery . however , there is no difference in the duration of disease - free survival between laparoscopic and open surgery . whatever the causative factor , the increase in postoperative wbcs affects the metastatic microenvironment and eventually contributes to cancer recurrences . this fact shows that complicated colorectal cancer was not correlative with the normalization of wbc count unless the lesion persists . in addition , the wbc count normalization period after surgery did not correlate with the presence of combined resection , but rather correlated with the duration of postoperative hospital stay . in this study , three factors ( surgical approach method , preoperative radiation therapy and postoperative hospital stay ) were found to be associated with the difference in time of the period of normalization of wbc count . in summary , postoperative early inflammatory phase and preoperative monocyte count correlate with the poor prognosis of colon cancer even after the calibration of stage . thus , we can conclude that preoperativeand postoperative inflammatory response and period unfavorably affect the metastatic microenvironment .

chronic obstructive pulmonary disease ( copd ) is a chronic respiratory disease characterized by a progressive decline in lung function and accompanied by respiratory symptoms , primarily dyspnea , cough , and sputum production.1,2 given the progressive nature of the disease , the aim of treatments is to reduce symptoms and exacerbations , thereby improving health - related quality of life . currently , the global initiative for chronic obstructive lung disease ( gold ) guidelines recommend initiation with a short - acting bronchodilator , followed by the addition of long - acting bronchodilators as symptoms and/or risk of exacerbations increase , and eventually adding inhaled steroids and/or roflumilast.2 commonly used bronchodilators include inhaled long - acting beta2-agonists ( labas ) ( eg , twice - daily formoterol , twice - daily salmeterol , and more recently once - daily indacaterol ) , inhaled long - acting antimuscarinic agents ( lamas ) ( eg , once - daily tiotropium , and recently twice - daily aclidinium and once - daily glycopyrronium ) , and oral methylxanthines ( eg , theophylline ) . bronchodilators improve lung function , reduce symptoms and exacerbations , and improve health - related quality of life.2 bronchodilators are frequently used in combination when higher effect sizes are warranted . in trials where a laba was administered in addition to a lama , the observed effect sizes were greater than those with either laba or lama alone,3,4 and in some cases the effects of the monotherapies have been shown to be additive when used in combination.5 however , effect sizes of bronchodilator monotherapies are not always additive , as seen in published trials of bronchodilator combinations . most recently , this was demonstrated with indacaterol , where the effect size of indacaterol administered with tiotropium compared with tiotropium monotherapy was much lower than the effect sizes observed in studies that compared indacaterol monotherapy with placebo.6 furthermore , more recent trials have found that monotherapy treatments have shown lower increases in forced expiratory volume in 1 second ( fev1 ) , compared to trials of the same investigational drug performed 5 or 10 years earlier . this may be due to evolution in trial designs , where , for example , later trials are more likely to permit concomitant treatment with other bronchodilators as part of usual care.7 additionally , there is evidence that the effect of bronchodilators varies depending on the severity of the patient s copd . patients with very severe copd show lower improvement of fev1 when treated with bronchodilators , compared with patients with moderate or severe copd.8,9 the laba olodaterol has been developed as a once - daily long - acting bronchodilator for maintenance treatment of copd . phase iii clinical trials of olodaterol have been completed , and the product , at the 5 mcg per day dose , has been approved in a number of european countries and canada , and is currently under regulatory review in the united states by the food and drug administration ( fda)10 and other regulatory agencies . to date , indacaterol is the only other once - daily laba approved and made available in many countries worldwide . indacaterol has been licensed in the united states and canada at a dose of 75 mcg per day.11,12 in most other countries , two doses are licensed : 150 mcg per day and 300 mcg per day for those patients for whom 150 mcg is not sufficient.13 in the absence of head - to - head , randomized controlled clinical trials ( rcts ) conducted on labas , the primary objective of this systematic literature review and meta - analysis was to provide estimates of relative efficacy of two new once - daily labas , olodaterol and indacaterol . a network meta - analysis was performed to indirectly compare the two treatments , linked through common treatment comparators . the review made two specific treatment comparisons : olodaterol 5 mcg compared with indacaterol 150 mcg , and olodaterol 5 mcg compared with indacaterol 75 mcg . a comprehensive and systematic literature review to identify rcts studying olodaterol and indacaterol in copd was undertaken to establish the evidence base for use in the meta - analyses . a systematic literature review was performed according to a prespecified protocol , to comply with the requirements of the national institute for health and care excellence single technology assessment.14 the following electronic databases were searched : 1 ) the cochrane library , including the cochrane database of systematic reviews , the cochrane central register of controlled trials , and the database of abstracts of reviews of effectiveness ; 2 ) medline and medline in - process ; and 3 ) embase . in addition , the following additional sources were searched to identify conference abstracts and published and unpublished studies : web sites for conference abstracts from the following organizations : american thoracic society ( 2010 and 2011 ) , european respiratory society ( 2009 and 2010 ) , and british thoracic society ( winter 2009 and winter 2010 ) ; bibliographic reference lists of the included studies and reviews ( searched for other relevant published studies ) ; trialtrove ( http://www.citeline.com/products/trialtrove/overview/ ) ; clinicaltrials.gov ( http://www.clinicaltrials.gov/ ) ; web sites for the fda and european medicines agency ; and the boehringer ingelheim trial database for olodaterol studies . the search strategies relied on three sets of terms : health condition of interest ( copd ) , study type ( rcts ) , and intervention ( olodaterol and/or indacaterol ) . appropriate terms were combined , and iterative searches were performed using other relevant terms and concepts . the searches were limited to articles published from 1 january 1990 , through 5 august 2011 , given the development timelines of the two compounds under study . articles in languages other than english were excluded . during systematic review and meta - analysis planning and execution , data for olodaterol were not publicly available , therefore , data on file with boehringer ingelheim were used . most data are now publicly available in the primary publications and/or on the fda web site , in the briefing documents for a pulmonary and allergy drug advisory committee meeting held in january 2013.10,1518 all data , including data not published in the pulmonary and allergy drug advisory committee document , are listed in the supplementary material . any disagreements were discussed with the research team to reach a consensus about study inclusion . during screening , further exclusions of studies were based on lack of outcome data , lack of a link into the treatment network , and study length ( ie , studies lasting less than 6 weeks ) . data extraction was performed by an experienced literature reviewer and quality checked by the statistician responsible for the meta - analyses . quality assessments were conducted for each included trial , using quality criteria recommended by the centre for reviews and dissemination.19 full details of this assessment can be found in the supplementary material . similar trough fev1 data at week 6 were permitted in the absence of week 12 data . other lung - function outcomes were considered for analysis , but trough fev1 was selected because it was the only outcome consistently reported across the evidence base . while the area under the fev1-time curve ( 03 hours post - dose ) was reported in all olodaterol trials , it was not reported for the indacaterol 150 or 75 mcg studies and so could not be included in the meta - analysis . st george s respiratory questionnaire ( sgrq ) total score was analyzed as change from baseline at week 12 . the proportion of responders based on sgrq total score was analyzed at week 12 , response was defined as a decrease ( improvement ) in sgrq total score of at least 4 points . transition dyspnea index ( tdi ) , by definition a change from baseline measure , was analyzed at week 12 . use of rescue medication ( eg , as - needed salbutamol ) was captured and analyzed as change from baseline in average number of puffs per day , using the maximum observed time for available data . finally , the proportion of patients experiencing at least one exacerbation , captured as a copd worsening adverse event , was analyzed for trials with a treatment duration of 24 weeks or longer reporting data for this outcome . some data imputations were required for standard errors , to maximize inclusion of change - from - baseline data into the meta - analyses . these imputations , together with further details on endpoint definitions are provided , where appropriate , in the data tables in the supplementary material . for network meta - analyses of dichotomous outcomes , a mixed log - binomial model was fit to estimate relative risks and confidence intervals . for network meta - analyses of continuous outcomes , a mixed normal - response model was fit to estimate mean differences and confidence intervals . these models included fixed treatment effects , fixed study effects , and random effects for the interaction between treatment and study . the random effects were included to allow the treatment effects to vary from study to study . the models were fit adopting an estimation approach using proc glimmix in sas version 9.2 ( sas institute inc . , cary , nc , usa ) and the methodology outlined by jones et al.20 in addition to network meta - analyses , direct meta - analyses were performed using standard techniques to assess heterogeneity within the network and to support the analysis that required use of the adjusted indirect comparison technique.2123 heterogeneity was investigated in several ways . for heterogeneity in trial designs , the a priori desire is to include a homogeneous set of trials in the network . to this end , trials were included / excluded based on permitted respiratory comedication during the trials ; patient subgroups of data were used where patient - level data were available ; and two additional indacaterol studies identified after completion of the systematic review were included,24 which allowed comparison of indacaterol with olodaterol when given with concomitant bronchodilation . for heterogeneity in analyses / results , the generalized chi - squared statistic divided by the remaining degrees of freedom ( should produce values close to 1.0 for a well - fitting model ) was calculated and heterogeneity tests for direct meta - analyses were performed where appropriate . a systematic literature review was performed according to a prespecified protocol , to comply with the requirements of the national institute for health and care excellence single technology assessment.14 the following electronic databases were searched : 1 ) the cochrane library , including the cochrane database of systematic reviews , the cochrane central register of controlled trials , and the database of abstracts of reviews of effectiveness ; 2 ) medline and medline in - process ; and 3 ) embase . in addition , the following additional sources were searched to identify conference abstracts and published and unpublished studies : web sites for conference abstracts from the following organizations : american thoracic society ( 2010 and 2011 ) , european respiratory society ( 2009 and 2010 ) , and british thoracic society ( winter 2009 and winter 2010 ) ; bibliographic reference lists of the included studies and reviews ( searched for other relevant published studies ) ; trialtrove ( http://www.citeline.com/products/trialtrove/overview/ ) ; clinicaltrials.gov ( http://www.clinicaltrials.gov/ ) ; web sites for the fda and european medicines agency ; and the boehringer ingelheim trial database for olodaterol studies . the search strategies relied on three sets of terms : health condition of interest ( copd ) , study type ( rcts ) , and intervention ( olodaterol and/or indacaterol ) . appropriate terms were combined , and iterative searches were performed using other relevant terms and concepts . the searches were limited to articles published from 1 january 1990 , through 5 august 2011 , given the development timelines of the two compounds under study . articles in languages other than english were excluded . during systematic review and meta - analysis planning and execution , data for olodaterol were not publicly available , therefore , data on file with boehringer ingelheim were used . most data are now publicly available in the primary publications and/or on the fda web site , in the briefing documents for a pulmonary and allergy drug advisory committee meeting held in january 2013.10,1518 all data , including data not published in the pulmonary and allergy drug advisory committee document , are listed in the supplementary material . any disagreements were discussed with the research team to reach a consensus about study inclusion . during screening , further exclusions of studies were based on lack of outcome data , lack of a link into the treatment network , and study length ( ie , studies lasting less than 6 weeks ) . data extraction was performed by an experienced literature reviewer and quality checked by the statistician responsible for the meta - analyses . quality assessments were conducted for each included trial , using quality criteria recommended by the centre for reviews and dissemination.19 full details of this assessment can be found in the supplementary material . similar trough fev1 data at week 6 were permitted in the absence of week 12 data . other lung - function outcomes were considered for analysis , but trough fev1 was selected because it was the only outcome consistently reported across the evidence base . while the area under the fev1-time curve ( 03 hours post - dose ) was reported in all olodaterol trials , it was not reported for the indacaterol 150 or 75 mcg studies and so could not be included in the meta - analysis . st george s respiratory questionnaire ( sgrq ) total score was analyzed as change from baseline at week 12 . the proportion of responders based on sgrq total score was analyzed at week 12 , response was defined as a decrease ( improvement ) in sgrq total score of at least 4 points . transition dyspnea index ( tdi ) , by definition a change from baseline measure , was analyzed at week 12 . use of rescue medication ( eg , as - needed salbutamol ) was captured and analyzed as change from baseline in average number of puffs per day , using the maximum observed time for available data . finally , the proportion of patients experiencing at least one exacerbation , captured as a copd worsening adverse event , was analyzed for trials with a treatment duration of 24 weeks or longer reporting data for this outcome . some data imputations were required for standard errors , to maximize inclusion of change - from - baseline data into the meta - analyses . these imputations , together with further details on endpoint definitions are provided , where appropriate , in the data tables in the supplementary material . for network meta - analyses of dichotomous outcomes , a mixed log - binomial model was fit to estimate relative risks and confidence intervals . for network meta - analyses of continuous outcomes , a mixed normal - response model was fit to estimate mean differences and confidence intervals . these models included fixed treatment effects , fixed study effects , and random effects for the interaction between treatment and study . the random effects were included to allow the treatment effects to vary from study to study . the models were fit adopting an estimation approach using proc glimmix in sas version 9.2 ( sas institute inc . , cary , nc , usa ) and the methodology outlined by jones et al.20 in addition to network meta - analyses , direct meta - analyses were performed using standard techniques to assess heterogeneity within the network and to support the analysis that required use of the adjusted indirect comparison technique.2123 heterogeneity was investigated in several ways . for heterogeneity in trial designs , the a priori desire is to include a homogeneous set of trials in the network . to this end , trials were included / excluded based on permitted respiratory comedication during the trials ; patient subgroups of data were used where patient - level data were available ; and two additional indacaterol studies identified after completion of the systematic review were included,24 which allowed comparison of indacaterol with olodaterol when given with concomitant bronchodilation . for heterogeneity in analyses / results , the generalized chi - squared statistic divided by the remaining degrees of freedom ( should produce values close to 1.0 for a well - fitting model ) was calculated and heterogeneity tests for direct meta - analyses were performed where appropriate . a total of 142 titles were retrieved through database searches and a further 31 through other sources such as conference web sites . after removal of duplicates , 110 titles and abstracts were screened . after applying the screening inclusion and exclusion criteria to both the titles / abstracts and the full - text articles , 23 publications , which reported data from a total of ten indacaterol trials ( b1302,25 b2201,26 study b2354,27 study b2355,28 inhance,29 inlight-1,30 inlight-2,31 insist,32 intensity,33 and involve34 ) , were eligible for meta - analysis , outlined in the prisma diagram in figure 1.35 from the boehringer ingelheim trial database search , eight olodaterol trials were identified that met the inclusion criteria ( study numbers 1222.11 [ nct00782210],15 1222.12 [ nct00782509],15 1222.13 [ nct00793624],16 1222.14 [ nct00796653],16 1222.24 [ nct00931385],17 1222.25 [ nct00932646],17 1222.39 [ nct01040689],18 and 1222.40 [ nct01040728]).18 during the meta - analysis phase of the project , two further publications were identified and included in the evidence base.24,36 cope et al36 presented a network meta - analysis of four indacaterol trials ( involve , inhance , inlight-1 , and inlight-2 ) already included in the evidence base . mahler et al24 presented results for two indacaterol trials ( intrust-1 and intrust-2 ) that were not otherwise identified at the time of the literature searches . table 1 lists the 20 included studies ( 18 from the systematic review and 2 identified after the review ) and provides details on the included treatments , sample sizes , concomitant bronchodilator use , and whether the trials permitted patients with very severe copd ( gold stage iv).37 further trial / baseline characteristics are presented in the supplementary material . the supplementary material also presents data for each of the outcomes for analysis and the descriptions of how the data were captured from each trial . the resulting treatment network for this evidence base is presented in the supplementary material ( figure a1 ) . the different doses of olodaterol and indacaterol were considered as unique entities in this network and in these meta - analyses . figure 2 presents an abbreviated version of the complete network and highlights the trials contributing to the indirect comparisons of primary interest ( ie , the comparison of olodaterol 5 mcg with indacaterol 75 mcg and with indacaterol 150 mcg ) . an important difference among the studies included in the evidence base is whether concomitant use of noninvestigational copd treatments was permitted in the trials by design . specifically , the olodaterol trials allowed concomitant use of lamas ( tiotropium ) , short - acting muscarinic antagonists ( samas ) ( ipratropium ) , inhaled corticosteroids , and xanthines ( theophylline ) . four of the eight included olodaterol trials used stratified randomization according to lama use to ensure a balance in tiotropium users across treatment arms . two olodaterol trials did not permit concomitant lama treatment , because tiotropium was one of the investigational drugs . none of the indacaterol trials originally identified in the systematic literature review permitted any concomitant bronchodilator use ( ie , samas and lamas were not allowed ) or xanthine use ; all of the indacaterol trials permitted use of inhaled corticosteroids . in contrast , the two intrust trials required the co - administration of indacaterol with tiotropium;24 thus , 100% of patients in those trials received tiotropium in addition to indacaterol or placebo ( inhaled steroids were also allowed , but not xanthines ) . as - needed short - acting beta - agonists were permitted as rescue medication in all olodaterol and indacaterol trials , and their use was captured via the rescue medication outcome . in addition , there were differences in the patient populations tested in the trials ; while the olodaterol trials included patients with moderate to very severe copd ( gold ii to gold iv),37 the indacaterol trials included only patients with moderate to severe copd ( ie , excluded patients with very severe copd [ gold stage iv]).37 given the findings from the trial characteristics summary ( ie , to take into account heterogeneity in trial design , specifically concomitant bronchodilator use ) , two analyses were performed using subgroups of trials or patients that could be considered to be subject to similar trial conditions and therefore comparable . given the data availability , these analyses could only be performed for the change from baseline in trough fev1 at week 12 . lama - free therapy and included all trials in which patients did not receive concomitant lama ( ie , all indacaterol trials identified in the original systematic review and the olodaterol trials 1222.39 and 1222.40 ) . lama add - on therapy and included all trials / subgroups in which all patients received concomitant or co - administered lama ( ie , stratified subgroups of olodaterol trials 1222.11 , 1222.12 , 1222.13 , and 1222.14 ; intrust trials for indacaterol ) . because intrust had only placebo as a common comparator , direct meta - analysis and the adjusted indirect comparison technique were used for this analysis . a third analysis population incorporated all trials ( full network ) identified by the systematic review and was considered a sensitivity analysis for the trough fev1 outcome . given data availability , this population was the only possible analysis population for the other outcomes . change from baseline in trough fev1 for each of the analysis populations . for the lama - free analysis , which included only trials explicitly excluding concomitant use of lama treatment , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg . for the lama add - on analysis , which included only trials / subgroups receiving either concomitant or co - administered lama treatment , no differences were seen between olodaterol 5 mcg and indacaterol 150 mcg . no data were available for indacaterol 75 mcg for the lama add - on analysis . for the sensitivity analysis of the full network , the indirect comparisons favored indacaterol , statistically significantly for the 150 mcg dose . figure 4 presents the indirect treatment comparison results for the continuous outcomes tdi , change from baseline in sgrq total score , and change from baseline in rescue medication puffs per day for the full network analysis . despite the known heterogeneity among the trials included in the full network , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg . figure 5 presents the indirect treatment comparison results for the dichotomous outcomes sgrq response and proportion of patients with exacerbations for the full network analysis . again , despite the known heterogeneity among the trials , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg , where data were available . as shown in the separate analyses for change from baseline in trough fev1 , the identified heterogeneity in trial design with respect to concomitant bronchodilator use impacted the results and conclusions of the meta - analyses . table 3 presents generalized chi - squared divided by the remaining degrees of freedom statistics for each network meta - analysis . because the estimates were all fairly close to 1 , the models could be assumed to fit the data well , but this does not eradicate the issues of known trial heterogeneity . table 4 presents direct meta - analyses heterogeneity test results for comparisons with placebo ( where possible ) . these estimates do not display significant and consistent heterogeneity of trial results within each arm of the network diagram , but it should be noted that such heterogeneity tests can lack statistical power with small trial numbers . additionally , information regarding the consistency assumption within the network as a whole is not provided due to the lack of head - to - head data between the selected treatments of interest . forest plots for the direct meta - analyses are presented in the supplementary material ( figure a2 through figure a21 ) . a total of 142 titles were retrieved through database searches and a further 31 through other sources such as conference web sites . after removal of duplicates , 110 titles and abstracts were screened . after applying the screening inclusion and exclusion criteria to both the titles / abstracts and the full - text articles , 23 publications , which reported data from a total of ten indacaterol trials ( b1302,25 b2201,26 study b2354,27 study b2355,28 inhance,29 inlight-1,30 inlight-2,31 insist,32 intensity,33 and involve34 ) , were eligible for meta - analysis , outlined in the prisma diagram in figure 1.35 from the boehringer ingelheim trial database search , eight olodaterol trials were identified that met the inclusion criteria ( study numbers 1222.11 [ nct00782210],15 1222.12 [ nct00782509],15 1222.13 [ nct00793624],16 1222.14 [ nct00796653],16 1222.24 [ nct00931385],17 1222.25 [ nct00932646],17 1222.39 [ nct01040689],18 and 1222.40 [ nct01040728]).18 during the meta - analysis phase of the project , two further publications were identified and included in the evidence base.24,36 cope et al36 presented a network meta - analysis of four indacaterol trials ( involve , inhance , inlight-1 , and inlight-2 ) already included in the evidence base . mahler et al24 presented results for two indacaterol trials ( intrust-1 and intrust-2 ) that were not otherwise identified at the time of the literature searches . table 1 lists the 20 included studies ( 18 from the systematic review and 2 identified after the review ) and provides details on the included treatments , sample sizes , concomitant bronchodilator use , and whether the trials permitted patients with very severe copd ( gold stage iv).37 further trial / baseline characteristics are presented in the supplementary material . the supplementary material also presents data for each of the outcomes for analysis and the descriptions of how the data were captured from each trial . the resulting treatment network for this evidence base is presented in the supplementary material ( figure a1 ) . the different doses of olodaterol and indacaterol were considered as unique entities in this network and in these meta - analyses . figure 2 presents an abbreviated version of the complete network and highlights the trials contributing to the indirect comparisons of primary interest ( ie , the comparison of olodaterol 5 mcg with indacaterol 75 mcg and with indacaterol 150 mcg ) . an important difference among the studies included in the evidence base is whether concomitant use of noninvestigational copd treatments was permitted in the trials by design . specifically , the olodaterol trials allowed concomitant use of lamas ( tiotropium ) , short - acting muscarinic antagonists ( samas ) ( ipratropium ) , inhaled corticosteroids , and xanthines ( theophylline ) . four of the eight included olodaterol trials used stratified randomization according to lama use to ensure a balance in tiotropium users across treatment arms . two olodaterol trials did not permit concomitant lama treatment , because tiotropium was one of the investigational drugs . none of the indacaterol trials originally identified in the systematic literature review permitted any concomitant bronchodilator use ( ie , samas and lamas were not allowed ) or xanthine use ; all of the indacaterol trials permitted use of inhaled corticosteroids . in contrast , the two intrust trials required the co - administration of indacaterol with tiotropium;24 thus , 100% of patients in those trials received tiotropium in addition to indacaterol or placebo ( inhaled steroids were also allowed , but not xanthines ) . as - needed short - acting beta - agonists were permitted as rescue medication in all olodaterol and indacaterol trials , and their use was captured via the rescue medication outcome . in addition , there were differences in the patient populations tested in the trials ; while the olodaterol trials included patients with moderate to very severe copd ( gold ii to gold iv),37 the indacaterol trials included only patients with moderate to severe copd ( ie , excluded patients with very severe copd [ gold stage iv]).37 given the findings from the trial characteristics summary ( ie , to take into account heterogeneity in trial design , specifically concomitant bronchodilator use ) , two analyses were performed using subgroups of trials or patients that could be considered to be subject to similar trial conditions and therefore comparable . given the data availability , these analyses could only be performed for the change from baseline in trough fev1 at week 12 . lama - free therapy and included all trials in which patients did not receive concomitant lama ( ie , all indacaterol trials identified in the original systematic review and the olodaterol trials 1222.39 and 1222.40 ) . the second analysis population was lama add - on therapy and included all trials / subgroups in which all patients received concomitant or co - administered lama ( ie , stratified subgroups of olodaterol trials 1222.11 , 1222.12 , 1222.13 , and 1222.14 ; intrust trials for indacaterol ) . because intrust had only placebo as a common comparator , direct meta - analysis and the adjusted indirect comparison technique were used for this analysis . a third analysis population incorporated all trials ( full network ) identified by the systematic review and was considered a sensitivity analysis for the trough fev1 outcome . given data availability , this population was the only possible analysis population for the other outcomes . figure 3 presents the indirect treatment comparison results for the outcome change from baseline in trough fev1 for each of the analysis populations . for the lama - free analysis , which included only trials explicitly excluding concomitant use of lama treatment , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg . for the lama add - on analysis , which included only trials / subgroups receiving either concomitant or co - administered lama treatment , no data were available for indacaterol 75 mcg for the lama add - on analysis . for the sensitivity analysis of the full network , the indirect comparisons favored indacaterol , statistically significantly for the 150 mcg dose . figure 4 presents the indirect treatment comparison results for the continuous outcomes tdi , change from baseline in sgrq total score , and change from baseline in rescue medication puffs per day for the full network analysis . despite the known heterogeneity among the trials included in the full network , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg . figure 5 presents the indirect treatment comparison results for the dichotomous outcomes sgrq response and proportion of patients with exacerbations for the full network analysis . again , despite the known heterogeneity among the trials , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg , where data were available . as shown in the separate analyses for change from baseline in trough fev1 , the identified heterogeneity in trial design with respect to concomitant bronchodilator use impacted the results and conclusions of the meta - analyses . table 3 presents generalized chi - squared divided by the remaining degrees of freedom statistics for each network meta - analysis . because the estimates were all fairly close to 1 , the models could be assumed to fit the data well , but this does not eradicate the issues of known trial heterogeneity . table 4 presents direct meta - analyses heterogeneity test results for comparisons with placebo ( where possible ) . these estimates do not display significant and consistent heterogeneity of trial results within each arm of the network diagram , but it should be noted that such heterogeneity tests can lack statistical power with small trial numbers . additionally , information regarding the consistency assumption within the network as a whole is not provided due to the lack of head - to - head data between the selected treatments of interest . forest plots for the direct meta - analyses are presented in the supplementary material ( figure a2 through figure a21 ) . in the absence of a direct head - to - head comparison , indirect comparisons can provide exploratory insights into the relative effectiveness of olodaterol and indacaterol . the current study indirectly compares olodaterol and indacaterol , while accounting for the considerable systematic heterogeneity in trial design among olodaterol and indacaterol trials . evaluating lung function , trials / subgroups were selected in order to form indirect treatment comparisons based on patients subject to similar trial conditions , in which no evident differences between olodaterol and indacaterol were found . there were important differences among the olodaterol trials and indacaterol trials with respect to allowed concomitant copd medications . although all trials allowed concomitant inhaled steroid use and allowed the use of short - acting beta - agonists as rescue medication on an as - needed basis , the olodaterol trials also allowed concomitant use of lamas ( tiotropium ) , samas ( ipratropium ) , and xanthines ( theophylline ) , whereas the indacaterol trials did not . another notable difference was that olodaterol trials enrolled patients across the full disease severity spectrum , from moderate to very severe copd ( gold ii - iv),37 whereas the indacaterol trials were restricted to patients with moderate to severe copd ( gold ii / iii).37 this difference in patient populations , and therefore potential lung function response,8 could not be addressed in this meta - analysis because olodaterol trials were not stratified for disease severity and therefore using the subgroup excluding gold iv patients for the meta - analysis would not be a randomized comparison . the comparative efficacy of indacaterol and olodaterol differed depending on the subset of trials and patients included in the analyses . change from baseline in trough fev1 was similar among treatments when analyzing trials or data deemed to be comparable . in contrast , analyses not taking into consideration differences in allowed concomitant therapies can lead to inappropriate conclusions from the indirect comparisons . thus , selecting studies of similar design for the drugs of interest is important in order to estimate the true differences in effect size . acknowledging the known differences of the patient populations recruited to the trials ( very severe copd patients included in trials of olodaterol , but not indacaterol ) , it is interesting to note that the absolute difference seen in the sensitivity analysis for trough fev1 outcome is not replicated in either sgrq endpoint evaluating health - related quality of life , where there are no differences among the treatments , or in the other endpoints investigated ( exacerbations , rescue medication use , and tdi ) . assessing the results for external validity with previously published meta - analyses,36,3841 the direct meta - analyses matched closely with the estimated treatment effects for indacaterol ; however , because olodaterol data have only recently been published , the indirect comparisons have not been externally validated . the smaller effect sizes of laba administered in addition to a lama , compared with the effect sizes of a laba alone , were replicated . specifically , for indacaterol 150 mcg versus placebo , the effect size for trough fev1 change from baseline was 0.07 liters when studied in combination with concomitant lama ( supplementary material , figure a6 ) , and 0.17 liters when studied in the absence of concomitant lama ( supplementary material , figure a4 ) . as with all meta - analyses , certain limitations should be considered when interpreting the results of this meta - analysis . some between - trial differences in inclusion and exclusion criteria that influence the meta - analytic results were identified , especially with regard to the use of concomitant respiratory medication . the olodaterol trials could be considered to be studying treatment effects likely to be seen in real - world settings , whereas the indacaterol trials provide an estimate of absolute treatment effect . a limitation in the lama - add on analysis of the lung - function endpoint is that only add - on tiotropium , and no other important bronchodilators ( eg , samas or xanthines ) , could be investigated , thus limiting the generalizability of the findings . another limitation was that the add - on tiotropium in the olodaterol trials was concomitant medication , whereas in the indacaterol trials it was part of the investigational treatment . this limitation could lead to differences in application of , monitoring of , and compliance with the treatment . a limitation with regard to the lama - free analysis for the lung - function endpoint was that the included olodaterol trials were cross - over trials lasting 6 weeks , whereas the indacaterol trials were parallel - group trials lasting 12 weeks . however , this trial heterogeneity may not influence the relative treatment effects and therefore indirect comparison results , because the relative treatment differences are maintained between week 6 and week 12 , based on studies comparing olodaterol 5 mcg and formoterol 12 mcg.10,1518 finally , analysis of endpoints other than change from baseline in trough fev1 were possible only on the evidence base of trials that are inherently dissimilar with respect to concomitant bronchodilator use . thus , these analyses should be interpreted with caution . in conclusion , based on the analyses of change from baseline in trough fev1 , when compared under similar trial conditions , olodaterol 5 mcg and indacaterol 75 mcg or 150 mcg seem to be equally effective in the treatment of patients with copd . only head - to - head studies would be able to confirm the equal effectiveness of olodaterol and indacaterol . this research highlights the importance of concomitant copd medication and study population when estimating treatment effects in copd clinical trials . mr roskell is a full - time employee of bresmed health solutions , but performed this work while employed by rti health solutions . he has the following conflicts of interest : he is a member of the gold scientific committees and supports the implementation of the gold guidelines recommendations . he has also received honoraria , consulting fees , and advisory board and speaking fees from bayer , pfizer , glaxosmithkline , boehringer ingelheim , and forest laboratories . he is the principal investigator for research grants at the university of texas health science center at san antonio and was paid for participating in multicenter clinical trials sponsored by glaxosmithkline and the national institutes of health . dr hamilton , dr disse , and dr becker are full - time employees of boehringer ingelheim gmbh .

purposein the absence of head - to - head clinical trials comparing the once - daily , long - acting beta2-agonists olodaterol and indacaterol for the treatment of chronic obstructive pulmonary disease ( copd ) , an indirect treatment comparison by systematic review and synthesis of the available clinical evidence was conducted.methodsa systematic literature review of randomized , controlled clinical trials in patients with copd was performed to evaluate the efficacy and safety of olodaterol and indacaterol . network meta - analysis and adjusted indirect comparison methods were employed to evaluate treatment efficacy , using outcomes based on trough forced expiratory volume in 1 second ( fev1 ) , transition dyspnea index , st george s respiratory questionnaire total score and response , rescue medication use , and proportion of patients with exacerbations.resultseighteen trials were identified for meta - analysis ( eight , olodaterol ; ten , indacaterol ) . olodaterol trials included patients of all severities , whilst indacaterol trials excluded patients with very severe copd . concomitant maintenance bronchodilator use was allowed in most olodaterol trials , but not in indacaterol trials . when similarly designed trials / data were analyzed for change from baseline in trough fev1 ( liters ) , the following mean differences ( 95% confidence interval ) were observed : trials excluding concomitant bronchodilator : indacaterol 75 mcg versus olodaterol 5 mcg , 0.005 ( 0.077 to 0.067 ) , and indacaterol 150 mcg versus olodaterol 5 mcg , 0.020 ( 0.036 to 0.077 ) ; trials with concomitant tiotropium : indacaterol 150 mcg versus olodaterol 5 mcg , 0.000 ( 0.043 to 0.042 ) . in sensitivity analyses of the full network , results for change from baseline in trough fev1 favored indacaterol , but this dataset suffered from trial design heterogeneity . for the other endpoints investigated , no statistically significant differences were found when analyzed in the full network.conclusionwhen compared under similar trial conditions , olodaterol and indacaterol have similar efficacy in patients with copd . this research highlights the importance of considering the concomitant copd medication when evaluating treatment effects in copd .

Introduction Methods Systematic review process Outcomes Statistical analyses Results Literature search results Trial characteristics, treatment network, and data availability Meta-analysis strategy Indirect comparison results Heterogeneity Discussion and conclusion Disclosure

chronic obstructive pulmonary disease ( copd ) is a chronic respiratory disease characterized by a progressive decline in lung function and accompanied by respiratory symptoms , primarily dyspnea , cough , and sputum production.1,2 given the progressive nature of the disease , the aim of treatments is to reduce symptoms and exacerbations , thereby improving health - related quality of life . currently , the global initiative for chronic obstructive lung disease ( gold ) guidelines recommend initiation with a short - acting bronchodilator , followed by the addition of long - acting bronchodilators as symptoms and/or risk of exacerbations increase , and eventually adding inhaled steroids and/or roflumilast.2 commonly used bronchodilators include inhaled long - acting beta2-agonists ( labas ) ( eg , twice - daily formoterol , twice - daily salmeterol , and more recently once - daily indacaterol ) , inhaled long - acting antimuscarinic agents ( lamas ) ( eg , once - daily tiotropium , and recently twice - daily aclidinium and once - daily glycopyrronium ) , and oral methylxanthines ( eg , theophylline ) . most recently , this was demonstrated with indacaterol , where the effect size of indacaterol administered with tiotropium compared with tiotropium monotherapy was much lower than the effect sizes observed in studies that compared indacaterol monotherapy with placebo.6 furthermore , more recent trials have found that monotherapy treatments have shown lower increases in forced expiratory volume in 1 second ( fev1 ) , compared to trials of the same investigational drug performed 5 or 10 years earlier . patients with very severe copd show lower improvement of fev1 when treated with bronchodilators , compared with patients with moderate or severe copd.8,9 the laba olodaterol has been developed as a once - daily long - acting bronchodilator for maintenance treatment of copd . indacaterol has been licensed in the united states and canada at a dose of 75 mcg per day.11,12 in most other countries , two doses are licensed : 150 mcg per day and 300 mcg per day for those patients for whom 150 mcg is not sufficient.13 in the absence of head - to - head , randomized controlled clinical trials ( rcts ) conducted on labas , the primary objective of this systematic literature review and meta - analysis was to provide estimates of relative efficacy of two new once - daily labas , olodaterol and indacaterol . the review made two specific treatment comparisons : olodaterol 5 mcg compared with indacaterol 150 mcg , and olodaterol 5 mcg compared with indacaterol 75 mcg . a comprehensive and systematic literature review to identify rcts studying olodaterol and indacaterol in copd was undertaken to establish the evidence base for use in the meta - analyses . a systematic literature review was performed according to a prespecified protocol , to comply with the requirements of the national institute for health and care excellence single technology assessment.14 the following electronic databases were searched : 1 ) the cochrane library , including the cochrane database of systematic reviews , the cochrane central register of controlled trials , and the database of abstracts of reviews of effectiveness ; 2 ) medline and medline in - process ; and 3 ) embase . in addition , the following additional sources were searched to identify conference abstracts and published and unpublished studies : web sites for conference abstracts from the following organizations : american thoracic society ( 2010 and 2011 ) , european respiratory society ( 2009 and 2010 ) , and british thoracic society ( winter 2009 and winter 2010 ) ; bibliographic reference lists of the included studies and reviews ( searched for other relevant published studies ) ; trialtrove ( http://www.citeline.com/products/trialtrove/overview/ ) ; clinicaltrials.gov ( http://www.clinicaltrials.gov/ ) ; web sites for the fda and european medicines agency ; and the boehringer ingelheim trial database for olodaterol studies . while the area under the fev1-time curve ( 03 hours post - dose ) was reported in all olodaterol trials , it was not reported for the indacaterol 150 or 75 mcg studies and so could not be included in the meta - analysis . st george s respiratory questionnaire ( sgrq ) total score was analyzed as change from baseline at week 12 . a systematic literature review was performed according to a prespecified protocol , to comply with the requirements of the national institute for health and care excellence single technology assessment.14 the following electronic databases were searched : 1 ) the cochrane library , including the cochrane database of systematic reviews , the cochrane central register of controlled trials , and the database of abstracts of reviews of effectiveness ; 2 ) medline and medline in - process ; and 3 ) embase . in addition , the following additional sources were searched to identify conference abstracts and published and unpublished studies : web sites for conference abstracts from the following organizations : american thoracic society ( 2010 and 2011 ) , european respiratory society ( 2009 and 2010 ) , and british thoracic society ( winter 2009 and winter 2010 ) ; bibliographic reference lists of the included studies and reviews ( searched for other relevant published studies ) ; trialtrove ( http://www.citeline.com/products/trialtrove/overview/ ) ; clinicaltrials.gov ( http://www.clinicaltrials.gov/ ) ; web sites for the fda and european medicines agency ; and the boehringer ingelheim trial database for olodaterol studies . while the area under the fev1-time curve ( 03 hours post - dose ) was reported in all olodaterol trials , it was not reported for the indacaterol 150 or 75 mcg studies and so could not be included in the meta - analysis . st george s respiratory questionnaire ( sgrq ) total score was analyzed as change from baseline at week 12 . after applying the screening inclusion and exclusion criteria to both the titles / abstracts and the full - text articles , 23 publications , which reported data from a total of ten indacaterol trials ( b1302,25 b2201,26 study b2354,27 study b2355,28 inhance,29 inlight-1,30 inlight-2,31 insist,32 intensity,33 and involve34 ) , were eligible for meta - analysis , outlined in the prisma diagram in figure 1.35 from the boehringer ingelheim trial database search , eight olodaterol trials were identified that met the inclusion criteria ( study numbers 1222.11 [ nct00782210],15 1222.12 [ nct00782509],15 1222.13 [ nct00793624],16 1222.14 [ nct00796653],16 1222.24 [ nct00931385],17 1222.25 [ nct00932646],17 1222.39 [ nct01040689],18 and 1222.40 [ nct01040728]).18 during the meta - analysis phase of the project , two further publications were identified and included in the evidence base.24,36 cope et al36 presented a network meta - analysis of four indacaterol trials ( involve , inhance , inlight-1 , and inlight-2 ) already included in the evidence base . table 1 lists the 20 included studies ( 18 from the systematic review and 2 identified after the review ) and provides details on the included treatments , sample sizes , concomitant bronchodilator use , and whether the trials permitted patients with very severe copd ( gold stage iv).37 further trial / baseline characteristics are presented in the supplementary material . figure 2 presents an abbreviated version of the complete network and highlights the trials contributing to the indirect comparisons of primary interest ( ie , the comparison of olodaterol 5 mcg with indacaterol 75 mcg and with indacaterol 150 mcg ) . none of the indacaterol trials originally identified in the systematic literature review permitted any concomitant bronchodilator use ( ie , samas and lamas were not allowed ) or xanthine use ; all of the indacaterol trials permitted use of inhaled corticosteroids . as - needed short - acting beta - agonists were permitted as rescue medication in all olodaterol and indacaterol trials , and their use was captured via the rescue medication outcome . in addition , there were differences in the patient populations tested in the trials ; while the olodaterol trials included patients with moderate to very severe copd ( gold ii to gold iv),37 the indacaterol trials included only patients with moderate to severe copd ( ie , excluded patients with very severe copd [ gold stage iv]).37 given the findings from the trial characteristics summary ( ie , to take into account heterogeneity in trial design , specifically concomitant bronchodilator use ) , two analyses were performed using subgroups of trials or patients that could be considered to be subject to similar trial conditions and therefore comparable . change from baseline in trough fev1 for each of the analysis populations . for the lama - free analysis , which included only trials explicitly excluding concomitant use of lama treatment , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg . for the lama add - on analysis , which included only trials / subgroups receiving either concomitant or co - administered lama treatment , no differences were seen between olodaterol 5 mcg and indacaterol 150 mcg . for the sensitivity analysis of the full network , the indirect comparisons favored indacaterol , statistically significantly for the 150 mcg dose . figure 4 presents the indirect treatment comparison results for the continuous outcomes tdi , change from baseline in sgrq total score , and change from baseline in rescue medication puffs per day for the full network analysis . despite the known heterogeneity among the trials included in the full network , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg . figure 5 presents the indirect treatment comparison results for the dichotomous outcomes sgrq response and proportion of patients with exacerbations for the full network analysis . again , despite the known heterogeneity among the trials , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg , where data were available . as shown in the separate analyses for change from baseline in trough fev1 , the identified heterogeneity in trial design with respect to concomitant bronchodilator use impacted the results and conclusions of the meta - analyses . after applying the screening inclusion and exclusion criteria to both the titles / abstracts and the full - text articles , 23 publications , which reported data from a total of ten indacaterol trials ( b1302,25 b2201,26 study b2354,27 study b2355,28 inhance,29 inlight-1,30 inlight-2,31 insist,32 intensity,33 and involve34 ) , were eligible for meta - analysis , outlined in the prisma diagram in figure 1.35 from the boehringer ingelheim trial database search , eight olodaterol trials were identified that met the inclusion criteria ( study numbers 1222.11 [ nct00782210],15 1222.12 [ nct00782509],15 1222.13 [ nct00793624],16 1222.14 [ nct00796653],16 1222.24 [ nct00931385],17 1222.25 [ nct00932646],17 1222.39 [ nct01040689],18 and 1222.40 [ nct01040728]).18 during the meta - analysis phase of the project , two further publications were identified and included in the evidence base.24,36 cope et al36 presented a network meta - analysis of four indacaterol trials ( involve , inhance , inlight-1 , and inlight-2 ) already included in the evidence base . table 1 lists the 20 included studies ( 18 from the systematic review and 2 identified after the review ) and provides details on the included treatments , sample sizes , concomitant bronchodilator use , and whether the trials permitted patients with very severe copd ( gold stage iv).37 further trial / baseline characteristics are presented in the supplementary material . figure 2 presents an abbreviated version of the complete network and highlights the trials contributing to the indirect comparisons of primary interest ( ie , the comparison of olodaterol 5 mcg with indacaterol 75 mcg and with indacaterol 150 mcg ) . none of the indacaterol trials originally identified in the systematic literature review permitted any concomitant bronchodilator use ( ie , samas and lamas were not allowed ) or xanthine use ; all of the indacaterol trials permitted use of inhaled corticosteroids . as - needed short - acting beta - agonists were permitted as rescue medication in all olodaterol and indacaterol trials , and their use was captured via the rescue medication outcome . in addition , there were differences in the patient populations tested in the trials ; while the olodaterol trials included patients with moderate to very severe copd ( gold ii to gold iv),37 the indacaterol trials included only patients with moderate to severe copd ( ie , excluded patients with very severe copd [ gold stage iv]).37 given the findings from the trial characteristics summary ( ie , to take into account heterogeneity in trial design , specifically concomitant bronchodilator use ) , two analyses were performed using subgroups of trials or patients that could be considered to be subject to similar trial conditions and therefore comparable . lama - free therapy and included all trials in which patients did not receive concomitant lama ( ie , all indacaterol trials identified in the original systematic review and the olodaterol trials 1222.39 and 1222.40 ) . figure 3 presents the indirect treatment comparison results for the outcome change from baseline in trough fev1 for each of the analysis populations . for the lama - free analysis , which included only trials explicitly excluding concomitant use of lama treatment , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg . for the lama add - on analysis , which included only trials / subgroups receiving either concomitant or co - administered lama treatment , no data were available for indacaterol 75 mcg for the lama add - on analysis . for the sensitivity analysis of the full network , the indirect comparisons favored indacaterol , statistically significantly for the 150 mcg dose . figure 4 presents the indirect treatment comparison results for the continuous outcomes tdi , change from baseline in sgrq total score , and change from baseline in rescue medication puffs per day for the full network analysis . despite the known heterogeneity among the trials included in the full network , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg . figure 5 presents the indirect treatment comparison results for the dichotomous outcomes sgrq response and proportion of patients with exacerbations for the full network analysis . again , despite the known heterogeneity among the trials , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg , where data were available . as shown in the separate analyses for change from baseline in trough fev1 , the identified heterogeneity in trial design with respect to concomitant bronchodilator use impacted the results and conclusions of the meta - analyses . in the absence of a direct head - to - head comparison , indirect comparisons can provide exploratory insights into the relative effectiveness of olodaterol and indacaterol . the current study indirectly compares olodaterol and indacaterol , while accounting for the considerable systematic heterogeneity in trial design among olodaterol and indacaterol trials . evaluating lung function , trials / subgroups were selected in order to form indirect treatment comparisons based on patients subject to similar trial conditions , in which no evident differences between olodaterol and indacaterol were found . although all trials allowed concomitant inhaled steroid use and allowed the use of short - acting beta - agonists as rescue medication on an as - needed basis , the olodaterol trials also allowed concomitant use of lamas ( tiotropium ) , samas ( ipratropium ) , and xanthines ( theophylline ) , whereas the indacaterol trials did not . another notable difference was that olodaterol trials enrolled patients across the full disease severity spectrum , from moderate to very severe copd ( gold ii - iv),37 whereas the indacaterol trials were restricted to patients with moderate to severe copd ( gold ii / iii).37 this difference in patient populations , and therefore potential lung function response,8 could not be addressed in this meta - analysis because olodaterol trials were not stratified for disease severity and therefore using the subgroup excluding gold iv patients for the meta - analysis would not be a randomized comparison . acknowledging the known differences of the patient populations recruited to the trials ( very severe copd patients included in trials of olodaterol , but not indacaterol ) , it is interesting to note that the absolute difference seen in the sensitivity analysis for trough fev1 outcome is not replicated in either sgrq endpoint evaluating health - related quality of life , where there are no differences among the treatments , or in the other endpoints investigated ( exacerbations , rescue medication use , and tdi ) . specifically , for indacaterol 150 mcg versus placebo , the effect size for trough fev1 change from baseline was 0.07 liters when studied in combination with concomitant lama ( supplementary material , figure a6 ) , and 0.17 liters when studied in the absence of concomitant lama ( supplementary material , figure a4 ) . however , this trial heterogeneity may not influence the relative treatment effects and therefore indirect comparison results , because the relative treatment differences are maintained between week 6 and week 12 , based on studies comparing olodaterol 5 mcg and formoterol 12 mcg.10,1518 finally , analysis of endpoints other than change from baseline in trough fev1 were possible only on the evidence base of trials that are inherently dissimilar with respect to concomitant bronchodilator use . in conclusion , based on the analyses of change from baseline in trough fev1 , when compared under similar trial conditions , olodaterol 5 mcg and indacaterol 75 mcg or 150 mcg seem to be equally effective in the treatment of patients with copd . only head - to - head studies would be able to confirm the equal effectiveness of olodaterol and indacaterol . this research highlights the importance of concomitant copd medication and study population when estimating treatment effects in copd clinical trials .

reports indicate that cervical pathology is common in mounted warfighters ( i.e. warfighters that are crewmembers or operators of ground- or air - based vehicles)1,2,3,4,5,6 . investigators have proposed that frequent bouts of ride induced forces ( such as vibration , shock , or + gz forces ) may increase the risk of pathology in the spine6,7,8,9 . the pain mounted warfighters experience from an underlying cervical pathology can be debilitating , compromise mission efficacy and completion , result in profiles that limit warfighter duty , or be career ending5 , 7 , 10,11,12,13 . studies14 , 15 examining fighter pilots specifically have reported a prevalence of cervical pathology ranging from 18.9% to 63.6% over a 12-month reporting period ( termed 1 yr prevalence period ) . studies using diagnostic examinations to determine the presence of cervical pathologies reported no significant difference between fighter pilots and controls , although fighter pilots appear to have a higher prevalence of neck pain16,17,18 . a comparison of cervical spine radiological changes in f-16 fighter pilots and matched controls found no significant difference between the groups ( 8% vs. 10% prevalence)16 . similar findings were also reported in a 13 yr longitudinal study of fighter aircraft personnel indicating occupational exposure to acceleration did not cause significant radiological changes in the spinal column when compared to matched controls17 . in contrast , a study revealed 3% of fighter pilots and 80% of transport pilots assessed by magnetic resonance imaging presented with cervical intervertebral disc degeneration18 . the investigators noted that age differences between the fighter and transport pilot group may play a role in the higher prevalence of pathology reported in the transport pilots . cervical pathology also appears to be prevalent in helicopter personnel , particularly pilots , with 43 to 57% of military helicopter pilots reporting cervical pain12 , 19 , 20 . the cervical pain experienced by pilots includes acute bouts of pain , as well as regular and continuous pain that hampers performance in both work - related duties and leisure time activities20 . the prevalence values were based on large sample sizes , however , many of these studies did not compare their findings to non - flying military control groups making it difficult to determine if the issue is related to flight or an outcome of military duty ( or life ) . in a study that did compare helicopter pilots to nonflying controls ( air traffic personnel ) , 5.7% of helicopter pilots ( n=1599 ) reported frequent and continuous cervical pain as compared to 20% of air traffic personnel ( n=123)2 . however , since the data were collected via clinical examination it is possible that fewer pilots , as compared to nonflying controls , reported feeling cervical pain out of fear of being grounded . investigation of the prevalence of cervical pathology in ground - based military vehicle personnel is lacking in the literature . the one report available21 indicates that danish main battle tank personnel were not at greater risk than other types of units ( infantry , signal , combat service support , engineers , and artillery ) for cervical spine pathology . however , there are many types of ground - based vehicles such as main battle tanks , humvees ( i.e. hmmwv ) and bradley fighting vehicle , each presenting different ride characteristics . for instance , the bradley fighting vehicles resembles a tank , but rides like an off - road vehicle . in civilian studies , investigators have reported both vibration and mechanical shock exposure as strongly associated with the prevalence of neck pain in populations that regularly use off - road all - terrain vehicles to help perform daily occupational tasks9 , 22 . more generally , a recent study23 exploring the incidence of neck and shoulder pain in civilian drivers of earth moving machines , forklifts , public buses and garbage machines reported a cumulative of incidences for neck and shoulder pain of 31.9% and 21.4% , respectfully . differences in results between the danish main battle tank and the study observing civilian drivers may in part due to age and level of fitness . in potentially less fit or older civilian population undergoing similar types of vehicle - ride exposure thus , the purpose of our study was to determine if evidence exists in the literature indicating a definite effect of vehicle - ride exposure on cervical pathology . this investigation utilized meta - analyses to synthesize quantitatively the available literature and to determine where future research is required . an online search using medline nioshtic , military & government collection ( ebsco ) , pubmed , and web of science identified articles published between january 1980 and november 2014 investigating the effects of vehicle - ride exposure on the occurrence of cervical pathology . the database search used multiple combinations of the following terms : vibration , spinal pathology , neck pain , intervertebral disc degeneration , degeneration , fracture , herniated disc , shock , loading , impact , repeated loading , industrial vibration , vehicle vibration , radicular , radiculopathy , neck strain , spine , mechanical shock , and g forces . in addition , we examined the reference list of the articles acquired through the search to find additional pertinent articles . the inclusion criteria were determined prior to the start of the database search in order to reduce any possible selection bias . to be included , studies needed to have a clear sampling method , control group , clearly defined exposure time , focus on cervical injuries resulting from vehicle - ride exposure , and explicit pathology . after screening the titles and abstracts , two reviewers ( r.k . and k.g . ) the reviewers resolved disagreements concerning article eligibility by consensus or by arbitration of a third reviewer ( j.s . ) if disagreement persisted . imagej ( rasband , w. , national institutes of health , usa ) enabled extraction of pertinent data contained in figures . all data were entered into a custom microsoft excel spreadsheet ( microsoft corporation , redmond , washington , usa ) and later transferred to the statistical software comprehensive meta - analysis version 2 ( biostat , englewood , nj , usa ) for statistical analyses . the meta - analysis utilized an unweighted random effects model using an odds ratio centered on 1.00 as the effect metric . ninety - five percent confidence intervals greater than 1.00 indicate that vehicle - ride exposure had a significant effect on cervical pathology . the sample sizes and number of injuries recorded for the treatment and control groups for each study were used in the analyses . subsequent sub - analyses were conducted using data collection method [ imaging ( i.e. magnetic resonance imaging , radiograph , computed tomography ) or questionnaire ] , vehicle type ( i.e. , ground - based vehicles , fixed - wing aircraft and rotary - wing aircraft ) and operator / other personnel as moderators . for the operator / other personnel moderator , the investigators defined operator as anyone flying ( i.e. fighter jet , jet , transport , or helicopter pilots ) or driving ( i.e. wheeled- or tracked - vehicle drivers ) a vehicle . other personnel represented anyone other than the operator on the vehicle ( such as a weapons systems operators , flight surgeon , flight engineer , tank commander , loader , or gunner ) . risk of publication bias was assessed using a funnel plot and a fail - safe number identified any presence of selective reporting within the studies . the scottish intercollegiate guidelines network ( sign ) grading system was used to evaluate the quality of evidence for each article included in the meta - analysis ( table 1table 1.study description and level of evidencestudysample populationmethod of assessmentlevel of evidenceaydog et al.16732 turkish military pilotsradiographs2 ( )byeon et al.26186 military helicopter korea air forceradiograph2 ( + ) drew et al.1035 u.s . high performance aircraft pilotsquestionnaire2 ( )hamalainen et al.312 finnish air force fighter pilot magnetic resonance imaging2 ( )hendriksen et al.1188 royal netherlands air force pilotsradiograph2 ( + ) mcbride et al.25516 locomotive engineers employed by the national rail carrier in new zealandquestionnaire2 ( + + ) nissen et al.21317 main battle tank personnelquestionnaire2 ( + ) petren - mallmin and linder2729 swedish air force pilotsmagnetic resonance imaging2 ( + + ) pippig et al.2286 pilots examined at the german air force institute of medicineclinical examination , peripheral neurological examination , computerized tomography , magnetic resonance imaging2 ( )rehn et al.24431 drivers of all - terrain vehiclesquestionnaire2 ( )solvelius et al.1712 finnish air force pilot cadetsmagnetic resonance imaging2 ( )tachibana and hanada2840 japanese air defense force pilotsmagnetic resonance imaging2 ( 0 ) ) . an online search using medline nioshtic , military & government collection ( ebsco ) , pubmed , and web of science identified articles published between january 1980 and november 2014 investigating the effects of vehicle - ride exposure on the occurrence of cervical pathology . the database search used multiple combinations of the following terms : vibration , spinal pathology , neck pain , intervertebral disc degeneration , degeneration , fracture , herniated disc , shock , loading , impact , repeated loading , industrial vibration , vehicle vibration , radicular , radiculopathy , neck strain , spine , mechanical shock , and g forces . in addition , we examined the reference list of the articles acquired through the search to find additional pertinent articles . the inclusion criteria were determined prior to the start of the database search in order to reduce any possible selection bias . to be included , studies needed to have a clear sampling method , control group , clearly defined exposure time , focus on cervical injuries resulting from vehicle - ride exposure , and explicit pathology . after screening the titles and abstracts , two reviewers ( r.k . and k.g . ) the reviewers resolved disagreements concerning article eligibility by consensus or by arbitration of a third reviewer ( j.s . ) if disagreement persisted . imagej ( rasband , w. , national institutes of health , usa ) enabled extraction of pertinent data contained in figures . all data were entered into a custom microsoft excel spreadsheet ( microsoft corporation , redmond , washington , usa ) and later transferred to the statistical software comprehensive meta - analysis version 2 ( biostat , englewood , nj , usa ) for statistical analyses . the meta - analysis utilized an unweighted random effects model using an odds ratio centered on 1.00 as the effect metric . ninety - five percent confidence intervals greater than 1.00 indicate that vehicle - ride exposure had a significant effect on cervical pathology . the sample sizes and number of injuries recorded for the treatment and control groups for each study were used in the analyses . subsequent sub - analyses were conducted using data collection method [ imaging ( i.e. magnetic resonance imaging , radiograph , computed tomography ) or questionnaire ] , vehicle type ( i.e. , ground - based vehicles , fixed - wing aircraft and rotary - wing aircraft ) and operator / other personnel as moderators . for the operator / other personnel moderator , the investigators defined operator as anyone flying ( i.e. fighter jet , jet , transport , or helicopter pilots ) or driving ( i.e. wheeled- or tracked - vehicle drivers ) a vehicle . other personnel represented anyone other than the operator on the vehicle ( such as a weapons systems operators , flight surgeon , flight engineer , tank commander , loader , or gunner ) . risk of publication bias was assessed using a funnel plot and a fail - safe number identified any presence of selective reporting within the studies . the scottish intercollegiate guidelines network ( sign ) grading system was used to evaluate the quality of evidence for each article included in the meta - analysis ( table 1table 1.study description and level of evidencestudysample populationmethod of assessmentlevel of evidenceaydog et al.16732 turkish military pilotsradiographs2 ( )byeon et al.26186 military helicopter korea air forceradiograph2 ( + ) drew et al.1035 u.s . high performance aircraft pilotsquestionnaire2 ( )hamalainen et al.312 finnish air force fighter pilot magnetic resonance imaging2 ( )hendriksen et al.1188 royal netherlands air force pilotsradiograph2 ( + ) mcbride et al.25516 locomotive engineers employed by the national rail carrier in new zealandquestionnaire2 ( + + ) nissen et al.21317 main battle tank personnelquestionnaire2 ( + ) petren - mallmin and linder2729 swedish air force pilotsmagnetic resonance imaging2 ( + + ) pippig et al.2286 pilots examined at the german air force institute of medicineclinical examination , peripheral neurological examination , computerized tomography , magnetic resonance imaging2 ( )rehn et al.24431 drivers of all - terrain vehiclesquestionnaire2 ( )solvelius et al.1712 finnish air force pilot cadetsmagnetic resonance imaging2 ( )tachibana and hanada2840 japanese air defense force pilotsmagnetic resonance imaging2 ( 0 ) ) . twelve studies1,2,3 , 10 , 16 , 17 , 21 , 24,25,26,27,28 met all inclusion criteria and were used for later analyses ( fig . the data extracted for the analysis included : number participants in the treatment and control groups ; total number of injuries in the treatment and control groups ; vehicle type ( fixed - wing , rotary - wing , or ground ) ; and exposure time in hours . table 2table 2.effect of vehicle - ride exposure on cervical pathologystudy namevehicle descriptiondata collection methododds ratiolower limitupper limitp - valueaydog et al.16fw : fighter radiographs1.310.612.790.49aydog et al . 16fw : transportradiographs0.710.311.590.40byeon et al.26rw : helicoptermri3.191.715.950.001drew et al.10fw : fighter questionnaire1.920.725.100.19hamalainen et al.3fw : fighter mri1.670.2212.350.62hamalainen et al.3fw : fighter mri5.500.5159.010.16hamalainen et al.3fw : fighter mri2.000.3810.410.41hamalainen et al.3fw : fighter mri0.600.084.450.62hamalainen et al.3fw : fighter mri1.500.258.840.65hamalainen et al.3fw : fighter mri1.670.2212.350.62hendriksen et al.1fw : fighter radiographs4.321.4612.810.01hendriksen et al.1fw : fighter radiographs2.171.323.570.01hendriksen et al.1fw : fighter radiographs2.101.153.800.02hendriksen et al.1fw : fighter radiographs1.310.573.050.52hendriksen et al.1fw : fighter radiographs2.291.304.050.01hendriksen et al.1fw : fighter radiographs1.660.863.200.13hendriksen et al.1fw : fighter radiographs1.280.612.690.51hendriksen et al.1fw : fighter radiographs3.081.019.380.05hendriksen et al.1fw : fighter radiographs1.410.842.370.19hendriksen et al.1fw : fighter radiographs1.850.487.120.37hendriksen et al.1fw : fighter radiographs1.801.063.030.03hendriksen et al.1fw : fighter radiographs4.321.4612.810.01hendriksen et al.1fw : fighter radiographs1.230.602.530.57hendriksen et al.1fw : fighter radiographs2.800.5813.410.20hendriksen et al.1fw : fighter radiographs6.992.0723.560.01hendriksen et al.1fw : fighter radiographs3.691.2311.070.02hendriksen et al.1fw : fighter radiographs2.691.325.500.01hendriksen et al.1fw : fighter radiographs2.791.335.860.01hendriksen et al.1fw : fighter radiographs6.362.1918.460.001hendriksen et al.1fw : fighter radiographs3.281.089.940.04mcbride et al.25locomotivequestionnaire1.891.432.500.001nissen et al.21 gb : main battle tankquestionnaire0.990.681.450.96petren - mallmin and linder27fw : fightermri14.830.73295.970.08pippig et al.2fw : fighterct / mri0.080.050.140.001pippig et al.2rw : helicopterct / mri0.060.030.120.001pippig et al.2fw : transportct / mri0.210.120.390.001rehn et al.24 gb : forest machinequestionnaire4.232.736.550.001rehn et al.24 gb : snow groomerquestionnaire3.782.156.630.001rehn et al.24 gb : snow mobilequestionnaire2.521.564.070.001solvelius et al.17fw : fightermri1.140.216.370.88tachibana and hanada28fw : fightermri0.590.172.050.40tachibana and hanada28fw : fightermri1.260.324.930.74tachibana and hanada28fw : fightermri0.330.101.100.07tachibana and hanada28fw : fightermri1.700.495.930.40summary effect1.591.162.170.01fw : fixed - wing , rw : rotary - wing , gb : ground - based , ct : computerized tomography , mri : magnetic resonance imaging displays the effect estimates and confidence intervals for each datum point selected . in some cases in addition , a fail - safe n determined that 573 negative data points would be required to increase the p - value for the overall meta - analysis to above 0.05 . fw : fixed - wing , rw : rotary - wing , gb : ground - based , ct : computerized tomography , mri : magnetic resonance imaging funnel plot of included studies . the results of the meta - analysis indicated that vehicle - ride exposure is more likely to increase cervical pathology ( p=0.01 , odds ratio=1.59 , 95% ci=1.162.17 ) as compared to controls . follow - up analysis using data collection method ( i.e. imaging or questionnaire ) as a moderator revealed studies using some type of imaging for the method ( i.e. magnetic resonance imaging , radiograph , computed tomography ) of collecting their injury data indicated that vehicle - ride exposure ( p=0.04 , odds ratio=1.50 , 95% ci=1.022.20 ) was significantly likely to increase cervical pathology . in studies that used a questionnaire to collect their injury vehicle - ride exposure data , we also observed that vehicle - ride exposure ( p=0.001 , odds ratio=2.28 , 95% ci=1.443.60 ) was significantly likely to increase cervical pathology . using vehicle type ( i.e. ground - based vehicles , fixed - wing and rotary - wing ) as a moderator revealed that vehicle - ride exposure in ground - based vehicles ( p=0.01 , odds ratio=2.33 , 95% ci=1.413.85 ) and fixed - wing aircraft ( p=0.01 , odds ratio=1.59 , 95% ci=1.132.23 ) were significantly likely to increase cervical pathology ( fig . 3fig . wso : weapons systems operator ; ( * p<0.05 ) . ) . vehicle - ride exposure in rotary - wing aircraft ( p=0.82 , odds ratio=0.75 , 95% ci=0.068.65 ) was not likely to increase cervical pathology ( fig . 3 ) . analyzing the data using vehicle operator / other personnel type ( e.g. pilot and crewmembers ) as a moderator , the results revealed that being a fighter jet pilot or weapons systems operator was significantly likely to increase cervical pathology ( p<0.001 , odds ratio=1.78 , 95% ci=1.262.50 ) ( fig . the results also revealed that being a transport pilot ( p=0.10 , odds ratio=0.38 , 95% ci=0.121.21 ) was not significantly likely to increase the development of cervical pathology ( fig . wso : weapons systems operator ; ( * p<0.05 ) . due to the lack of ground - based studies , we could not perform separate analyses using the operator / other personnel as a moderator for the different classes of ground - based ( e.g. wheeled- or tracked ) vehicle operators as previously performed with fixed - wing aircraft ( e.g. fighter jet and transport plane ) pilots . in addition , we were not able to use operator / other personnel as a moderator in the rotary - wing aircraft group because the studies used in the analysis only reported data on helicopter pilots . furthermore , only one of the included studies identified the type of helicopter ( e.g. attack or transport helicopter ) flown by the pilot reporting the cervical pathology ; thus , further sub - categorization of the rotary - wing group was not possible . the purpose of this study was to determine if the literature provides definitive evidence that vehicle - ride exposure influences the incidence of cervical spine pathology . our main finding is that the current literature suggests vehicle - ride exposure increases cervical pathology . most studies utilized radiological and magnetic resonance imaging to diagnose the presence of cervical pathology . five out of the seven studies using diagnostic testing indicated radiologists , neuroradiologists or other medical doctors examined the diagnostic imaging1 , 3 , 16 , 17 , 26 . only four studies ( 6 out of 46 data points ) collected data on cervical pathology using a questionnaire10 , 21 , 25 , 29 . the imaging and questionnaire studies both indicated vehicle - ride exposure is likely to increase cervical pathology , suggesting that both evaluation approaches provide similar outcomes . examination of vehicle type ( i.e. ground - based vehicles , fixed - wing aircrafts and rotary - wing aircraft ) suggests that vehicle - ride exposure in ground - based vehicles and fixed - wing aircraft increases cervical pathology . however , we observed that fixed - wing aircraft accounted for 38 of 46 data points , with ground - based and rotary - wing vehicles accounting for 5 of 46 data points and 3 of 46 data points , respectively . the minimal number of data points for ground - based vehicles and rotary - wing aircraft makes it difficult to assess accurately the effect of ride exposure in these vehicles . additional studies in rotary - wing and ground - based vehicles need to be completed in order to understand how these types of vehicles influence cervical pathology . fixed - wing aircraft fighter pilots and fighter jet weapons systems operators were significantly more likely than controls to report a cervical pathology when vehicle operator / other personnel was used as a moderator . our results suggest that transport pilots are not likely to incur a cervical pathology ; however , a limited number of studies including transport pilots met our inclusion criteria . the analysis using transport plane operator / other personnel as a moderator represented only two data points from two separate studies . this lack of research is also evident in both the ground - based vehicle and rotary - wing aircraft literature . more research is required within the ground - based group investigating the effects of vehicle - ride exposure on cervical pathology in wheeled- and tracked - vehicle drivers and other personnel . further research is also required to understand the influence of ride exposure on cervical spine pathology in rotary - wing aircraft , specifically type of helicopters , given their importance in military operations . only three studies2 , 16 , 26 with helicopter data met the inclusion criteria outlined a priori in the present analysis . of the included studies , only one2 identified the type of helicopters and that study did not report the prevalence of cervical pathology per type of helicopter . in addition , the observed confidence intervals of this particular moderated analysis were very large ( p=0.82 , odds ratio=0.75 , 95% ci=0.068.65)2 . this finding may be due to the very low number of participants in the control group ( n=123 ) as compared to helicopter pilots ( n=1,599 ) of one of the included studies . the authors of the study2 reported 20% of the control group ( n=25/123 ) was identified with a cervical pathology as compared to about 2% of the helicopter group ( n=32/1,599 ) . this is in contrast to the other two studies2 , 16 included in the moderated analysis . aydog et al.16 reported that reported 10% of the control group ( n=21/202 ) and 19% of the helicopter group ( n=31/159 ) , while byeon et al.26 reported 18.2% of the control group ( n=16/88 ) and 41.5% of the helicopter group ( n=68/164 ) were identified as having developed a cervical pathology . we believe that the lower number of participants in the control group of the pippig and kriebel2 study may have artificially inflated the reported value of 20% in the control group ; thus impacting the 95% ci of the moderated analysis exploring the influence of vehicle exposure on helicopter pilots . additional research is needed to assess the impact of vehicle exposure in helicopter populations as compared to nonflying controls . future research should also seek to distinguish between various types of helicopters ( e.g. ah-64 apache longbow attack helicopter as compared to ch-47 chinook transport helicopter ) to better identify the effects of flight exposure in pilots as well as other flight personnel . this information is vital to gaining a better understanding of how vehicle specific ride ( or flight ) exposure influences the incidence of cervical pathology in mounted warfighters . ground - based vehicles experience vibration between 0.2 to 20 hz30 with superimposed episodes of mechanical shock30 ; while fighter jets expose a pilot to repeated + gz forces6 . helicopter pilots and other helicopter personnel contend with largely vibratory forces as compared to the frequent + g environment experienced by fighter pilots and fighter jet weapons systems operators8 , 31 . helicopters experience lower vibratory amplitude than armored vehicles , but higher dominant frequencies30 . using the iso 2631 - 1 standards for health criteria researchers of an early study8 indicated that helicopter mean vibration measures were approximately 0.54 ms at the seat , with some flight values exceeding 0.60 ms . a more recent study32 that evaluated helicopter vibration under different flight profiles ( e.g. hovering , cruising at normal speed , max speed , and descent ) found that weighted vibration measures ( calculated according to iso 2631 - 1 standard ) in the z - axis ranged from 0.32 ms to 0.51 ms depending on the type of helicopter . the differences in ride exposures combined with the lack of vehicle specific information and vibratory and shock profiles for current vehicles suggests researchers should direct their attention to both ground - based vehicle and helicopter personnel in future studies where currently a lack of controlled design studies exist . for example , several studies12 , 18 , 20 have reported that helicopter pilots have an increased prevalence of cervical pathology ; however , these studies have failed to compare the findings to non - flight personnel with comparable age and career work experience . both ground - based vehicle and aircraft studies should seek to compare vehicle operators and other vehicle personnel to active duty and civilian non - vehicle or flight personnel . research also needs to investigate the injury prevalence in all crewmembers , as the ride posture and exposures vary significantly from position to position ( tank driver compared to tank commander for example ) . to date , most of the literature has focused on pilots and drivers with minimal attention given to other crewmembers . a better understanding of how vehicle exposure influences cervical pathology in other personnel with different seating positions and locations is required . the use of helmets with and without additional head - supported mass also deserves further investigation . none of the studies meeting the inclusion criteria included specification on the weight of the helmet and other gear supported by the head and cervical spine . this will allow the helmet and associated gear to be used a moderator allowing for a clearer interpretation of the effects of vehicle - ride exposure forces on cervical pathology . future investigations need to determine if a dose - response relationship exists between vehicle - ride exposure time and development of cervical pathology . we were unable to explore this question through a predictive model in the present study due to lack of data . however , we did observe an interesting pattern within our career vehicle - ride ( flight ) time data in studies that collected injury data via questionnaire . 4.graph of odds ratio and log of time ( hours ) for the studies using a questionnaire . shows that as exposure time increases , the report of a cervical pathology is more likely until a saturation point is reached at approximately 4.00 log ( time ) , which is equivalent to 10,000 h. clearly , more data are required to generate any reasonable conclusions . we did not observe this same type of pattern in a scatter plot between career vehicle - ride ( flight ) time and effect size derived from studies using imaging ( e.g. radiography ) to identify pathology . imaging is an objective indicator of changes to soft or bony tissues ; however , it can not identify the presence of pain . questionnaires allow for subjective measures ( i.e. pain ) , which may increase with increased vehicle - ride exposure even in the absence of confirmed tissue changes via imaging . graph of odds ratio and log of time ( hours ) for the studies using a questionnaire . this study suggests that populations exposed to frequent + g forces require injury prevention interventions . investigators have proposed several injury risk factors such as repeated exposure to accelerative + gz forces6 ; unanticipated high + gz accelerative forces37 ; decrease endurance at the neck musculature3 ; and head3 , 38 and seat positioning10 during high + gz maneuvering . intervention strategies and or programs proposed in the literature to help mitigate some of these risks include : having pilots brace their heads against the seat ( or canopy ) prior to high + gz load38,39,40 ; performing pre - flight warm - ups that include stretching and isometrics5 , 15 , 39 ; unloading ( decreasing the + gz ) prior to moving the head while in flight39 ; and neck strengthening exercises5 , 15 , 39 , 40 . in order to develop effective intervention protocols for mounted warfighters in all military occupational specialties ( mos ) , more work must be completed to further understand ride exposure and the development of cervical spine pathology . although the results of the present study suggest that vehicle - ride exposure likely increases cervical pathology , there are several limitations . first , there remains a possibility that not all available data ( published or unpublished ) were included . however , the calculated fail - safe n indicated that 573 negative data points are required to negate the positive general effect of vehicle - ride exposure on the development of cervical pathology . second , one study represented 20 out of 39 data points ; thus , this study may have influenced the results of the present study even though no indication of a publication bias as determined via a funnel plot . third , many of the studies did not include or explicitly state the number females included in their study ; thus , whether or not the same outcomes would be present in female populations remains to be determined . finally , in an attempt to include all published and unpublished data within the present meta - analysis meeting the inclusion criteria there is a potential that one or more studies used in the analyses were poorly performed studies . this may have adversely influenced the outcome of the analyses ; however , we wanted to be inclusive of all relevant studies regardless of quality . in addition , because there were a limited number of studies meeting the inclusion criteria a possibility exists that the point estimates and confidence intervals may provide false assurances when using an unweighted random effects model41 . therefore , as per borenstein41 the authors have provided the separate effects for each datum point included ( table 2 ) . the results of our meta - analysis provide evidence that overall vehicle - ride exposure is likely to increase cervical pathology in vehicle operators and other personnel compared to controls . the results of the present analysis suggest that exposure in ground - based vehicles and fixed - wing aircraft is likely to increase cervical pathology . in fixed - wing aircraft , fighter pilots and fighter jet weapons systems operators are more likely to develop a cervical pathology than control groups , while transport pilots were less likely to develop cervical pathology than control groups . personnel- and vehicle - specific data in ground - based vehicle and rotary - wing aircraft that would allow the determination of the likelihood of cervical pathology in personnel other than pilots was not available at time of analysis .

research to date on the effect vehicle - ride exposure has on the development of cervical pathologies in mounted warfighters is conflicting . the purpose of this study was to determine if the literature suggests a definite effect of vehicle - ride exposure on cervical pathology . databases were searched using multiple combinations of select terms . twelve studies meeting the inclusion criteria were included in the meta - analysis . the results of the meta - analysis revealed that overall vehicle - ride exposure was likely to increase cervical pathology ( p=0.01 , odds ratio=1.59 , 95% ci=1.162.17 ) . using vehicle type as a moderator it was found that vehicle - ride exposure in ground - based vehicles ( p=0.01 , odds ratio=2.33 , 95% ci=1.413.85 ) and fixed - wing aircraft ( p=0.01 , odds ratio = 1.59 , 95% ci=1.132.23 ) were likely to increase cervical pathology . using operator / other personnel moderator it was found that in the populations tested , fighter pilots or fighter jet weapons systems operators were more likely to develop a cervical pathology ( p<0.001 , odds ratio=1.78 , 95% ci=1.262.50 ) . the available studies indicate an increase in cervical pathology for personnel exposed to ground - based vehicles and fixed - wing aircraft .

Introduction Methods Identification, study selection and data extraction Data analysis Results Discussion Conclusion

reports indicate that cervical pathology is common in mounted warfighters ( i.e. studies using diagnostic examinations to determine the presence of cervical pathologies reported no significant difference between fighter pilots and controls , although fighter pilots appear to have a higher prevalence of neck pain16,17,18 . investigation of the prevalence of cervical pathology in ground - based military vehicle personnel is lacking in the literature . however , there are many types of ground - based vehicles such as main battle tanks , humvees ( i.e. in potentially less fit or older civilian population undergoing similar types of vehicle - ride exposure thus , the purpose of our study was to determine if evidence exists in the literature indicating a definite effect of vehicle - ride exposure on cervical pathology . an online search using medline nioshtic , military & government collection ( ebsco ) , pubmed , and web of science identified articles published between january 1980 and november 2014 investigating the effects of vehicle - ride exposure on the occurrence of cervical pathology . the inclusion criteria were determined prior to the start of the database search in order to reduce any possible selection bias . to be included , studies needed to have a clear sampling method , control group , clearly defined exposure time , focus on cervical injuries resulting from vehicle - ride exposure , and explicit pathology . the meta - analysis utilized an unweighted random effects model using an odds ratio centered on 1.00 as the effect metric . ninety - five percent confidence intervals greater than 1.00 indicate that vehicle - ride exposure had a significant effect on cervical pathology . , ground - based vehicles , fixed - wing aircraft and rotary - wing aircraft ) and operator / other personnel as moderators . for the operator / other personnel moderator , the investigators defined operator as anyone flying ( i.e. other personnel represented anyone other than the operator on the vehicle ( such as a weapons systems operators , flight surgeon , flight engineer , tank commander , loader , or gunner ) . the scottish intercollegiate guidelines network ( sign ) grading system was used to evaluate the quality of evidence for each article included in the meta - analysis ( table 1table 1.study description and level of evidencestudysample populationmethod of assessmentlevel of evidenceaydog et al.16732 turkish military pilotsradiographs2 ( )byeon et al.26186 military helicopter korea air forceradiograph2 ( + ) drew et al.1035 u.s . an online search using medline nioshtic , military & government collection ( ebsco ) , pubmed , and web of science identified articles published between january 1980 and november 2014 investigating the effects of vehicle - ride exposure on the occurrence of cervical pathology . the database search used multiple combinations of the following terms : vibration , spinal pathology , neck pain , intervertebral disc degeneration , degeneration , fracture , herniated disc , shock , loading , impact , repeated loading , industrial vibration , vehicle vibration , radicular , radiculopathy , neck strain , spine , mechanical shock , and g forces . the inclusion criteria were determined prior to the start of the database search in order to reduce any possible selection bias . to be included , studies needed to have a clear sampling method , control group , clearly defined exposure time , focus on cervical injuries resulting from vehicle - ride exposure , and explicit pathology . the meta - analysis utilized an unweighted random effects model using an odds ratio centered on 1.00 as the effect metric . ninety - five percent confidence intervals greater than 1.00 indicate that vehicle - ride exposure had a significant effect on cervical pathology . , ground - based vehicles , fixed - wing aircraft and rotary - wing aircraft ) and operator / other personnel as moderators . for the operator / other personnel moderator , the investigators defined operator as anyone flying ( i.e. other personnel represented anyone other than the operator on the vehicle ( such as a weapons systems operators , flight surgeon , flight engineer , tank commander , loader , or gunner ) . the scottish intercollegiate guidelines network ( sign ) grading system was used to evaluate the quality of evidence for each article included in the meta - analysis ( table 1table 1.study description and level of evidencestudysample populationmethod of assessmentlevel of evidenceaydog et al.16732 turkish military pilotsradiographs2 ( )byeon et al.26186 military helicopter korea air forceradiograph2 ( + ) drew et al.1035 u.s . the data extracted for the analysis included : number participants in the treatment and control groups ; total number of injuries in the treatment and control groups ; vehicle type ( fixed - wing , rotary - wing , or ground ) ; and exposure time in hours . table 2table 2.effect of vehicle - ride exposure on cervical pathologystudy namevehicle descriptiondata collection methododds ratiolower limitupper limitp - valueaydog et al.16fw : fighter radiographs1.310.612.790.49aydog et al . 16fw : transportradiographs0.710.311.590.40byeon et al.26rw : helicoptermri3.191.715.950.001drew et al.10fw : fighter questionnaire1.920.725.100.19hamalainen et al.3fw : fighter mri1.670.2212.350.62hamalainen et al.3fw : fighter mri5.500.5159.010.16hamalainen et al.3fw : fighter mri2.000.3810.410.41hamalainen et al.3fw : fighter mri0.600.084.450.62hamalainen et al.3fw : fighter mri1.500.258.840.65hamalainen et al.3fw : fighter mri1.670.2212.350.62hendriksen et al.1fw : fighter radiographs4.321.4612.810.01hendriksen et al.1fw : fighter radiographs2.171.323.570.01hendriksen et al.1fw : fighter radiographs2.101.153.800.02hendriksen et al.1fw : fighter radiographs1.310.573.050.52hendriksen et al.1fw : fighter radiographs2.291.304.050.01hendriksen et al.1fw : fighter radiographs1.660.863.200.13hendriksen et al.1fw : fighter radiographs1.280.612.690.51hendriksen et al.1fw : fighter radiographs3.081.019.380.05hendriksen et al.1fw : fighter radiographs1.410.842.370.19hendriksen et al.1fw : fighter radiographs1.850.487.120.37hendriksen et al.1fw : fighter radiographs1.801.063.030.03hendriksen et al.1fw : fighter radiographs4.321.4612.810.01hendriksen et al.1fw : fighter radiographs1.230.602.530.57hendriksen et al.1fw : fighter radiographs2.800.5813.410.20hendriksen et al.1fw : fighter radiographs6.992.0723.560.01hendriksen et al.1fw : fighter radiographs3.691.2311.070.02hendriksen et al.1fw : fighter radiographs2.691.325.500.01hendriksen et al.1fw : fighter radiographs2.791.335.860.01hendriksen et al.1fw : fighter radiographs6.362.1918.460.001hendriksen et al.1fw : fighter radiographs3.281.089.940.04mcbride et al.25locomotivequestionnaire1.891.432.500.001nissen et al.21 gb : main battle tankquestionnaire0.990.681.450.96petren - mallmin and linder27fw : fightermri14.830.73295.970.08pippig et al.2fw : fighterct / mri0.080.050.140.001pippig et al.2rw : helicopterct / mri0.060.030.120.001pippig et al.2fw : transportct / mri0.210.120.390.001rehn et al.24 gb : forest machinequestionnaire4.232.736.550.001rehn et al.24 gb : snow groomerquestionnaire3.782.156.630.001rehn et al.24 gb : snow mobilequestionnaire2.521.564.070.001solvelius et al.17fw : fightermri1.140.216.370.88tachibana and hanada28fw : fightermri0.590.172.050.40tachibana and hanada28fw : fightermri1.260.324.930.74tachibana and hanada28fw : fightermri0.330.101.100.07tachibana and hanada28fw : fightermri1.700.495.930.40summary effect1.591.162.170.01fw : fixed - wing , rw : rotary - wing , gb : ground - based , ct : computerized tomography , mri : magnetic resonance imaging displays the effect estimates and confidence intervals for each datum point selected . fw : fixed - wing , rw : rotary - wing , gb : ground - based , ct : computerized tomography , mri : magnetic resonance imaging funnel plot of included studies . the results of the meta - analysis indicated that vehicle - ride exposure is more likely to increase cervical pathology ( p=0.01 , odds ratio=1.59 , 95% ci=1.162.17 ) as compared to controls . magnetic resonance imaging , radiograph , computed tomography ) of collecting their injury data indicated that vehicle - ride exposure ( p=0.04 , odds ratio=1.50 , 95% ci=1.022.20 ) was significantly likely to increase cervical pathology . in studies that used a questionnaire to collect their injury vehicle - ride exposure data , we also observed that vehicle - ride exposure ( p=0.001 , odds ratio=2.28 , 95% ci=1.443.60 ) was significantly likely to increase cervical pathology . ground - based vehicles , fixed - wing and rotary - wing ) as a moderator revealed that vehicle - ride exposure in ground - based vehicles ( p=0.01 , odds ratio=2.33 , 95% ci=1.413.85 ) and fixed - wing aircraft ( p=0.01 , odds ratio=1.59 , 95% ci=1.132.23 ) were significantly likely to increase cervical pathology ( fig . vehicle - ride exposure in rotary - wing aircraft ( p=0.82 , odds ratio=0.75 , 95% ci=0.068.65 ) was not likely to increase cervical pathology ( fig . analyzing the data using vehicle operator / other personnel type ( e.g. pilot and crewmembers ) as a moderator , the results revealed that being a fighter jet pilot or weapons systems operator was significantly likely to increase cervical pathology ( p<0.001 , odds ratio=1.78 , 95% ci=1.262.50 ) ( fig . the results also revealed that being a transport pilot ( p=0.10 , odds ratio=0.38 , 95% ci=0.121.21 ) was not significantly likely to increase the development of cervical pathology ( fig . due to the lack of ground - based studies , we could not perform separate analyses using the operator / other personnel as a moderator for the different classes of ground - based ( e.g. wheeled- or tracked ) vehicle operators as previously performed with fixed - wing aircraft ( e.g. in addition , we were not able to use operator / other personnel as a moderator in the rotary - wing aircraft group because the studies used in the analysis only reported data on helicopter pilots . attack or transport helicopter ) flown by the pilot reporting the cervical pathology ; thus , further sub - categorization of the rotary - wing group was not possible . the purpose of this study was to determine if the literature provides definitive evidence that vehicle - ride exposure influences the incidence of cervical spine pathology . our main finding is that the current literature suggests vehicle - ride exposure increases cervical pathology . the imaging and questionnaire studies both indicated vehicle - ride exposure is likely to increase cervical pathology , suggesting that both evaluation approaches provide similar outcomes . ground - based vehicles , fixed - wing aircrafts and rotary - wing aircraft ) suggests that vehicle - ride exposure in ground - based vehicles and fixed - wing aircraft increases cervical pathology . however , we observed that fixed - wing aircraft accounted for 38 of 46 data points , with ground - based and rotary - wing vehicles accounting for 5 of 46 data points and 3 of 46 data points , respectively . the minimal number of data points for ground - based vehicles and rotary - wing aircraft makes it difficult to assess accurately the effect of ride exposure in these vehicles . additional studies in rotary - wing and ground - based vehicles need to be completed in order to understand how these types of vehicles influence cervical pathology . fixed - wing aircraft fighter pilots and fighter jet weapons systems operators were significantly more likely than controls to report a cervical pathology when vehicle operator / other personnel was used as a moderator . our results suggest that transport pilots are not likely to incur a cervical pathology ; however , a limited number of studies including transport pilots met our inclusion criteria . the analysis using transport plane operator / other personnel as a moderator represented only two data points from two separate studies . this lack of research is also evident in both the ground - based vehicle and rotary - wing aircraft literature . more research is required within the ground - based group investigating the effects of vehicle - ride exposure on cervical pathology in wheeled- and tracked - vehicle drivers and other personnel . further research is also required to understand the influence of ride exposure on cervical spine pathology in rotary - wing aircraft , specifically type of helicopters , given their importance in military operations . only three studies2 , 16 , 26 with helicopter data met the inclusion criteria outlined a priori in the present analysis . in addition , the observed confidence intervals of this particular moderated analysis were very large ( p=0.82 , odds ratio=0.75 , 95% ci=0.068.65)2 . the authors of the study2 reported 20% of the control group ( n=25/123 ) was identified with a cervical pathology as compared to about 2% of the helicopter group ( n=32/1,599 ) . aydog et al.16 reported that reported 10% of the control group ( n=21/202 ) and 19% of the helicopter group ( n=31/159 ) , while byeon et al.26 reported 18.2% of the control group ( n=16/88 ) and 41.5% of the helicopter group ( n=68/164 ) were identified as having developed a cervical pathology . we believe that the lower number of participants in the control group of the pippig and kriebel2 study may have artificially inflated the reported value of 20% in the control group ; thus impacting the 95% ci of the moderated analysis exploring the influence of vehicle exposure on helicopter pilots . this information is vital to gaining a better understanding of how vehicle specific ride ( or flight ) exposure influences the incidence of cervical pathology in mounted warfighters . helicopter pilots and other helicopter personnel contend with largely vibratory forces as compared to the frequent + g environment experienced by fighter pilots and fighter jet weapons systems operators8 , 31 . none of the studies meeting the inclusion criteria included specification on the weight of the helmet and other gear supported by the head and cervical spine . this will allow the helmet and associated gear to be used a moderator allowing for a clearer interpretation of the effects of vehicle - ride exposure forces on cervical pathology . future investigations need to determine if a dose - response relationship exists between vehicle - ride exposure time and development of cervical pathology . pain ) , which may increase with increased vehicle - ride exposure even in the absence of confirmed tissue changes via imaging . in order to develop effective intervention protocols for mounted warfighters in all military occupational specialties ( mos ) , more work must be completed to further understand ride exposure and the development of cervical spine pathology . although the results of the present study suggest that vehicle - ride exposure likely increases cervical pathology , there are several limitations . however , the calculated fail - safe n indicated that 573 negative data points are required to negate the positive general effect of vehicle - ride exposure on the development of cervical pathology . second , one study represented 20 out of 39 data points ; thus , this study may have influenced the results of the present study even though no indication of a publication bias as determined via a funnel plot . finally , in an attempt to include all published and unpublished data within the present meta - analysis meeting the inclusion criteria there is a potential that one or more studies used in the analyses were poorly performed studies . in addition , because there were a limited number of studies meeting the inclusion criteria a possibility exists that the point estimates and confidence intervals may provide false assurances when using an unweighted random effects model41 . the results of our meta - analysis provide evidence that overall vehicle - ride exposure is likely to increase cervical pathology in vehicle operators and other personnel compared to controls . the results of the present analysis suggest that exposure in ground - based vehicles and fixed - wing aircraft is likely to increase cervical pathology . in fixed - wing aircraft , fighter pilots and fighter jet weapons systems operators are more likely to develop a cervical pathology than control groups , while transport pilots were less likely to develop cervical pathology than control groups . personnel- and vehicle - specific data in ground - based vehicle and rotary - wing aircraft that would allow the determination of the likelihood of cervical pathology in personnel other than pilots was not available at time of analysis .

endosymbionts are normally asexual and transmitted by uniparental vertical inheritance ( sachs et al . , 2011 ) . multicellular organisms thus have a single mitochondrial genotype and those that have photosynthesis rely on a single clone of plastids . the evolution of such obligate symbiotic mutualisms has strong elements of partner commitment driven by kin selection , because exclusive association of hosts with a single symbiont genotype ensures that its services to growth and survival of the host will benefit clone mates that are vertically transmitted when hosts reproduce ( frank , 1994 , doebeli and knowlton , 1998 , sachs et al . , 2004 , the same logic implies that hosts and symbionts are potentially in conflict over the mode of symbiont transmission ( frank , 1996 , douglas , 2008 ) , as symbionts would always benefit from additional horizontal transmission . however , hosts might suffer fitness losses from this form of commitment - disloyalty and therefore suppress symbiont investments in sexual reproduction ( frank , 1996 , leigh , 2010 ) . when , despite such host efforts , symbiont lineages manage to co - infect hosts and compete for resources , hosts will be under selection to monitor symbiont genetic diversity and eliminate additional symbiont lineages when such competition implies a net loss of cumulative symbiont service to the host ( frank , 1996 ) . the classical mitochondrial and plastid endosymbioses are so integrated with their host cells that their reduced genomes preclude any form of non - symbiotic life , and the same is true for many obligate and facultative endosymbionts with less reduced genomes ( mccutcheon and moran , 2012 ) . many of these interactions likely represent adaptive endpoints of host - symbiont coevolution , where host - symbiont conflicts were resolved in favor of the hosts ( mccutcheon and moran , 2012 , wernegreen , 2012 ) or symbiont ( werren et al . , 2008 ) , but their advanced stage of symbiosis normally precludes direct tests of evolutionary conflict theory over symbiont mixing because co - evolved symbionts can often not be reared in vitro . the fungus - growing ants offer a feasible model system to do such tests , because they have multiple obligate mutualisms , including fungus gardens ( schultz and brady , 2008 , mikheyev et al . , 2010 ) and cuticular actinobacteria ( cafaro et al . , 2011 , andersen et al . , 2013 ) that are ectosymbionts for individual ants , but endosymbionts for the ant colonies . this implies that partners can be reared in vitro without each other 's interference for sufficient periods of time to quantify antagonism between symbiont clones , monitor host reactions to alternative symbionts , and relate observed differences to the genetic characteristics of the interactants ( armitage et al . , 2011 , bot et al . attine ant colonies have never been found to rear a multi - clone fungus garden ( apterostigma , dentinger et al . , 2009 ; cyphomyrmex , green et al . , 2002 and mehdiabadi et al . , 2012 ; atta , mueller et al . , there was substantial genetic variation among fungus - garden clones across sympatric colonies , consistent with vertical transmission and normal patterns of variation of mitochondrial and plastid organelles across individual animals and plants ( e.g. embley and martin , 2006 ) . however , in contrast to these cellular endosymbionts , there may be considerable horizontal transfer of symbionts when territories of founding attine colonies overlap , consistent with species belonging to the same genus often sharing clades of symbionts ( green et al . , 2002 , poulsen and boomsma , 2005 , de fine licht and boomsma , 2011 , de fine licht and boomsma , 2014 , mehdiabadi et al . , 2012 ) , while sympatric attine ant genera normally rear distinct fungal symbiont clades ( mueller and gerardo , 2002 , dentinger et al . , 2009 , vo et al . , 2009 , . horizontal swaps of fungus - garden symbionts between colonies of the same or closely related attine ant species may reduce the efficiency of co - evolutionary adaptation at the lowest taxonomic level , but allows ant lineages to replace an asexual crop symbiont that is compromised by genetic load or another form of maladaptation to prevailing ecological conditions ( mueller , 2002 ) . however , as long as fungus - garden clones are thriving , they will also be under selection to actively defend their ant - care monopoly ( bot et al . such defenses are expected to evolve when the threat to be replaced is real , i.e. , any hostility of this kind should target non - self symbiont genotypes belonging to the clade of symbionts that can in fact partake in a viable symbiosis with a focal attine ant species . the acromyrmex echinatior and acromyrmex octospinosus populations in gamboa , panama appear to co - exploit the same clade of fungus - garden symbionts in sympatry ( bot et al . resident fungus gardens of these ant species have been shown to maintain their clonal integrity by a combination of behavioral adaptations in the ants to remove and kill alternative fungus clones ( bot et al . , 2001 , , 2009 ) and by the expression of somatic incompatibility reactions between clones from different acromyrmex colonies reared together on the same agar plates ( poulsen and boomsma , 2005 ) . these incompatibilities correlated with amplified fragment length polymorphism ( aflp ) genetic distances between pairs of fungal symbionts , a pattern that also applied to the fecal fluid of acromyrmex large workers fed with fungus from their own and other colonies ( poulsen and boomsma , 2005 ) . however , founding a. octospinosus queens readily accept fungal clones from other colonies , suggesting that this stage offers a special window for horizontal transfers even though signs of reduced performance with a novel symbiont taken from a mature colony were also found ( poulsen et al . , 2009 ) . this study suggested that incompatibility mechanisms might be different for sympatric atta leaf - cutting ants , which tend to rear different fungal symbionts ( mikheyev et al . , 2007 , kooij et al . , 2015 ) and whose queens never forage during colony founding and therefore have negligible likelihood of encountering alternative symbionts . somatic ( in)compatibilities between plated fungi of panamanian acromyrmex species are expressed in a gradual manner that correlates with aflp genetic distances between pairs of clones ( poulsen and boomsma , 2005 ) . in basidiomycetes , somatic incompatibilities are generally induced by allorecognition so that strains are increasingly likely to be incompatible when they are more genetically different ( may , 1988 , worrall , 1997 ) . rayner , 1991 , worrall , 1997 ) and usually involve dark pigmentation in the interaction zone ( rayner et al . , 1984 ) , changes in septal maintenance , or blockage of septa precluding the movement of cytoplasm , and can lead to programmed cell death ( rayner , 1991 ) . the underlying genetic mechanisms remain largely unknown , but multiple loci appear to be involved ( worrall , 1997 ) and their expression may be linked to sexual incompatibility genes ( van der nest et al . , 2009 , if sex occurs at all , it is extremely rare in the fungal symbionts of higher attine ants ( fisher et al . , 1994a , fisher et al . , 1994b , pagnocca et al . , 2001 , mueller , 2002 , mikheyev et al . , 2006 ) recent work further indicated that the symbionts of panamanian leaf - cutting ants are multi - genomic chimeras ( kooij et al . , 2015 ) , which likely explains why incompatibility patterns between panamanian acromyrmex symbionts appear to be gradual ( poulsen and boomsma , 2005 ) , we aimed to further our understanding of the biological factors governing somatic incompatibility among strains of attine ant fungal symbionts . we focused on comparing sympatric mature colonies of panamanian atta colombica and acromyrmex echinatior leaf - cutting ants to address the following questions : ( 1 ) do plated fungal symbionts of a. echinatior and a. colombica express similar somatic incompatibility reactions when confronted with symbionts from other colonies ? ( 2 ) to what extent does the intensity of these reactions differ within and between the two genera ? ( 3 ) to what extent is the intensity of these reactions correlated with genetic distance between the fungal symbionts ? ( 4 ) are sympatric colonies of a. echinatior and a. colombica rearing the same or overlapping set(s ) of fungal symbionts or are they associated with distinct lineages of the leucoagaricus gongylophorus symbiont ? the same set of symbionts would be expected if horizontal symbiont transmission between the two ant genera were more frequent than natural divergence of lineages via mutation and genetic drift . in contrast , segregated lineages would be expected when horizontal transmission between genera is absent because co - adaptations in l. gongylophorus strains reared by a. echinatior and a. colombica would preclude that horizontal symbiont swaps between genera are viable . finally , we also compared two different sets of genetic markers ( aflps and microsatellites ) and different time spans between plate inoculation and scoring of incompatibilities to evaluate the robustness of our conclusions . fungal cultivars were isolated from nine acromyrmex echinatior colonies ( ae150a , ae160 , ae168 , ae263 , ae266 , ae322 , ae356 , ae394 , ae488 ) and nine atta colombica colonies ( ac-2006 - 27 , ac-2009 - 42 , ac-2009 - 46 , ac-2011 - 2 , ac-2011 - 3 , ac-2012 - 1 , ac-2012 - 2 , ac-2012 - 8 , ac-2012 - 31 ) living sympatrically in gamboa , panama , and grown on 39 g l potato dextrose agar ( sigma aldrich , st louis , mo , usa ) with the addition of 5 g l yeast extract , 15 mg l tetracycline and 12 mg l streptomycin . for each of the colonies a single isolate was obtained as it has been shown before that both acromyrmex ( poulsen and boomsma , 2005 ) and atta ( mueller et al . , 2010 ) maintain their fungal symbiont in a monoculture . dna of each fungal strain was extracted using the qiagen ( venlo , the netherlands ) dneasy plant tissue extraction kit and stored at 20 c until further analysis . the gamboa sampling site was the same as where most previous colonies of the copenhagen fungus - growing ant research program have been collected , including the acromyrmex colonies studied by bot et al . , 2001 , poulsen and boomsma , 2005 , mikheyev et al . , 2007 , richard et al . ( 2009 ) . to calculate the genetic distance between each of the fungal strains we used two different methods : aflp and ten microsatellite markers ( a128 , a1030 , a1132 , a1151 , b12 , b447 , c101 , c126 , c647 and d115 developed by scott et al . ( 1995 ) with two selective primer combinations ( eco - acc + mse - cat and eco - acc + mse - cac ) . microsatellite markers were analyzed using pcr with 5 l vwr red taq dna polymerase master mix ( vwr international , haasrode , belgium ) , 0.25 l forward and reverse primer each , 1.5 l ddh2o and 1 l dna , and a program of 5 min denaturing at 95 c , followed by 14 cycles of 30 s denaturing at 95 c , 30 s annealing at 6858 c with a touchdown of 0.5 c per cycle , and 30 s extension at 72 c followed by 20 cycles of 30 s denaturing at 95 c , 30 s annealing at 58 c and 30 s extension at 72 c , and finally a 15 min extension at 72 c . both aflp and microsatellite amplification products were analyzed on an abi 3130xl ( applied biosystems , nrum , denmark ) sequencer . specific allele scorings ( aflp : table s1 ; microsatellites : table s2 ) were obtained by analyzing chromatograms in genemapper 4.0 ( applied biosystems , nrum , denmark ) . the program populations 1.2.32 ( langella , 2001 ) was used to calculate fst values for the microsatellite data and nei 's standard genetic distance ( ds ) for the aflp data , followed by neighbor joining phylogenetic analyses with 500 bootstrap replicates for each of the two types of markers . was used to analyze population structure for the aflp data with the following settings : an optimized k = 3 tested with the online program structure harvester ( earl and vonholdt , 2011 ) , a burn - in period of 1,000,000 iterations followed by 10,000,000 mcmc iterations , an admixture model , and independent allele frequencies among populations with = 0.78 . the 20 runs that we obtained were merged with the greedy analysis with 1,000,000 repeats in the clumpp software ( jakobsson and rosenberg , 2007 ) , and visualized with distruct ( rosenberg , 2004 ) . microsatellite data were further analyzed using the package poppr ( kamvar et al . , 2014 ) in r ( r core team , 2013 ) , to evaluate the discriminatory power of our markers and verify clonality of the symbionts . to test whether plated fungi showed ( in)compatibility , cultures of all 18 fungi were paired in all possible ( 171 ) combinations with four replicate pairings for each combination . for each pair , small tufts of mycelium ( ca . 2 mm ) were placed at a distance of 1.5 cm from each other on a 5 cm petri dish with 39 g l potato dextrose agar with the addition of 5 g l yeast extract and 35 g l agar . the growth medium was selected in a pilot study testing somatic incompatibility reactions for control ( self ) encounters on this and three alternative media , which showed that the used pdya medium most consistently avoided discolorations in controls ( fig s1 ) . incompatibility reactions were assessed after 6 , 8 , and 10 weeks and scored using the semi - quantitative scale described by poulsen and boomsma ( 2005 ) : 0 = demarcation zone absent , 1 = demarcation zone weak but present , 2 = demarcation zone broad and distinct , and 3 = strong demarcation zone with consistent brown or black coloration of mycelium . these four scores are consistent with the variation in somatic ( in)compatibility reactions that are typically found in free - living basidiomycetes , where they usually occur in a more stepwise manner as explained above ( worrall , 1997 ) . all scorings were done blindly by randomly assigning numbers to each plate , after which two of the authors did the initial assessment and a third author blindly checked combinations for which the first two authors did not agree on the score . degrees of ( in)compatibility were subsequently compared with genetic distances between pairs of fungal clones using mantel and partial mantel tests for dissimilarity matrices ( mantel ) ( r core team , 2013 ) with 99,999 permutations in the community ecology package : ordination , diversity and dissimilarities vegan correlations were forced through the origin based on the fact that the controls were ( 0,0 ) . figures were created using plot with repeated symbols by size ( sizeplot ) in the plotrix package ( lemon , 2006 ) . for acromyrmex symbionts , consistent scorings of somatic incompatibility were obtained 8 weeks after agar plates were inoculated . coloration contrasts were not fully developed after 6 weeks , so mantel correlation coefficients between incompatibility and genetic distance after 6 weeks remained low ( fig s2 ) . beyond 8 weeks , mantel correlation coefficients continued to improve , but contaminations and medium desiccation problems affected scoring accuracy 10 weeks after inoculation ( fig s2 ) so that almost 5% of the replicates were lost . as scoring results at 8 weeks were approximately the same as the 2-months between inoculation and scoring in poulsen and boomsma ( 2005 ) , our main results for the 8 weeks observation period are presented , to remain as comparable as possible with that previous study on somatic incompatibilities between a. echinatior and a. octospinosus fungal symbionts collected at the same sampling site more than 10 yr earlier . however , for atta symbionts a comparable result was only obtained when using microsatellite markers , as aflp markers produced mantel correlation coefficients close to zero for all observation periods ( fig s2 ) . using the 8 weeks data , somatic incompatibilities increased with increasing aflp genetic distances between fungi ( mantel r = 0.463 , p = 0.003 , fig 1a ) in acromyrmex symbionts , but not in atta symbionts ( mantel r = 0.164 , p = 0.792 , fig 1c ) . when using genetic distances based on microsatellite markers , both acromyrmex ( mantel r = 0.469 , p = 0.003 , fig 1b ) and atta ( mantel r = 0.312 , p = 0.032 , fig 1d ) symbionts had incompatibilities that increased with genetic distance , but less of the incompatibility variance was explained in atta than in acromyrmex . the a. echinatior results were consistent with the results obtained by poulsen and boomsma ( 2005 ) , but in that study the variance explained by the mantel coefficient was larger ( r = 0.855 ; p < 0.0001 ) . however , when the 10-weeks scorings for acromyrmex symbiont pairings was used there was a correlation closer to the one obtained after 2 months by poulsen and boomsma ( 2005 ) ( microsatellites : mantel r = 0.596 , p < 0.001 ; aflp : mantel r = 0.654 , p < 0.001 ) . overall , the atta fungi had smaller genetic distances when calculated from the microsatellite marker data ( 0.09 0.01 se ) , but larger genetic distances when using aflp markers ( 0.26 0.03 se ) compared to acromyrmex ( 0.13 0.01 se and 0.17 0.02 se , respectively ) , which is reflected in the average number of aflp bands observed ( atta : 37.3 1.8 se ; acromyrmex 30.9 1.5 se ; this implied that aflp and microsatellite genetic distances ( fst ) were only comparable for acromyrmex symbionts ( r = 0.932 ; fig s3 ) , whereas correlations decreased in comparisons between acromyrmex and atta symbionts and became very low in comparisons involving only colonies of atta colombica ( fig s3 ) . independent of the type of genetic marker used , mean genetic distances were higher in comparisons between acromyrmex and atta ( microsatellites : 0.20 0.01 se ; aflp : 0.36 0.02 se ) than in comparisons within the ant genera ( means ses given above ; microsatellite markers : f2,168 = 45.127 , p < 0.0001 ; aflp : f2,168 = 22 , also the average incompatibility scores were different for comparisons within and across ant species ( genera ) ( = 12.236 ; df = 2 ; p < 0.01 ) . this difference was mostly due to less strongly expressed somatic incompatibilities between atta symbionts , because the stronger mean reactions among acromyrmex symbionts alone and between acromyrmex and atta symbionts were not significantly different from each other ( w = 1635 , p = 0.338 ) . after pooling data across the entire range of genetic distances , the increase in somatic incompatibility with genetic distance was completely absent ( aflp : mantel r = 0.045 , p = 0.595 , fig 2a ) or no longer significant ( microsatellites : mantel r = 0.153 , p = 0.213 , fig 2b ) . specific evaluation of the microsatellite genetic differences between the fungal symbionts of atta and acromyrmex colonies showed that they were completely separated ( fig 3 , fig s4 and supplementary information ) , consistent with earlier findings by mikheyev et al . calculations for the standardized indexes of association , a linkage test ( rd , with 999 permutations ) , accounting for the number of loci sampled , were significant for the acromyrmex symbionts ( ia = 1.451 , p = 0.001 ; rd = 0.184 , p = 0.001 ) and for the atta symbionts ( ia = 1.923 , p = 0.001 ; rd = 0.319 , p = 0.001 ) . however , because only seven out of nine atta symbionts had independent multi - locus genotypes , with three being identical ( see supplementary information for details ) , a clone - correction was applied . this showed that the standardized index of association for the atta symbionts remained highly significant ( ia = 0.744 , p = 0.037 ; rd = 0.119 , p = 0.009 ) , consistent with all multi - locus genotypes being clonal . rooting the symbiont tree with a sympatric fungal symbiont of trachymyrmex zeteki ( fig s5 ) and by constructing a minimum spanning network ( fig 3 ) and a upgma tree ( supplementary information ) , both based on bruvo genetic distances , confirmed that the symbionts belonged to separate clades , although bootstrap values for the rooted tree were low . mirror imaging of trees obtained by microsatellite and aflp markers showed almost complete congruence for the acromyrmex fungi , but more noisy correspondence for the atta fungal symbionts ( fig s6 ) , confirming that these markers were less reliable predictors of somatic incompatibilities for atta symbionts . furthermore , structure analyses of the aflp data showed that four of the nine acromyrmex symbionts were similar to the atta symbionts ( fig 4 ) , and these were the same four symbionts that were most closely related to atta symbionts in the rooted tree based on microsatellites ( fig s5 ) . the results of our study show that sympatric panamanian colonies of a. echinatior and a. colombica rear genetically different lineages of the leaf - cutting ant garden symbiont l. gongylophorus and that microsatellite markers appear to predict genetic ( in)compatibility better than aflp markers . however , even when using microsatellite markers , the correlation between somatic incompatibility and neutral - marker - based genetic distance in atta is noisier than in acromyrmex . this shows that incompatibility reactions correlate only with genetic distances among fungal strains that have a realistic probability of being horizontally transferred , and not between more distant clades that are apparently unsuitable as symbionts for the sister genus of leaf - cutting ants . the results of our study confirm the earlier findings by poulsen and boomsma ( 2005 ) showing that somatic ( in)compatibility of panamanian acromyrmex symbionts is predictable from pairwise genetic distances for aflp markers ( figs 1a , c ) and that the same result can be obtained with more specific microsatellite markers ( figs 1b , d ) . they also indicate that incompatibility reactions between separate clades of fungal symbionts , maintained by the two different genera of leaf - cutting ants , can no longer be predicted by neutral genetic markers . this lack of genetic signal may be due to incompatibility being ultimately caused by allelic variation at unknown loci ( worrall , 1997 ) that only correlate with neutral markers when there is recent common ancestry . this is only likely for local fungal symbiont lineages that are exploited by a single metapopulation of attine ants that share a joint pool of symbionts because each ant colony can in principle establish a viable symbiosis with each of these fungal genotypes . we expect somatic incompatibility to be actively maintained by selection only in populations where the ants have the possibility to acquire multiple genetically different symbionts that are viable alternatives . such selection would then be driven by resident fungus - garden symbionts being under selection to defend their monopoly against alternative strains that might be secondarily introduced , and with the active support of the farming ants that would lose fitness when maintaining multiple lineages of the same symbiont that compete for their attention rather than serving their hosts unconditionally ( frank , 1996 , bot et al . , 2001 ) . when symbionts belong to different clades that no longer mix or exchange genes , each symbiont lineage is only a viable symbiont for one lineage of ant farmers or the other , but not for both . our finding that a. colombica and a. echinatior maintain separated clades of fungal symbionts ( figs 3 and s5 ) suggests that l. gongylophorus has , in fact , been split into an atta and acromyrmex clade after its monophyletic origin 23 mya ( mikheyev et al . , 2010 ) . the two species of leaf - cutting ants that we investigated rear representatives of these symbiont lineages that appear adapted to being , respectively , an atta and acromyrmex symbiont . the fact that the fungal symbionts of panamanian leaf - cutting ants appear to have split in two monophyletic clades , is consistent with the results of a recent study that experimentally swapped fungus - garden symbionts between sympatric trachymyrmex septentrionalis and atta texana from texas , usa ( seal et al . , 2012 ) . although these ants are at the northern edge of the attine ant distribution ( mueller et al . , 2011a ) , and likely to have lower genetic variation among their symbionts , they share even less common ancestry than the panamanian fungal symbionts of our present study ( mueller et al . , 2011b ) . the swapped fungal symbionts thus could only serve as viable mutualists for either trachymyrmex or atta , but not both , which was confirmed in the published experiments showing that : ( 1 ) alternative fungus gardens were not always rejected by the ants but were never adopted as a viable alternative symbiont , because the ants were able to grow their original symbiont back from minuscule remnants that the authors had been unable to remove . ( 2 ) none of the t. septentrionalis colonies ever produced virgin queens when they maintained an a. texana fungal symbiont , consistent with the new combination being non - viable for transmitting ant or fungal genes to future generations . a follow - up study transplanting a. texana fungus to colonies of both t. septentrionalis and trachymyrmex turrifex confirmed that these trachymyrmex species can not enter into viable symbiosis with l. gongylophorus symbionts and that the virgin queens produced on swapped gardens had poor fat reserves making it unlikely that they could successfully found colonies ( seal and mueller , 2013 ) . to make further progress , it would be desirable to identify the genes that are directly responsible for somatic incompatibility , as this would allow direct studies on the signatures of selection and specialization across the clades of higher attine ant symbionts . in another , non - eusocial model system of fungus - growing insects , the sirex wood wasp , a range of genes are involved in somatic incompatibility reactions among lineages of the associated fungus , including fusion and recognition genes and genes that mediate cellular damage , stress response , and programmed cell death ( van der nest et al . , 2011 ) . whether these genes have homologs or analogs in attine ant fungal symbionts remains to be explored , as the sirex symbiont amylostereum areolatum belongs to a distantly related clade of basidiomycetes ( binder and hibbett , 2002 ) , and their respective domestication histories may have implied that recognition systems were lost and gained over evolutionary time . incompatibility reactions among atta symbionts were significantly weaker and less predictable from neutral fst marker values than similar reactions between acromyrmex - associated fungi . one possible explanation for this difference could be that there is a fundamental difference in colony founding in the sense that acromyrmex queens forage during colony founding similar to all more basal attine ants , whereas atta queens have secondarily evolved claustral colony founding . this implies that newly - mated atta queens close off their nest cavity to raise the first worker cohort purely on their body reserves , so that new colonies will only be opened by these workers 80100 d after they were founded ( weber , 1969 , fernandez - marin and wcislo , 2005 ) . however , acromyrmex queens not only forage for leaf fragments to manure their incipient fungus garden but , particularly when they have lost their garden , also for a replacement garden of another incipient colony whose queen is out foraging ( poulsen et al . , 2009 ) , an option that is unavailable for founding atta queens . although swapping of incipient fungus gardens with a fungus garden fragment from a mature acromyrmex colony was relatively unconstrained during early colony founding , it is likely that stronger mutual commitment between a founding queen and her resident fungus garden builds up in a matter of weeks , including stronger incompatibility reactions in case one of the first workers brings in an unrelated fungus garden fragment from a neighboring nest ( poulsen et al . , 2009 ) . this can never happen in the 80100 d during which atta colonies remain closed , removing selection for expressing incompatibility mechanisms during colony founding . why neutral markers should be weaker predictors of somatic incompatibility in atta colonies after workers start foraging remains unclear . imprinting of workers on the odor of a resident fungus garden is a possibility ( seal et al . , 2012 ) , but it seems unclear why such mechanisms should differ between atta and acromyrmex symbionts and why that should reduce selection for more direct defenses by fungal symbionts against being replaced . fungus gardens of panamanian acromyrmex colonies differ in chemical profiles ( richard et al . these differences do not correlate with genetic distances and comparable data for sympatric atta colonies are lacking . another hypothesis may be that mature atta colonies have hundreds of fungus gardens , whereas sympatric mature acromyrmex colonies have one or a few at best . this may make a difference in the likelihood of a resident fungus garden symbiont being replaced by an accidentally imported small fragment of fungus garden from a neighboring colony , so that less accurate recognition systems suffice in mature colonies of atta but not in acromyrmex . finally , there could also be a technical explanation for the incompatibility differences between the fungal symbionts of atta and acromyrmex . we found similar variation for the symbiont - specific microsatellite markers obtained from panamanian symbiont samples ( scott et al . , 2009 ) and the general aflp markers for acromyrmex symbionts , but enhanced variation in aflp peaks for atta symbionts , relative to acromyrmex symbionts ( fig s3 ) . this suggests that dna from other organisms may have been amplified with the aflps and that such other organisms were only present in atta symbiont cultures . in principle , this could be viral ( pearson et al . , 2009 ) , bacterial ( suen et al . , 2010 ) or prion ( wickner et al .

obligate mutualistic symbioses rely on mechanisms that secure host - symbiont commitments to maximize host benefits and prevent symbiont cheating . previous studies showed that somatic incompatibilities correlate with neutral - marker - based genetic distances between fungal symbionts of panamanian acromyrmex leaf - cutting ants , but the extent to which this relationship applies more generally remained unclear . here we showed that genetic distances accurately predicted somatic incompatibility for acromyrmex echinatior symbionts irrespective of whether neutral microsatellites or aflp markers were used , but that such correlations were weaker or absent in sympatric atta colombica colonies . further analysis showed that the symbiont clades maintained by a. echinatior and a. colombica were likely to represent separate gene pools , so that neutral markers were unlikely to be similarly correlated with incompatibility loci that have experienced different selection regimes . we suggest that evolutionarily derived claustral colony founding by atta queens may have removed selection for strong incompatibility in atta fungi , as this condition makes the likelihood of symbiont swaps much lower than in acromyrmex , where incipient nests stay open because queens have to forage until the first workers emerge .

Introduction Materials and methods Results Discussion

multicellular organisms thus have a single mitochondrial genotype and those that have photosynthesis rely on a single clone of plastids . , 2004 , the same logic implies that hosts and symbionts are potentially in conflict over the mode of symbiont transmission ( frank , 1996 , douglas , 2008 ) , as symbionts would always benefit from additional horizontal transmission . many of these interactions likely represent adaptive endpoints of host - symbiont coevolution , where host - symbiont conflicts were resolved in favor of the hosts ( mccutcheon and moran , 2012 , wernegreen , 2012 ) or symbiont ( werren et al . , 2008 ) , but their advanced stage of symbiosis normally precludes direct tests of evolutionary conflict theory over symbiont mixing because co - evolved symbionts can often not be reared in vitro . , 2013 ) that are ectosymbionts for individual ants , but endosymbionts for the ant colonies . the acromyrmex echinatior and acromyrmex octospinosus populations in gamboa , panama appear to co - exploit the same clade of fungus - garden symbionts in sympatry ( bot et al . these incompatibilities correlated with amplified fragment length polymorphism ( aflp ) genetic distances between pairs of fungal symbionts , a pattern that also applied to the fecal fluid of acromyrmex large workers fed with fungus from their own and other colonies ( poulsen and boomsma , 2005 ) . this study suggested that incompatibility mechanisms might be different for sympatric atta leaf - cutting ants , which tend to rear different fungal symbionts ( mikheyev et al . , 2015 ) and whose queens never forage during colony founding and therefore have negligible likelihood of encountering alternative symbionts . somatic ( in)compatibilities between plated fungi of panamanian acromyrmex species are expressed in a gradual manner that correlates with aflp genetic distances between pairs of clones ( poulsen and boomsma , 2005 ) . in basidiomycetes , somatic incompatibilities are generally induced by allorecognition so that strains are increasingly likely to be incompatible when they are more genetically different ( may , 1988 , worrall , 1997 ) . the underlying genetic mechanisms remain largely unknown , but multiple loci appear to be involved ( worrall , 1997 ) and their expression may be linked to sexual incompatibility genes ( van der nest et al . , 2009 , if sex occurs at all , it is extremely rare in the fungal symbionts of higher attine ants ( fisher et al . , 2006 ) recent work further indicated that the symbionts of panamanian leaf - cutting ants are multi - genomic chimeras ( kooij et al . , 2015 ) , which likely explains why incompatibility patterns between panamanian acromyrmex symbionts appear to be gradual ( poulsen and boomsma , 2005 ) , we aimed to further our understanding of the biological factors governing somatic incompatibility among strains of attine ant fungal symbionts . we focused on comparing sympatric mature colonies of panamanian atta colombica and acromyrmex echinatior leaf - cutting ants to address the following questions : ( 1 ) do plated fungal symbionts of a. echinatior and a. colombica express similar somatic incompatibility reactions when confronted with symbionts from other colonies ? ( 3 ) to what extent is the intensity of these reactions correlated with genetic distance between the fungal symbionts ? ( 4 ) are sympatric colonies of a. echinatior and a. colombica rearing the same or overlapping set(s ) of fungal symbionts or are they associated with distinct lineages of the leucoagaricus gongylophorus symbiont ? in contrast , segregated lineages would be expected when horizontal transmission between genera is absent because co - adaptations in l. gongylophorus strains reared by a. echinatior and a. colombica would preclude that horizontal symbiont swaps between genera are viable . fungal cultivars were isolated from nine acromyrmex echinatior colonies ( ae150a , ae160 , ae168 , ae263 , ae266 , ae322 , ae356 , ae394 , ae488 ) and nine atta colombica colonies ( ac-2006 - 27 , ac-2009 - 42 , ac-2009 - 46 , ac-2011 - 2 , ac-2011 - 3 , ac-2012 - 1 , ac-2012 - 2 , ac-2012 - 8 , ac-2012 - 31 ) living sympatrically in gamboa , panama , and grown on 39 g l potato dextrose agar ( sigma aldrich , st louis , mo , usa ) with the addition of 5 g l yeast extract , 15 mg l tetracycline and 12 mg l streptomycin . microsatellite markers were analyzed using pcr with 5 l vwr red taq dna polymerase master mix ( vwr international , haasrode , belgium ) , 0.25 l forward and reverse primer each , 1.5 l ddh2o and 1 l dna , and a program of 5 min denaturing at 95 c , followed by 14 cycles of 30 s denaturing at 95 c , 30 s annealing at 6858 c with a touchdown of 0.5 c per cycle , and 30 s extension at 72 c followed by 20 cycles of 30 s denaturing at 95 c , 30 s annealing at 58 c and 30 s extension at 72 c , and finally a 15 min extension at 72 c . the growth medium was selected in a pilot study testing somatic incompatibility reactions for control ( self ) encounters on this and three alternative media , which showed that the used pdya medium most consistently avoided discolorations in controls ( fig s1 ) . all scorings were done blindly by randomly assigning numbers to each plate , after which two of the authors did the initial assessment and a third author blindly checked combinations for which the first two authors did not agree on the score . degrees of ( in)compatibility were subsequently compared with genetic distances between pairs of fungal clones using mantel and partial mantel tests for dissimilarity matrices ( mantel ) ( r core team , 2013 ) with 99,999 permutations in the community ecology package : ordination , diversity and dissimilarities vegan correlations were forced through the origin based on the fact that the controls were ( 0,0 ) . for acromyrmex symbionts , consistent scorings of somatic incompatibility were obtained 8 weeks after agar plates were inoculated . beyond 8 weeks , mantel correlation coefficients continued to improve , but contaminations and medium desiccation problems affected scoring accuracy 10 weeks after inoculation ( fig s2 ) so that almost 5% of the replicates were lost . as scoring results at 8 weeks were approximately the same as the 2-months between inoculation and scoring in poulsen and boomsma ( 2005 ) , our main results for the 8 weeks observation period are presented , to remain as comparable as possible with that previous study on somatic incompatibilities between a. echinatior and a. octospinosus fungal symbionts collected at the same sampling site more than 10 yr earlier . however , for atta symbionts a comparable result was only obtained when using microsatellite markers , as aflp markers produced mantel correlation coefficients close to zero for all observation periods ( fig s2 ) . using the 8 weeks data , somatic incompatibilities increased with increasing aflp genetic distances between fungi ( mantel r = 0.463 , p = 0.003 , fig 1a ) in acromyrmex symbionts , but not in atta symbionts ( mantel r = 0.164 , p = 0.792 , fig 1c ) . when using genetic distances based on microsatellite markers , both acromyrmex ( mantel r = 0.469 , p = 0.003 , fig 1b ) and atta ( mantel r = 0.312 , p = 0.032 , fig 1d ) symbionts had incompatibilities that increased with genetic distance , but less of the incompatibility variance was explained in atta than in acromyrmex . the a. echinatior results were consistent with the results obtained by poulsen and boomsma ( 2005 ) , but in that study the variance explained by the mantel coefficient was larger ( r = 0.855 ; p < 0.0001 ) . overall , the atta fungi had smaller genetic distances when calculated from the microsatellite marker data ( 0.09 0.01 se ) , but larger genetic distances when using aflp markers ( 0.26 0.03 se ) compared to acromyrmex ( 0.13 0.01 se and 0.17 0.02 se , respectively ) , which is reflected in the average number of aflp bands observed ( atta : 37.3 1.8 se ; acromyrmex 30.9 1.5 se ; this implied that aflp and microsatellite genetic distances ( fst ) were only comparable for acromyrmex symbionts ( r = 0.932 ; fig s3 ) , whereas correlations decreased in comparisons between acromyrmex and atta symbionts and became very low in comparisons involving only colonies of atta colombica ( fig s3 ) . independent of the type of genetic marker used , mean genetic distances were higher in comparisons between acromyrmex and atta ( microsatellites : 0.20 0.01 se ; aflp : 0.36 0.02 se ) than in comparisons within the ant genera ( means ses given above ; microsatellite markers : f2,168 = 45.127 , p < 0.0001 ; aflp : f2,168 = 22 , also the average incompatibility scores were different for comparisons within and across ant species ( genera ) ( = 12.236 ; df = 2 ; p < 0.01 ) . this difference was mostly due to less strongly expressed somatic incompatibilities between atta symbionts , because the stronger mean reactions among acromyrmex symbionts alone and between acromyrmex and atta symbionts were not significantly different from each other ( w = 1635 , p = 0.338 ) . after pooling data across the entire range of genetic distances , the increase in somatic incompatibility with genetic distance was completely absent ( aflp : mantel r = 0.045 , p = 0.595 , fig 2a ) or no longer significant ( microsatellites : mantel r = 0.153 , p = 0.213 , fig 2b ) . specific evaluation of the microsatellite genetic differences between the fungal symbionts of atta and acromyrmex colonies showed that they were completely separated ( fig 3 , fig s4 and supplementary information ) , consistent with earlier findings by mikheyev et al . this showed that the standardized index of association for the atta symbionts remained highly significant ( ia = 0.744 , p = 0.037 ; rd = 0.119 , p = 0.009 ) , consistent with all multi - locus genotypes being clonal . rooting the symbiont tree with a sympatric fungal symbiont of trachymyrmex zeteki ( fig s5 ) and by constructing a minimum spanning network ( fig 3 ) and a upgma tree ( supplementary information ) , both based on bruvo genetic distances , confirmed that the symbionts belonged to separate clades , although bootstrap values for the rooted tree were low . mirror imaging of trees obtained by microsatellite and aflp markers showed almost complete congruence for the acromyrmex fungi , but more noisy correspondence for the atta fungal symbionts ( fig s6 ) , confirming that these markers were less reliable predictors of somatic incompatibilities for atta symbionts . the results of our study show that sympatric panamanian colonies of a. echinatior and a. colombica rear genetically different lineages of the leaf - cutting ant garden symbiont l. gongylophorus and that microsatellite markers appear to predict genetic ( in)compatibility better than aflp markers . however , even when using microsatellite markers , the correlation between somatic incompatibility and neutral - marker - based genetic distance in atta is noisier than in acromyrmex . this shows that incompatibility reactions correlate only with genetic distances among fungal strains that have a realistic probability of being horizontally transferred , and not between more distant clades that are apparently unsuitable as symbionts for the sister genus of leaf - cutting ants . the results of our study confirm the earlier findings by poulsen and boomsma ( 2005 ) showing that somatic ( in)compatibility of panamanian acromyrmex symbionts is predictable from pairwise genetic distances for aflp markers ( figs 1a , c ) and that the same result can be obtained with more specific microsatellite markers ( figs 1b , d ) . they also indicate that incompatibility reactions between separate clades of fungal symbionts , maintained by the two different genera of leaf - cutting ants , can no longer be predicted by neutral genetic markers . this lack of genetic signal may be due to incompatibility being ultimately caused by allelic variation at unknown loci ( worrall , 1997 ) that only correlate with neutral markers when there is recent common ancestry . we expect somatic incompatibility to be actively maintained by selection only in populations where the ants have the possibility to acquire multiple genetically different symbionts that are viable alternatives . our finding that a. colombica and a. echinatior maintain separated clades of fungal symbionts ( figs 3 and s5 ) suggests that l. gongylophorus has , in fact , been split into an atta and acromyrmex clade after its monophyletic origin 23 mya ( mikheyev et al . the two species of leaf - cutting ants that we investigated rear representatives of these symbiont lineages that appear adapted to being , respectively , an atta and acromyrmex symbiont . the fact that the fungal symbionts of panamanian leaf - cutting ants appear to have split in two monophyletic clades , is consistent with the results of a recent study that experimentally swapped fungus - garden symbionts between sympatric trachymyrmex septentrionalis and atta texana from texas , usa ( seal et al . , 2011a ) , and likely to have lower genetic variation among their symbionts , they share even less common ancestry than the panamanian fungal symbionts of our present study ( mueller et al . the swapped fungal symbionts thus could only serve as viable mutualists for either trachymyrmex or atta , but not both , which was confirmed in the published experiments showing that : ( 1 ) alternative fungus gardens were not always rejected by the ants but were never adopted as a viable alternative symbiont , because the ants were able to grow their original symbiont back from minuscule remnants that the authors had been unable to remove . to make further progress , it would be desirable to identify the genes that are directly responsible for somatic incompatibility , as this would allow direct studies on the signatures of selection and specialization across the clades of higher attine ant symbionts . whether these genes have homologs or analogs in attine ant fungal symbionts remains to be explored , as the sirex symbiont amylostereum areolatum belongs to a distantly related clade of basidiomycetes ( binder and hibbett , 2002 ) , and their respective domestication histories may have implied that recognition systems were lost and gained over evolutionary time . one possible explanation for this difference could be that there is a fundamental difference in colony founding in the sense that acromyrmex queens forage during colony founding similar to all more basal attine ants , whereas atta queens have secondarily evolved claustral colony founding . this implies that newly - mated atta queens close off their nest cavity to raise the first worker cohort purely on their body reserves , so that new colonies will only be opened by these workers 80100 d after they were founded ( weber , 1969 , fernandez - marin and wcislo , 2005 ) . although swapping of incipient fungus gardens with a fungus garden fragment from a mature acromyrmex colony was relatively unconstrained during early colony founding , it is likely that stronger mutual commitment between a founding queen and her resident fungus garden builds up in a matter of weeks , including stronger incompatibility reactions in case one of the first workers brings in an unrelated fungus garden fragment from a neighboring nest ( poulsen et al . this can never happen in the 80100 d during which atta colonies remain closed , removing selection for expressing incompatibility mechanisms during colony founding . why neutral markers should be weaker predictors of somatic incompatibility in atta colonies after workers start foraging remains unclear . , 2012 ) , but it seems unclear why such mechanisms should differ between atta and acromyrmex symbionts and why that should reduce selection for more direct defenses by fungal symbionts against being replaced . fungus gardens of panamanian acromyrmex colonies differ in chemical profiles ( richard et al . these differences do not correlate with genetic distances and comparable data for sympatric atta colonies are lacking . this may make a difference in the likelihood of a resident fungus garden symbiont being replaced by an accidentally imported small fragment of fungus garden from a neighboring colony , so that less accurate recognition systems suffice in mature colonies of atta but not in acromyrmex . finally , there could also be a technical explanation for the incompatibility differences between the fungal symbionts of atta and acromyrmex . , 2009 ) and the general aflp markers for acromyrmex symbionts , but enhanced variation in aflp peaks for atta symbionts , relative to acromyrmex symbionts ( fig s3 ) . this suggests that dna from other organisms may have been amplified with the aflps and that such other organisms were only present in atta symbiont cultures .

sepsis is a major healthcare problem . despite advances in supportive care of critically ill patients , sepsis remains an important cause of death worldwide in adults and children [ 13 ] . the surviving sepsis campaign ( ssc ) , which standardized the approach to sepsis , was recently updated . several efforts have been made to improve adherence to ssc guidelines [ 5 , 6 ] . nevertheless , mortality and costs are still high [ 2 , 7 , 8 ] . first , the presence of microorganisms in the bloodstream causes an innate immune response characterized by the stimulation of monocytes and release of proinflammatory cytokines and the activation of a medley of different immune pathways . toll - like receptors ( tlrs ) play a key role in this initial immune activation , acting as innate immune system sensors through the recognition of highly conserved components of a variety of microorganisms . the activation of tlrs induces an inflammatory response to control the infection , which results in local vasodilatation , release of various cytotoxic chemicals , and , hopefully , destruction of the invading pathogen . many of these same components of inflammation that are beneficial in host defenses against infection can , under some circumstances , be deleterious , causing cell and tissue damage and hence multiple organ failure . endotoxin , also known as lipopolysaccharide , is a component of gram - negative bacteria and a strong activator of tlr4 . the recognition of endotoxin by immune cells is important in the pathogenesis of septic shock [ 10 , 11 ] . conventional therapy such as antibiotics and surgical procedures to remove the source of infection is crucial for treating sepsis , but these approaches can not reverse the effects of the bacterial toxins already released into blood or of the endogenous mediators produced by the host in response to bacteria . over recent years , numerous attempts have aimed to intervene in the inflammatory cascade . attempts to stop the inflammatory cascade using antiendotoxin strategies such as monoclonal antibodies or vaccines have failed [ 12 , 13 ] . a recent phase 2 trial found a nonsignificant trend toward better survival in patients with severe sepsis treated with eritoran tetrasodium , a tlr-4 antagonist . blood purification techniques including hemoperfusion , plasma exchange , and hemofiltration with hemoperfusion are associated with lower mortality in patients with sepsis as it has been demonstrated in a recent meta - analysis . devices to remove endotoxin or inflammatory cytokines have been designed as a strategy to reduce the morbidity and mortality associated with sepsis , especially with sepsis due to gram - negative bacteria . these devices have also been successfully used in patients with sepsis due to gram - positive microorganisms and in patients with acute respiratory distress syndrome ( ards ) , suggesting that they could have an immunomodulating action in addition to endotoxin elimination [ 1721 ] . indeed , this review aims to summarize the immune modulation actions of polymyxin b - immobilized cartridge to understand the potential usefulness of this device beyond endotoxin elimination . over recent years , devices to eliminate endotoxin , inflammatory molecules such as cytokines and immune cells , have been designed to mitigate the deleterious effects of the inflammatory cascade . most of these devices are designed to combine the effect of molecules removal with renal replacement therapies ( hemofiltration , dialysis , or hemodiafiltration ) . the biocompatibility of these devices is the main limitation for its use , thrombocytopenia and bleeding risk are the potential side effects . polymyxin b is a cationic polypeptide antibiotic with activity against gram - negative bacteria and a high affinity to endotoxin , but its intravenous use has been limited due to nephrotoxicity and neurotoxicity . since 1994 , polymyxin b has been fixed and immobilized with polystyrene fiber in a hemoperfusion column polymyxin b - immobilized cartridge ( pmx ) that allows endotoxin removal without the toxic effects of this antibiotic . this treatment has been widely used in japan for septic shock due to gram - negative bacteria , and its use was authorized in europe in 1998 . recent studies support the safety and efficacy of this treatment [ 16 , 2426 ] . this medical device designed for extracorporeal use contains a series of porous polyethylene plates coated with a peptide specific to endotoxin and has a high adsorption capacity . compared lps adsorber and pmx hemoperfusion in a small sample of patients with gram - negative sepsis . however , due to limitations of the study , the authors concluded that further studies were necessary to clarify the efficacy of lps adsorber . this an-69 ( polysulfone and polyacrylonitrile ) based membrane adsorbs a large spectrum of plasma inflammatory mediators such as endotoxin and cytokines [ 30 , 31 ] . to date , clinical experience with this device is limited , but two trials are underway in septic patients . the results of these two trials are crucial to determine its usefulness compared with the current standard of care . based on the endotoxin - binding abilities of human albumin , this adsorber contains human serum albumin immobilized on polymethacrylate beads . this extracorporeal treatment is based on nonspecific adsorption of cytokines and other proinflammatory mediators onto a specially designed resin cartridge , coupled with hemofiltration . this is a promising therapy , although further studies are necessary to determine its usefulness in septic patients . one clinical trial , compact 2 , is underway to clarify whether adding high doses of cpfa to current clinical practice can reduce hospital mortality in septic shock patients ( clinicaltrials.gov number nct01639664 ) . this extracorporeal device removes cytokines through adsorption to a high - surface - area biocompatible porous polymer sorbent . this device does not target endotoxin , but it does rapidly eliminate several key cytokines by adsorption in both in vitro and in vivo experiments [ 38 , 39 ] . this device is very promising , but more studies in septic patients are needed . due to the broad information existing about safety and efficacy of polymyxin b - immobilized cartridge , we will review the immunological mechanisms described in this treatment . whereas some of these mechanisms are derived from endotoxin elimination , others result from direct action on other inflammatory molecules and cells or from a combination of endotoxin elimination and direct action on these mediators . immune cells recognize endotoxin and other bacterial compounds through the tlr , a group of transmembrane proteins that play crucial roles in the host defense against invading pathogens . during a gram - negative infection , tlr-4 recognizes endotoxin and originates a systemic inflammatory response in sepsis with potentially fatal effects in hosts . as a consequence , proinflammatory molecules such as interleukin-1 ( il-1 ) and tumor necrosis factor alpha ( tnf ) are released and generate other cell responses in the inflammatory cascade ( figure 1 ) . this increase in cytokines is followed by a major expression of tissue factor , which activates coagulation , and by an increase in nitric oxide synthesis , which induces vasodilation . endotoxin levels are high in septic patients [ 43 , 44 ] , but they are also high in critical patients without sepsis , such as patients undergoing cardiopulmonary bypass and those with chronic heart failure , chronic kidney disease , and other medical conditions [ 4447 ] . in critical patients without gram - negative infection , elevated endotoxin levels are related to translocation of gut bacterial antigens and endotoxin into the bloodstream due to gut barrier dysfunction [ 4850 ] . polymyxin b 's hydrophobic amino acids ( phe , leu ) form hydrophobic bonds with lipid a fatty acid in endotoxin , and the amino groups of polymyxin b form ionic bonds with the negatively charged phosphate groups of lipid a . this binding results in an antibiotic - endotoxin complex that is highly effective in neutralizing the deleterious effects of endotoxin . many studies have reported diverse benefits of pmx hemoperfusion in septic patients , including improved hemodynamics [ 24 , 25 , 5260 ] , increased ratio of partial pressure arterial oxygen and fraction of inspired oxygen ( pao2/fio2 ) [ 24 , 54 , 55 , 57 , 58 , 60 , 61 ] , decreased 28-day mortality [ 24 , 52 , 53 , 59 ] , and decreased endotoxin levels [ 52 , 53 , 55 , 58 , 59 , 6264 ] . in a multicenter study , vincent et al . found that pmx hemoperfusion was safe and improved cardiac and renal function due to sepsis or septic shock ; however , they could not demonstrate a reduction in mortality or in endotoxin levels from baseline to the end of treatment . in a systematic review of 28 studies , cruz et al . concluded that pmx hemoperfusion was associated with lower mortality ( rr 0.53 , 95% ci : 0.430.65 ) and improvements in mean arterial pressure ( map ) , use of inotropes , and pao2/fio2 . in 17 of these studies in which endotoxin levels were measured , endotoxin levels decreased by 33% to 80% after pmx hemoperfusion . more recently , the same authors published the euphas study , a prospective multicenter randomized controlled trial that enrolled 64 patients with severe sepsis or septic shock who underwent emergency surgery for intra - abdominal infection . patients were randomized to conventional therapy or to conventional therapy plus two sessions of pmx hemoperfusion . after the results of the scheduled interim , analysis revealed that pmx hemoperfusion significantly improved hemodynamics and organ dysfunction and reduced 28-day mortality ; the study was discontinued because it was considered unethical to deprive high risk patients of a potentially beneficial therapy ; however , early discontinuation resulted in a modest sample size . two adequately powered prospective trials are underway , and the results of these trials should elucidate the benefit of endotoxin removal ( clinicaltrials.gov numbers nct01046669 and nct01222663 ) [ 66 , 67 ] . several studies report a reduction in cytokines and inflammatory molecules in patients ' plasma after pmx hemoperfusion [ 54 , 56 , 61 , 6870 ] . in patients with severe sepsis , tani et al . found reductions in endotoxin , tnf , il-6 , il-10 , and plasminogen activator inhibitor-1 ( pai-1 ) activities after pmx hemoperfusion . in patients with ards , kushi et al . found a reduction in blood levels of pai-1 , neutrophil elastase ( ne ) , and il-8 after pmx hemoperfusion . ne is a protease that hydrolyzes lung elastin . in these patients , pao2/fio2 increased significantly after the treatment , and the authors related this increase to the elimination of il-8 and ne . in another study , the same group reported a decrease in ne in 20 septic patients treated with pmx hemoperfusion . in 12 patients with septic shock receiving conventional treatment plus two sessions of pmx hemoperfusion , zagli et al . found a decrease in il6 , il10 , and tnf in patients ' serum after the treatment , especially in survivors . most authors attribute the decrease in cytokines and inflammatory molecules to the removal of endotoxin and to the effect of this removal on the inflammatory cascade . patients with sepsis have increased high mobility group box-1 protein ( hmgb1 ) , a cytokine secreted by immune cells that triggers inflammatory mediators . the receptor for advanced glycation end - products ( rage ) is involved in hmgb1 signaling . the inhibition of the hmgb-1-rage axis could be an effective therapeutic strategy for septic shock . nakamura et al . compared il-6 , hmgb1 , and rage in serum between 15 patients with septic shock treated with pmx hemoperfusion and healthy volunteers . the levels of the three molecules decreased after pmx hemoperfusion and correlated with a decrease in endotoxin . abe et al . studied the effects of pmx hemoperfusion on hmgb1 in patients with acute exacerbation of idiopathic pulmonary fibrosis . moreover , hmgb-1 was detected in washing medium from the pmx column , suggesting that the decrease in this molecule was not only secondary to endotoxin removal but also to direct removal by the device . a recent study exploring the meaning of hmgb-1 levels in 60 patients with septic shock treated with pmx hemoperfusion found a significant positive correlation between the sequential organ failure assessment ( sofa ) score and hmgb-1 level ( p < 0.05 ) . the authors concluded that hmgb-1 is a useful prognostic biomarker in sepsis - induced organ failure in patients undergoing pmx hemoperfusion , but formal establishment of the utility of hmgb-1 as a prognostic biomarker still remains to be performed . pai-1 , a marker of vascular endothelial cell activation elevated by endotoxin and cytokines , is one of the fibrinolysis inhibitory factors . pai-1 levels decrease after pmx hemoperfusion , decreasing the stimulation of vascular endothelial cells [ 54 , 55 , 61 ] . pmx hemoperfusion may have a role in modulating fibrinolysis and inhibiting the development of ischemic organ dysfunction in sepsis . -2 play a contributory role in the pathogenesis of acute lung injury ( ali ) in septic patients . vascular endothelial growth factor ( vegf ) is a pluripotent growth and permeability factor that has a broad impact on endothelial cell function . recently studied nine patients with acute exacerbation of idiopathic pulmonary fibrosis treated with conventional therapy and pmx hemoperfusion 6 hours / day on two successive days . they found a high concentration of cytokines and vegf in the eluate from used pmx cartridge fibers , and the clinical improvement in these patients correlated with the amount of vegf in the eluate . this is the first study to demonstrate that cytokines and vegf can be directly adsorbed by pmx hemoperfusion independently from endotoxin removal . the authors suggest that cytokines can bind to pmx hemoperfusion fibers directly through ionic / hydrophobic interactions like endotoxin or indirectly via heparin coated in the fibers . anandamide is an intrinsic cannabinoid that has been related with hypotension in septic shock , although currently its direct link to sepsis is only established in a small patient population . one study in 24 patients with septic shock treated with pmx hemoperfusion found that anandamide levels decreased after pmx hemoperfusion in the nine patients who survived ; the authors conclude that removal of anandamide by pmx hemoperfusion , whether directly or as a result of endotoxin elimination , could be key to successful septic shock treatment . further studies are necessary to elucidate the effect of pmx hemoperfusion on anandamide , in order to establish it as a useful treatment for hypotension . elevation of nitric oxide ( no ) plays an important role in septic patients , producing vasodilatation and hypotension . nakamura et al . compared no breakdown products in urine in 20 patients with pmx hemoperfusion , 15 patients with conventional therapy , and 20 healthy controls . they found that septic patients increased no production and that pmx hemoperfusion reduced no levels and thus increased blood pressure . troponin is a biomarker that may be elevated in septic patients as a result of subclinical myocardial cell damage . nakamura et al . found increased troponin t in septic patients compared to nonseptic patients and age - matched healthy controls ; interestingly , troponin t decreased after pmx hemoperfusion p < 0.05 . erythropoietin levels may be higher in patients with sepsis ; erythropoietin levels decrease after pmx hemoperfusion and could be a prognostic indicator in patients with septic shock . during sepsis , different populations of leukocytes are activated and change their adhesive phenotype . the capture of leukocytes through extracorporeal blood purification could alter the immune response to sepsis . after ex vivo perfusion of heparinized blood from patients with sepsis and septic shock through pmx hemoperfusion in a laboratory circuit , kumagai et al . found significant decreases in neutrophils ( 78% ) , monocytes ( 70% ) , and lymphocytes ( 10% ) . this marked reduction in white blood cells should be attributed mostly to the reduction in the circulation of proinflammatory cytokines that induce cell activation and proliferation , as opposed to a direct effect of removal of these cells by the cartridge . examined the pmx hemoperfusion filters after treating 4 patients with sepsis ; pmx hemoperfusion bound monocytes from the peripheral blood leucocytes . pmx hemoperfusion could produce a beneficial effect by reducing the interaction between monocytes and functionally associated cells , including endothelial cells . nakamura et al . studied the effect of pmx hemoperfusion on platelet activation , comparing 30 patients treated with conventional therapy plus pmx hemoperfusion and 20 patients with conventional therapy alone . survival was 60% in the group that received pmx hemoperfusion and 30% in the group that received only conventional treatment . septic patients had increased paf ( p - selectin , platelet factor 4 , and -thromboglobulin ) , and pmx hemoperfusion reduced the levels of paf . the human body undergoes a biphasic immunological reaction in sepsis . a proinflammatory reaction takes place , marked by the release of proinflammatory cytokines like tnf , as a reaction to the bacterial toxins . on the other hand , the persistence of a marked compensatory anti - inflammatory response is called immunoparalysis ( figure 1 ) . this pronounced immunosuppressive state adversely affects immune function , making the patient vulnerable to opportunistic infections . these two phases of sepsis may occur simultaneously with a lasting anti - inflammatory response in later phases . most septic patients survive the initial proinflammatory phase , but they die during this second stage . strategies to stimulate this immunoparalysis phase of sepsis as ifn- and granulocyte - macrophage colony - stimulating factor ( gm - csf ) have been developed in animals , but extensive clinical studies are needed to test their safety and efficacy . recently , ono et al . showed that the expression of the surface antigens , hla - dr on monocytes and cd16 on granulocytes , is extremely decreased in patients with septic shock and that pmx hemoperfusion beneficially increases this expression on these leukocytes . the regulatory t cells ( treg ) that express cd4 , cd25 , and foxp3 comprise a small percentage of the t - lymphocyte population in the immune system , but they are central to the maintenance of immunological homeostasis and tolerance . in septic patients , the percentage of treg is increased , and this presumably contributes to sepsis - induced immunosuppression . polymyxin - b induces treg cell death in mice through the modulation of the purinergic p2x7 receptor . studied the effect of pmx hemoperfusion on the recovery from the immunosuppression owing to septic shock . treg , il-6 , and il-10 were higher in patients with septic shock than in patients with sepsis . after pmx hemoperfusion , treg cells , il-6 , and il-10 decreased . in survivors , the decrease in treg cells was accompanied by an increase in cd4 + cells . although further studies are necessary to confirm a causative relationship between treg depletion and pmx hemoperfusion in septic patients , this mechanism could explain why pmx hemoperfusion can be useful in patients without endotoxemia or in those with gram - positive sepsis [ 89 , 90 ] . the authors also suggest that the second pmx hemoperfusion treatment might provide additional benefits for recovery from immunoparalysis . this study sheds new light on the benefits of treating septic patients with pmx hemoperfusion beyond endotoxin removal . endotoxin may cause an inappropriate activation of proapoptotic pathways in immune cells during sepsis , and this may contribute to the impaired immune response that characterizes sepsis . endotoxin can also cause apoptosis of renal tubular cells through fas - mediated and caspase - mediated pathways . tested the hypothesis that pmx hemoperfusion might prevent gram - negative sepsis - induced acute renal failure by reducing the activity of proapoptotic circulating factors . they randomized 16 patients with gram - negative sepsis to receive standard care or standard care plus pmx hemoperfusion . proapoptotic activity was significantly reduced in the plasma of the pmx hemoperfusion group , with decreases in fas upregulation and caspase activity , and these patients also had improved renal function . several studies have found that pmx hemoperfusion has beneficial effects on oxygenation in patients with sepsis [ 24 , 61 , 65 ] . moreover , pmx hemoperfusion has been successful in patients with influenza a infection [ 89 , 95 ] , ards in drug - induced injury [ 96 , 97 ] , interstitial pneumonia [ 19 , 20 , 98 ] , and idiopathic fibrosis [ 18 , 69 , 74 , 99 ] . mechanisms other than endotoxin removal could explain the beneficial effects of pmx hemoperfusion in patients with respiratory failure . chemical mediators have an important role in the pathogenesis of ards , and decreasing them through direct or indirect removal could be beneficial in ards patients . . found decreases in pai1 , neutrophil elastase ( ne ) , and il-8 in ards after pmx hemoperfusion . studied the role of decreases in hmgb1 after pmx hemoperfusion in patients with acute exacerbation of idiopathic fibrosis . when the same authors investigated the effects of pmx hemoperfusion in a retrospective multicenter study of 160 patients with acute exacerbation of idiopathic pulmonary fibrosis or interstitial pneumonia , they found that the pao2/fio2 ratio significantly increased after pmx hemoperfusion . they concluded that pmx hemoperfusion might be an effective adjunctive therapy for these patients , although the mechanisms underlying the benefits of the treatment are uncertain . likewise , hara et al . reported that pmx hemoperfusion resulted in improved pao2/fio2 ratio 72 hours and 1 week after treatment in 33 patients with acute exacerbation of interstitial pneumonia . tsushima et al . treated 20 patients with ards with pmx hemoperfusion and compared the outcomes with a historical control group . they found improved pao2/fio2 ratio and survival ; however , the methodology of the study limits its power to draw conclusions . collectively , the matrix metalloproteinases ( mmp ) are capable of degrading all kinds of extracellular matrix proteins . mmp-9 is a protease involved in the degradation of the basement membrane , a part of the extracellular matrix associated with the alveolar epithelium and vascular endothelium . mmp-9 is essential for the remodeling of basement membranes in various inflammatory lung diseases , including ards . increased amounts of mmp-9 in the vasculature are likely to enhance vascular permeability and to facilitate cell homing and inflammatory remodeling . nakamura et al . studied the effect of pmx hemoperfusion on mmp levels in ards patients by treating 12 ards patients with two sessions of pmx hemoperfusion and comparing their laboratory data with those of healthy controls . after pmx hemoperfusion , mmp-9 decreased and chest x - ray findings improved . however , the precise mechanism is still unclear . the authors suggest the need for more studies to elucidate the beneficial effect of pmx hemoperfusion in ards . in a pilot study of 16 patients , abe et al . studied the effects of pmx hemoperfusion for acute exacerbation of interstitial pneumonia and demonstrated neutrophil adsorption and a decrease in mmp-9 . in recent years takahashi et al . studied the changes in serum s100a12 and rage after pmx hemoperfusion in postoperative septic shock . they found a significant decrease in s100a12 in serum after pmx hemoperfusion and an improvement in pao2/fio2 ratio but no decrease in rage . as mentioned above , oishi et al . found cytokines and vegf in the eluate from pmx hemoperfusion cartridges used to treat patients with acute exacerbation of pulmonary fibrosis , suggesting a new explanation for the improvement in oxygenation in nonseptic patients treated with pmx hemoperfusion . in recent years , many studies have shown that pmx hemoperfusion is a promising strategy for immunomodulation in septic shock , and two ongoing clinical trials will be key in determining its usefulness . although most studies have focused on the removal of endotoxin as the principal mechanism through which pmx hemoperfusion improves outcome in sepsis , other studies have revealed mechanisms involving diverse immunological pathways through which pmx hemoperfusion could improve outcome not only in sepsis but also in non - septic respiratory failure . however , these studies are limited by their small samples , their observational and in some cases retrospective design , and the lack of control groups in many cases . it is interesting to note that the elimination of endotoxin brings about a reduction in many inflammatory molecules and cells involved in the inflammatory cascade . endotoxin removal devices act at the onset of this complex cascade , and their benefits in terms of immunomodulation are encouraging . only a part of the consequences of endotoxin elimination has been studied and summarized in this review . future studies might reveal other mediators and cells involved in sepsis that might be altered after endotoxin removal . some of the studies reviewed here found mediators and cells in the eluate from pmx hemoperfusion cartridges or by direct examination of the filter . further studies are necessary to elucidate how pmx hemoperfusion eliminates these molecules , whether through ionic / hydrophobic interactions like in endotoxin removal or indirectly via heparin that coats the fibers . additional mechanisms could potentially explain why pmx hemoperfusion can be beneficial in gram - positive sepsis or non - septic respiratory failure . endotoxin can be elevated in other medical conditions apart from gram - negative infections , and its removal could also partially explain this benefit . the immunomodulating effects of pmx hemoperfusion in patients with interstitial pneumonia and acute exacerbations of pulmonary fibrosis are especially interesting , given the high mortality associated with these conditions . however , well - designed clinical trials are needed to assess the efficacy of pmx hemoperfusion in these medical conditions . future potential directions such as combination of different hemoperfusion devices to treat septic patients , in order to alter the host inflammatory response in more than one step , are currently speculative . technically , it could be viable , but no experience has been reported to date . in summary , antimicrobial therapy , surgical treatment of the focus of infection , and hemodynamic stabilization are crucial in the treatment of severe sepsis . it seems that pmx hemoperfusion might have other beneficial immunological mechanisms in addition to endotoxin removal ; however , the limited evidence suggests that we must be cautious with other indications for pmx hemoperfusion , and future studies are necessary .

severe sepsis results in high morbidity and mortality . immunomodulation strategies could be an adjunctive therapy to treat sepsis . endotoxin is a component of gram - negative bacteria and plays an important role in the pathogenesis of septic shock when it is recognized by immune cells . removal of endotoxin could be an effective adjunctive approach to the management of sepsis . devices to adsorb endotoxin or inflammatory cytokines have been designed as a strategy to treat severe sepsis , especially sepsis caused by gram - negative bacteria . polymyxin b - immobilized cartridge has been successfully used to treat patients with sepsis of abdominal origin . although this cartridge was conceived to adsorb endotoxin , several other immunological mechanisms have been elucidated , and this device has also yielded promising results in patients with nonseptic respiratory failure . in this paper , we summarize the immune modulation actions of polymyxin b - immobilized cartridge to explore its potential usefulness beyond endotoxin elimination .

1. Introduction 2. Devices to Remove Endotoxin and Inflammatory Molecules 3. Immunological Mechanisms Described for Polymyxin B-Immobilized Cartridge 4. Usefulness in Acute Respiratory Failure 5. Conclusions

sepsis is a major healthcare problem . despite advances in supportive care of critically ill patients , sepsis remains an important cause of death worldwide in adults and children [ 13 ] . the surviving sepsis campaign ( ssc ) , which standardized the approach to sepsis , was recently updated . several efforts have been made to improve adherence to ssc guidelines [ 5 , 6 ] . toll - like receptors ( tlrs ) play a key role in this initial immune activation , acting as innate immune system sensors through the recognition of highly conserved components of a variety of microorganisms . the activation of tlrs induces an inflammatory response to control the infection , which results in local vasodilatation , release of various cytotoxic chemicals , and , hopefully , destruction of the invading pathogen . endotoxin , also known as lipopolysaccharide , is a component of gram - negative bacteria and a strong activator of tlr4 . the recognition of endotoxin by immune cells is important in the pathogenesis of septic shock [ 10 , 11 ] . conventional therapy such as antibiotics and surgical procedures to remove the source of infection is crucial for treating sepsis , but these approaches can not reverse the effects of the bacterial toxins already released into blood or of the endogenous mediators produced by the host in response to bacteria . over recent years , numerous attempts have aimed to intervene in the inflammatory cascade . a recent phase 2 trial found a nonsignificant trend toward better survival in patients with severe sepsis treated with eritoran tetrasodium , a tlr-4 antagonist . blood purification techniques including hemoperfusion , plasma exchange , and hemofiltration with hemoperfusion are associated with lower mortality in patients with sepsis as it has been demonstrated in a recent meta - analysis . devices to remove endotoxin or inflammatory cytokines have been designed as a strategy to reduce the morbidity and mortality associated with sepsis , especially with sepsis due to gram - negative bacteria . these devices have also been successfully used in patients with sepsis due to gram - positive microorganisms and in patients with acute respiratory distress syndrome ( ards ) , suggesting that they could have an immunomodulating action in addition to endotoxin elimination [ 1721 ] . indeed , this review aims to summarize the immune modulation actions of polymyxin b - immobilized cartridge to understand the potential usefulness of this device beyond endotoxin elimination . over recent years , devices to eliminate endotoxin , inflammatory molecules such as cytokines and immune cells , have been designed to mitigate the deleterious effects of the inflammatory cascade . polymyxin b is a cationic polypeptide antibiotic with activity against gram - negative bacteria and a high affinity to endotoxin , but its intravenous use has been limited due to nephrotoxicity and neurotoxicity . since 1994 , polymyxin b has been fixed and immobilized with polystyrene fiber in a hemoperfusion column polymyxin b - immobilized cartridge ( pmx ) that allows endotoxin removal without the toxic effects of this antibiotic . this treatment has been widely used in japan for septic shock due to gram - negative bacteria , and its use was authorized in europe in 1998 . compared lps adsorber and pmx hemoperfusion in a small sample of patients with gram - negative sepsis . this is a promising therapy , although further studies are necessary to determine its usefulness in septic patients . this device does not target endotoxin , but it does rapidly eliminate several key cytokines by adsorption in both in vitro and in vivo experiments [ 38 , 39 ] . this device is very promising , but more studies in septic patients are needed . due to the broad information existing about safety and efficacy of polymyxin b - immobilized cartridge , we will review the immunological mechanisms described in this treatment . whereas some of these mechanisms are derived from endotoxin elimination , others result from direct action on other inflammatory molecules and cells or from a combination of endotoxin elimination and direct action on these mediators . immune cells recognize endotoxin and other bacterial compounds through the tlr , a group of transmembrane proteins that play crucial roles in the host defense against invading pathogens . during a gram - negative infection , tlr-4 recognizes endotoxin and originates a systemic inflammatory response in sepsis with potentially fatal effects in hosts . as a consequence , proinflammatory molecules such as interleukin-1 ( il-1 ) and tumor necrosis factor alpha ( tnf ) are released and generate other cell responses in the inflammatory cascade ( figure 1 ) . endotoxin levels are high in septic patients [ 43 , 44 ] , but they are also high in critical patients without sepsis , such as patients undergoing cardiopulmonary bypass and those with chronic heart failure , chronic kidney disease , and other medical conditions [ 4447 ] . in critical patients without gram - negative infection , elevated endotoxin levels are related to translocation of gut bacterial antigens and endotoxin into the bloodstream due to gut barrier dysfunction [ 4850 ] . polymyxin b 's hydrophobic amino acids ( phe , leu ) form hydrophobic bonds with lipid a fatty acid in endotoxin , and the amino groups of polymyxin b form ionic bonds with the negatively charged phosphate groups of lipid a . this binding results in an antibiotic - endotoxin complex that is highly effective in neutralizing the deleterious effects of endotoxin . many studies have reported diverse benefits of pmx hemoperfusion in septic patients , including improved hemodynamics [ 24 , 25 , 5260 ] , increased ratio of partial pressure arterial oxygen and fraction of inspired oxygen ( pao2/fio2 ) [ 24 , 54 , 55 , 57 , 58 , 60 , 61 ] , decreased 28-day mortality [ 24 , 52 , 53 , 59 ] , and decreased endotoxin levels [ 52 , 53 , 55 , 58 , 59 , 6264 ] . found that pmx hemoperfusion was safe and improved cardiac and renal function due to sepsis or septic shock ; however , they could not demonstrate a reduction in mortality or in endotoxin levels from baseline to the end of treatment . concluded that pmx hemoperfusion was associated with lower mortality ( rr 0.53 , 95% ci : 0.430.65 ) and improvements in mean arterial pressure ( map ) , use of inotropes , and pao2/fio2 . more recently , the same authors published the euphas study , a prospective multicenter randomized controlled trial that enrolled 64 patients with severe sepsis or septic shock who underwent emergency surgery for intra - abdominal infection . two adequately powered prospective trials are underway , and the results of these trials should elucidate the benefit of endotoxin removal ( clinicaltrials.gov numbers nct01046669 and nct01222663 ) [ 66 , 67 ] . several studies report a reduction in cytokines and inflammatory molecules in patients ' plasma after pmx hemoperfusion [ 54 , 56 , 61 , 6870 ] . in patients with severe sepsis , tani et al . found reductions in endotoxin , tnf , il-6 , il-10 , and plasminogen activator inhibitor-1 ( pai-1 ) activities after pmx hemoperfusion . in patients with ards , kushi et al . found a reduction in blood levels of pai-1 , neutrophil elastase ( ne ) , and il-8 after pmx hemoperfusion . in these patients , pao2/fio2 increased significantly after the treatment , and the authors related this increase to the elimination of il-8 and ne . in 12 patients with septic shock receiving conventional treatment plus two sessions of pmx hemoperfusion , zagli et al . found a decrease in il6 , il10 , and tnf in patients ' serum after the treatment , especially in survivors . most authors attribute the decrease in cytokines and inflammatory molecules to the removal of endotoxin and to the effect of this removal on the inflammatory cascade . patients with sepsis have increased high mobility group box-1 protein ( hmgb1 ) , a cytokine secreted by immune cells that triggers inflammatory mediators . the inhibition of the hmgb-1-rage axis could be an effective therapeutic strategy for septic shock . compared il-6 , hmgb1 , and rage in serum between 15 patients with septic shock treated with pmx hemoperfusion and healthy volunteers . studied the effects of pmx hemoperfusion on hmgb1 in patients with acute exacerbation of idiopathic pulmonary fibrosis . a recent study exploring the meaning of hmgb-1 levels in 60 patients with septic shock treated with pmx hemoperfusion found a significant positive correlation between the sequential organ failure assessment ( sofa ) score and hmgb-1 level ( p < 0.05 ) . the authors concluded that hmgb-1 is a useful prognostic biomarker in sepsis - induced organ failure in patients undergoing pmx hemoperfusion , but formal establishment of the utility of hmgb-1 as a prognostic biomarker still remains to be performed . pmx hemoperfusion may have a role in modulating fibrinolysis and inhibiting the development of ischemic organ dysfunction in sepsis . -2 play a contributory role in the pathogenesis of acute lung injury ( ali ) in septic patients . vascular endothelial growth factor ( vegf ) is a pluripotent growth and permeability factor that has a broad impact on endothelial cell function . recently studied nine patients with acute exacerbation of idiopathic pulmonary fibrosis treated with conventional therapy and pmx hemoperfusion 6 hours / day on two successive days . they found a high concentration of cytokines and vegf in the eluate from used pmx cartridge fibers , and the clinical improvement in these patients correlated with the amount of vegf in the eluate . the authors suggest that cytokines can bind to pmx hemoperfusion fibers directly through ionic / hydrophobic interactions like endotoxin or indirectly via heparin coated in the fibers . anandamide is an intrinsic cannabinoid that has been related with hypotension in septic shock , although currently its direct link to sepsis is only established in a small patient population . one study in 24 patients with septic shock treated with pmx hemoperfusion found that anandamide levels decreased after pmx hemoperfusion in the nine patients who survived ; the authors conclude that removal of anandamide by pmx hemoperfusion , whether directly or as a result of endotoxin elimination , could be key to successful septic shock treatment . elevation of nitric oxide ( no ) plays an important role in septic patients , producing vasodilatation and hypotension . compared no breakdown products in urine in 20 patients with pmx hemoperfusion , 15 patients with conventional therapy , and 20 healthy controls . troponin is a biomarker that may be elevated in septic patients as a result of subclinical myocardial cell damage . erythropoietin levels may be higher in patients with sepsis ; erythropoietin levels decrease after pmx hemoperfusion and could be a prognostic indicator in patients with septic shock . the capture of leukocytes through extracorporeal blood purification could alter the immune response to sepsis . after ex vivo perfusion of heparinized blood from patients with sepsis and septic shock through pmx hemoperfusion in a laboratory circuit , kumagai et al . this marked reduction in white blood cells should be attributed mostly to the reduction in the circulation of proinflammatory cytokines that induce cell activation and proliferation , as opposed to a direct effect of removal of these cells by the cartridge . examined the pmx hemoperfusion filters after treating 4 patients with sepsis ; pmx hemoperfusion bound monocytes from the peripheral blood leucocytes . septic patients had increased paf ( p - selectin , platelet factor 4 , and -thromboglobulin ) , and pmx hemoperfusion reduced the levels of paf . a proinflammatory reaction takes place , marked by the release of proinflammatory cytokines like tnf , as a reaction to the bacterial toxins . these two phases of sepsis may occur simultaneously with a lasting anti - inflammatory response in later phases . strategies to stimulate this immunoparalysis phase of sepsis as ifn- and granulocyte - macrophage colony - stimulating factor ( gm - csf ) have been developed in animals , but extensive clinical studies are needed to test their safety and efficacy . showed that the expression of the surface antigens , hla - dr on monocytes and cd16 on granulocytes , is extremely decreased in patients with septic shock and that pmx hemoperfusion beneficially increases this expression on these leukocytes . the regulatory t cells ( treg ) that express cd4 , cd25 , and foxp3 comprise a small percentage of the t - lymphocyte population in the immune system , but they are central to the maintenance of immunological homeostasis and tolerance . in septic patients , the percentage of treg is increased , and this presumably contributes to sepsis - induced immunosuppression . studied the effect of pmx hemoperfusion on the recovery from the immunosuppression owing to septic shock . treg , il-6 , and il-10 were higher in patients with septic shock than in patients with sepsis . after pmx hemoperfusion , treg cells , il-6 , and il-10 decreased . although further studies are necessary to confirm a causative relationship between treg depletion and pmx hemoperfusion in septic patients , this mechanism could explain why pmx hemoperfusion can be useful in patients without endotoxemia or in those with gram - positive sepsis [ 89 , 90 ] . this study sheds new light on the benefits of treating septic patients with pmx hemoperfusion beyond endotoxin removal . endotoxin may cause an inappropriate activation of proapoptotic pathways in immune cells during sepsis , and this may contribute to the impaired immune response that characterizes sepsis . tested the hypothesis that pmx hemoperfusion might prevent gram - negative sepsis - induced acute renal failure by reducing the activity of proapoptotic circulating factors . they randomized 16 patients with gram - negative sepsis to receive standard care or standard care plus pmx hemoperfusion . proapoptotic activity was significantly reduced in the plasma of the pmx hemoperfusion group , with decreases in fas upregulation and caspase activity , and these patients also had improved renal function . several studies have found that pmx hemoperfusion has beneficial effects on oxygenation in patients with sepsis [ 24 , 61 , 65 ] . moreover , pmx hemoperfusion has been successful in patients with influenza a infection [ 89 , 95 ] , ards in drug - induced injury [ 96 , 97 ] , interstitial pneumonia [ 19 , 20 , 98 ] , and idiopathic fibrosis [ 18 , 69 , 74 , 99 ] . mechanisms other than endotoxin removal could explain the beneficial effects of pmx hemoperfusion in patients with respiratory failure . chemical mediators have an important role in the pathogenesis of ards , and decreasing them through direct or indirect removal could be beneficial in ards patients . studied the role of decreases in hmgb1 after pmx hemoperfusion in patients with acute exacerbation of idiopathic fibrosis . when the same authors investigated the effects of pmx hemoperfusion in a retrospective multicenter study of 160 patients with acute exacerbation of idiopathic pulmonary fibrosis or interstitial pneumonia , they found that the pao2/fio2 ratio significantly increased after pmx hemoperfusion . they concluded that pmx hemoperfusion might be an effective adjunctive therapy for these patients , although the mechanisms underlying the benefits of the treatment are uncertain . reported that pmx hemoperfusion resulted in improved pao2/fio2 ratio 72 hours and 1 week after treatment in 33 patients with acute exacerbation of interstitial pneumonia . mmp-9 is a protease involved in the degradation of the basement membrane , a part of the extracellular matrix associated with the alveolar epithelium and vascular endothelium . increased amounts of mmp-9 in the vasculature are likely to enhance vascular permeability and to facilitate cell homing and inflammatory remodeling . studied the effect of pmx hemoperfusion on mmp levels in ards patients by treating 12 ards patients with two sessions of pmx hemoperfusion and comparing their laboratory data with those of healthy controls . studied the changes in serum s100a12 and rage after pmx hemoperfusion in postoperative septic shock . found cytokines and vegf in the eluate from pmx hemoperfusion cartridges used to treat patients with acute exacerbation of pulmonary fibrosis , suggesting a new explanation for the improvement in oxygenation in nonseptic patients treated with pmx hemoperfusion . in recent years , many studies have shown that pmx hemoperfusion is a promising strategy for immunomodulation in septic shock , and two ongoing clinical trials will be key in determining its usefulness . although most studies have focused on the removal of endotoxin as the principal mechanism through which pmx hemoperfusion improves outcome in sepsis , other studies have revealed mechanisms involving diverse immunological pathways through which pmx hemoperfusion could improve outcome not only in sepsis but also in non - septic respiratory failure . however , these studies are limited by their small samples , their observational and in some cases retrospective design , and the lack of control groups in many cases . it is interesting to note that the elimination of endotoxin brings about a reduction in many inflammatory molecules and cells involved in the inflammatory cascade . endotoxin removal devices act at the onset of this complex cascade , and their benefits in terms of immunomodulation are encouraging . only a part of the consequences of endotoxin elimination has been studied and summarized in this review . some of the studies reviewed here found mediators and cells in the eluate from pmx hemoperfusion cartridges or by direct examination of the filter . additional mechanisms could potentially explain why pmx hemoperfusion can be beneficial in gram - positive sepsis or non - septic respiratory failure . endotoxin can be elevated in other medical conditions apart from gram - negative infections , and its removal could also partially explain this benefit . the immunomodulating effects of pmx hemoperfusion in patients with interstitial pneumonia and acute exacerbations of pulmonary fibrosis are especially interesting , given the high mortality associated with these conditions . future potential directions such as combination of different hemoperfusion devices to treat septic patients , in order to alter the host inflammatory response in more than one step , are currently speculative . technically , it could be viable , but no experience has been reported to date . in summary , antimicrobial therapy , surgical treatment of the focus of infection , and hemodynamic stabilization are crucial in the treatment of severe sepsis . it seems that pmx hemoperfusion might have other beneficial immunological mechanisms in addition to endotoxin removal ; however , the limited evidence suggests that we must be cautious with other indications for pmx hemoperfusion , and future studies are necessary .

promoting the level of education , entering jobs and employment and increasing the social participation are among the social changes resulting from the transition from old to new society that a significant portion of the iranian women have experienced in recent years and could achieve their own interests and aspirations along their other traditional roles and attain social positions . the origin of these changes as well as improved quality of life and social advancement of women have encountered them with new problems and challenges . in most internal and external research that has been done on women , waiver what perspective they view women with , two major roles , as wives and mothers for women is defined . two important and main roles , that is , spousal and maternal have been defined for women regarding the mental and physical characteristics of a woman , she will have to bear a special responsibility for them changes are formed in the traditional gender division of labor that had previously been defined for women to add a new role on their previous roles . in other words , occupation as an added role could involve obligations and responsibilities for working women , because they have an obligation to their housework duties and tasks in the field of employment outside the home as well . in other words , with their employment women face not only a role , but a series of interconnected roles that are requiring each other , such as the spousal , maternal , and occupational roles and similarly confront various expectations simultaneously . this particular concern for women that reconciles their traditional and modern roles is more challenging . since becoming a mother and the maternal role are complex processes affected by personal and social factors such as supporting systems , economic conditions , and cultural situations ; it seems that analyzing the experiences and elaborations of working mothers is a step towards promoting proper life of women , children , and family as the most important pillar in the society as a whole , to understand the needs , specific problems , and even the probable strong points . this research seeks to answer this important question as , what is the process of maternal role among women ? . the aim of this study was to clarify the process of maternal role among working women . in cases where the researcher has to consider the process of formation or exposure to certain social phenomena in question , the basic theory and method of grounded theory as a qualitative research method based on symbolic interaction are used . analyzing the existing social processes deal with the context of human relationships . since the maternal role is a dynamic process of interaction formed by the relation with others in particular fields , and the working women make themselves compatible with the changes in life , the grounded theory methodology was chosen for this study . participants in this study were among the women working in one of the islamic azad university branches , including faculty and staff members and all the participants who had an experience of being a mother , and had at least one child . initially , the targeted sampling was started and gradually continued by stabilization of conceptual classes during the data analysis . participants were selected based on theoretical sampling according to the codes and classes continued until data saturation . after the saturation level of the data was achieved , interviews were conducted with nine participants . participants based on text written consent , freely participated in the study . in depth interviews and field notes the duration of interviews was 30 - 45 min with an average of 20 min . data collection and data analysis in this study were done simultaneously and the data were analyzed according to strauss and corbin approach and divided into three stages including open coding , axial coding , and selective coding ; which led to identifying the variable core as the main class relating with other classes to describe them . the four criteria ; including credibility , dependability , conformability , and transferability or fittingness were considered for the validity and credibility of the study . the purpose of this is to ensure the exact experiences of participants in the research . for the credibility and validation of the findings , the method regarding the participants check was used that indicated whether the participants recognize the data and the results are in conformity with their experiences in terms of accuracy , completeness , and interpretation ; or not . scaling ideas were taken and the amount realized from the data was compared with the participants . the full texts of the first implemented interviews together with the related codes , concepts , and categories were sent to some colleagues and supervisors to be analyzed and verified . dependability is a criterion that is verified when the researcher determines the validity of the findings . the question that defines this criterion is about the dependency of the results to each otherto fulfill this criterion , the study 's findings were given to three working mother who did not participate in the study and they verified the relevance of them with their own experiences . conformability in qualitative research and is equivalent to reliability in quantitative research and represents stability and reliability of the data over time and under identical conditions . to develop the ability to approve or object to all of the survey data , researchers tried to fully explain all the stages of the research to ensure the integrity of the open and axial coding , the data and findings were given to other researchers familiar with qualitative studies to verify their accuracies . transferability or fitness means fitness and mobility of the outcomes to be used in similar conditions . discussing and comparing our findings with other studies is a method to evaluate the appropriateness of the data that has been used in this study . data obtained from analysis of the interviews included , 414 codes from the open coding phase , 42 category of the axial coding stage and 9 main themes of working mothers experiences which consisted of different pregnancy experience , returning concerns , supportive umbrella , role assignation , role overlap , role strain , gradual acceptance , satisfaction , and erosion that led to the identification of the core variable of role conflict that resulted from selective coding considered as the basic social psychological process , which is faced by working mothers in the study and developed over time , that is shown in figure 1 . the process of maternal role the following are some of the components of maternal role in the process described in this study : one of the major problems of working mothers in the study with their maternal role was their return to work after maternity leave . mental concerns and worries about finding the right person and place to care for a child , how to breast feed the infant , obligations in full - time cooperation in the organization , and inadequate supports of the organization are the concerns for most mothers participating in the study . most mothers agreed that they were scared from the beginning of their period of maternity leave . in this regard , i was very worried from the beginning to what i should do about my child after my maternity leave , where should i put him , what i should do for breast feeding him . this theme reflected maternal duties in the absence of mother , including relatives of mother , supportive resources in the community such as kindergartens , private institutions , and service or using family members or neighbors due to necessity because of the particular circumstances . in other words , working mothers in the study had to assign their maternal roles to other when they were at work in order to meet the diverse needs of their child with the help of others . in this i asked one of the relatives of one of my neighbors to take care of my children and i used to pay her monthly wages . i used to take my younger child to her before going to work and send my elder child by a school car to school and take him back by the afternoon to the nursery for my husband to take him back home after his work . it indicates that the additional pressure exerted on the mother accepting various roles and being in forceful conditions providing her to tolerate the situations : i make the food ready with all my fatigue , wash the dishes , do the house work , and used to control the homework done by my children up to primary school . some mothers were inconsistent in their roles or due to negligence or failure in performing their duties ; they were affected by negative emotions such as disgust , hatred , guilt , and blame : it was 9 oclock that my mother in law called and said that my daughter had fever and vomiting and was crying . i talked to my child and calmed her down and said i would come after she had a little rest . i was worried and told myself i should have been with my child by then . she was crying there and suffering and hurting her grandfather , and i was here . participant number ( 6 ) says : i am not satisfied with my condition at all . i think if i was a housewife , taking care of children , it would be better , from what it is now . i enjoy when i solve a problem from a student and when he / she smiles . it indicates reduction of mental and physical potentials of mother in passage of time , due to being inconsiderate to her own health , tolerating extensive forces including stress , anxiety , and depression that are due to unpleasant experiences during the growth of her children and because of the existing occupations . in this case i used to postpone treatments and it continued and my disease became chronic and acute . then i noticed that i neglected myself and my children were upset about the thing that why i was so concerned about my work and inconsiderate about my health . now i have intensive physical problems and my children blame me for having worked much . when i opened my eyes , i was in a hospital , had numb feet and hands . the data in this study shows that worries and mainly anxiety that are common in all the participants includes multiple stresses for family duties , work and existence of numerous roles , and also coordination of traditional and modern roles ; among which the maternal role and relevant duties are having priority and importance . on the other hand , accepting various roles increases expectations for different roles by the children , spouse , family , and the community . this obligated the mothers within this study to respond to the expectation and needs of family and working roles . certainly this affects the maternal role of the mothers either intentionally or unintentionally , causing negligence in doing their important duties or other roles , or causes a balance between their roles by the support of the spouse , family and the relatives , or to tolerate the stresses all by her own . the participants have experienced various approaches as stated in their interviews and have had proposal in these regards , which are to be pointed in the conclusion section . one of the major problems of working mothers in the study with their maternal role was their return to work after maternity leave . mental concerns and worries about finding the right person and place to care for a child , how to breast feed the infant , obligations in full - time cooperation in the organization , and inadequate supports of the organization are the concerns for most mothers participating in the study . most mothers agreed that they were scared from the beginning of their period of maternity leave . in this regard , the participant number ( 5 ) states : i was very worried from the beginning to what i should do about my child after my maternity leave , where should i put him , what i should do for breast feeding him . this theme reflected maternal duties in the absence of mother , including relatives of mother , supportive resources in the community such as kindergartens , private institutions , and service or using family members or neighbors due to necessity because of the particular circumstances . in other words , working mothers in the study had to assign their maternal roles to other when they were at work in order to meet the diverse needs of their child with the help of others . in this i asked one of the relatives of one of my neighbors to take care of my children and i used to pay her monthly wages . i used to take my younger child to her before going to work and send my elder child by a school car to school and take him back by the afternoon to the nursery for my husband to take him back home after his work . it indicates that the additional pressure exerted on the mother accepting various roles and being in forceful conditions providing her to tolerate the situations : i make the food ready with all my fatigue , wash the dishes , do the house work , and used to control the homework done by my children up to primary school . i used to cook the next day 's lunch and never leave anything over . it seeks to create an overlapped role . some mothers were inconsistent in their roles or due to negligence or failure in performing their duties ; they were affected by negative emotions such as disgust , hatred , guilt , and blame : it was 9 oclock that my mother in law called and said that my daughter had fever and vomiting and was crying . i talked to my child and calmed her down and said i would come after she had a little rest . i was worried and told myself i should have been with my child by then . she was crying there and suffering and hurting her grandfather , and i was here . participant number ( 6 ) says : i am not satisfied with my condition at all . i think if i was a housewife , taking care of children , it would be better , from what it is now . they would not have to , the problems they are facing now . participant number ( 2 ) states about her satisfaction , as : i enjoy when i solve a problem from a student and when he / she smiles . i take this energy with me to my house and look after my children . it indicates reduction of mental and physical potentials of mother in passage of time , due to being inconsiderate to her own health , tolerating extensive forces including stress , anxiety , and depression that are due to unpleasant experiences during the growth of her children and because of the existing occupations . in this case i used to postpone treatments and it continued and my disease became chronic and acute . then i noticed that i neglected myself and my children were upset about the thing that why i was so concerned about my work and inconsiderate about my health . now i have intensive physical problems and my children blame me for having worked much . when i opened my eyes , i was in a hospital , had numb feet and hands . the data in this study shows that worries and mainly anxiety that are common in all the participants includes multiple stresses for family duties , work and existence of numerous roles , and also coordination of traditional and modern roles ; among which the maternal role and relevant duties are having priority and importance . on the other hand , accepting various roles increases expectations for different roles by the children , spouse , family , and the community . this obligated the mothers within this study to respond to the expectation and needs of family and working roles . certainly this affects the maternal role of the mothers either intentionally or unintentionally , causing negligence in doing their important duties or other roles , or causes a balance between their roles by the support of the spouse , family and the relatives , or to tolerate the stresses all by her own . the participants have experienced various approaches as stated in their interviews and have had proposal in these regards , which are to be pointed in the conclusion section . considering the data origins and nature of the classes show that the working mothers have to bear extensive hardship in their experience from the starting point of their maternal role due to obligation in executing their work and providing the expected needs in social roles by the community without considering the special conditions of that period . results of the study by azgoli et al . , showed that 67.6% out of the total working mothers had moderate to high fatigue during pregnancies ; 90% used to stand on their feet for more than 3 h a day ; 58.2% expressed that despite fatigue , they continued their work ; 93% confronted tiring and fatigue resources such as long distance to work , having shifts , long working hours per week and standing for long periods ; 10% had early labor ; and 18.5% had children with less weight . these finding are compatible with the finding of this research , expressing hard conditions for the participating women during their pregnancy period . the thought of going back to work after the end of maternal leave and continuing occupation role as in the past is a worrying concern that mothers have about taking care of their children during their absence from home . the result of the investigation by ukwuani and suchindran in nigeria , on the work by women and its effects on the feeding situations of the infants , showed that existence of very limited facilities and lack of official policies in that country regarding child care and working women , especially by the time of returning to work , have confronted the women with their child care , depriving the children from breast feeding and infections . with regard to the present study , inadequate supports by the organizations from the working women during pregnancy have caused the mothers to be worried about their child care after the maternal leave . , showed existence of state and the working place supports , including the possibility for women to decide on their working hours , existence of a place to look after their infants and the possibility for the women to visit their children during the working hours and the supporting regulations have caused mothers to have more time in taking care of their children . the working women in the study noticed that they should provide the various needs of their children under the supports by other to continue their role in working position ; hence they had to temporarily assign replacements for themselves such as grandmothers , aunts , or kindergarten nurses or private companies instructors . the study by gibson and mace , shows that mothers relatives are more helpful to the girls than the fathers relatives in child care , in ethiopia . , from their research aiming in analysis of social supports from the working mothers in europe show that norwegian mother had the highest supports that needed less help by grand parents , in comparison to the working women in italy and spain.the findings are in conformity with the findings in this research . despite getting available aids from supporting sources and assigning the maternal role during the absence of the mother , it is expected that mothers immediately start their roles such as maternal , spousal and housing roles and provide the needs of the family members after their working and social roles . hence , what the mothers under study experienced was multiple roles and confrontation with different expected roles by others . the research result of rastgar khaled , shows that working women face numerous roles that are considered as the creating source of stress in women . according to the investigation by maghsodi and bostan , 46% of the working women stated that keeping up the child care affairs is their duty and 90% had difficulties in providing cleanliness and tidiness at home . in the present research , all the mothers stated that they do their traditional roles in the house and being occupied has not caused those roles to fade , but they regarded the maternal role to have great importance , among others . moreover , the younger the children are the more stress is occurred in mothers due to their variety of roles . it has been stated by the participants that by passage of time for the maternal and occupation roles , more efforts and required for the conformity and compatibility with different life conditions in this case , parker and arthur , believe that one important thing about working and the family is balancing the relevant role also becoming compatible with different conditions . in the final stages of maternal role , the mothers having the feelings of neglecting maternal duties due to variety and contradicting of the roles are not satisfied with their maternal role , although they stated that they did best for their occupation . also the mothers , who had supports , were satisfied with their social role as well as their maternal role . according to the qualitative study by mc carthy , all the women had intensive emotions regarding their maternal roles , while the professional role was also important for them and they regarded this role to be satisfactory . moreover , the finding showed that working women were capable of providing the working / family balance in their mid - professional age rather than the beginning of their work and they had better feelings in that age . not considering the physical and mental requirements such as related requirements to health due to shortage of time or giving priority to the children 's needs and also unpleasant stresses and experiences from maternal and working roles simultaneously and the conflicts due to them cause reduction of energy and emergence of mental and physical fatigue for the mothers and hence they are confronted with a phenomenon called erosion . most of the participants in this study had no proper feelings for being healthy and involved with light to moderate physical illnesses . but , the distance , number of children and family supports were also among the effective factors on the physical health of the anthers that should not be neglected . , working women are significantly in a higher level that the housewives , regarding their social health , but no significant difference was observed between them with regards to their mental health . the final result regarding the expressing of the process of maternal role in working women indicates that maternal roles is a concept that is rather exclusive , multilateral , gradual and affected by different factors . as stated , the different roles that women undertake may be in contradiction with each other . role conflict is a social conflict and occurs due to stresses on the person for her to have different roles simultaneously . therefore if the working mothers want to do their maternal roles , without considering their occupational work or being a housewife , they should tolerate difficulties and respond to various expectations and role conflicts . various studies have been done both in iran and in foreign countries about the contractions between working and family responsibilities and about role conflicts among which the studies by rastgar khaled , maghsodi and bostan , karatepe and kilich , kinnunena et al . , shockley and allen could be pointed . according to maghsodi and bostan , 90% of women had difficulties in cleaning and tiding responsibilities , 66% of them had to prepare food the day before and 39% of them were alone in their child care and had no aids from their spouses . 46% of women stated that child care and taking care of children were their responsibility . the note to point is that the working participants who had more working hours during a week had to bear higher pressure as compared to the participants who were the members of the scientific board , in doing their assigned roles . according to tavassoli , 60% of women emphasized on the importance of the traditional role in house keeping and considered it to be prior to working outside the house . in this study , there is a contradiction between working as an economic activity in the community and maintaining the family and child care . stating from carter and carter , arian believes that the most important conflict that women face in families , with two jobs is the role conflict and hence balancing the conflicts regarding working and house affairs is a difficult task . this task should be done by the aid and support of the family members and the working place authorities . in this regard , the result of the study by emanuel that was done in australia shows that 81% of the women believed that social supports are related to developing the maternal role . accordingly , the social support is the most important factor in developing maternal role , affecting successful compatibilities with maternal role , having self confidence and satisfaction in the ability for growing and taking care of the children . according to this study , the cooperation and supports from the working place with mothers were in executing official statements for executing maternal leaves and using hourly leaves for breast feeding and better collaboration was done with scientific board members in affairs requiring cooperation and supports of senior authorities about the participant . according to the performed studies , one way to reduce conflicts and pressure for the role of working mothers is full supports and cooperation with the working mother . this cooperation causes the mothers to have more time for the affairs regarding child care and performing their maternal role . in the present study , supports and cooperation with mothers have had direct relations with reducing role conflicts that they are facing . the final result regarding the expressing of the process of maternal role in working women indicates that maternal roles is a concept that is rather exclusive , multilateral , gradual and affected by different factors . as stated , the different roles that women undertake may be in contradiction with each other . role conflict is a social conflict and occurs due to stresses on the person for her to have different roles simultaneously . therefore if the working mothers want to do their maternal roles , without considering their occupational work or being a housewife , they should tolerate difficulties and respond to various expectations and role conflicts . various studies have been done both in iran and in foreign countries about the contractions between working and family responsibilities and about role conflicts among which the studies by rastgar khaled , maghsodi and bostan , karatepe and kilich , kinnunena et al . , shockley and allen could be pointed . according to maghsodi and bostan , 90% of women had difficulties in cleaning and tiding responsibilities , 66% of them had to prepare food the day before and 39% of them were alone in their child care and had no aids from their spouses . 46% of women stated that child care and taking care of children were their responsibility . the note to point is that the working participants who had more working hours during a week had to bear higher pressure as compared to the participants who were the members of the scientific board , in doing their assigned roles . according to tavassoli , 60% of women emphasized on the importance of the traditional role in house keeping and considered it to be prior to working outside the house . in this study , there is a contradiction between working as an economic activity in the community and maintaining the family and child care . stating from carter and carter , arian believes that the most important conflict that women face in families , with two jobs is the role conflict and hence balancing the conflicts regarding working and house affairs is a difficult task . this task should be done by the aid and support of the family members and the working place authorities . in this regard , the result of the study by emanuel that was done in australia shows that 81% of the women believed that social supports are related to developing the maternal role . accordingly , the social support is the most important factor in developing maternal role , affecting successful compatibilities with maternal role , having self confidence and satisfaction in the ability for growing and taking care of the children . according to this study , the cooperation and supports from the working place with mothers were in executing official statements for executing maternal leaves and using hourly leaves for breast feeding and better collaboration was done with scientific board members in affairs requiring cooperation and supports of senior authorities about the participant . according to the performed studies , one way to reduce conflicts and pressure for the role of working mothers is full supports and cooperation with the working mother . this cooperation causes the mothers to have more time for the affairs regarding child care and performing their maternal role . in the present study , supports and cooperation with mothers have had direct relations with reducing role conflicts that they are facing . emergence of social and economic evolution has increased the trend of women 's presence in the present community , from one side , and on the other side it has caused their confrontation with various family and social roles . therefore , working women have to put other roles into the margin by considering the essentials of one role , or they just have to fulfill the expectations . since maternal role is one of the most important roles for women , this could intentionally or unintentionally affect the execution of the role . but the inevitable fact is that wherever in the world are the working women , they have similar concerns and their most important challenge is their maternal role and the quality in their child care . but as the finding of this research showed , the thing that is contributed to the working mother from the social and family combining roles is the role conflict due to simultaneity of their various roles . this will affect the process of their maternal role and it has caused the execution of this important role to face difficulties , to have a completely different process for the non - working women . what is certain is that the level and amount of responsibilities and the expectation due to family roles are more than the duties in our community of the working roles , such that women should spend more energy and power on them . it is specially observed about maternal duties of working women , since iranian women are mainly emotional and dependent on their children and the mental involvement for proper execution of the duties are always accompanied by their feelings . hence , according to the finding of the present research and other relevant investigations , it seems that the most important practical approach for reducing the conflict in the maternal roles of the working women and better execution of these roles is having family and social supports . creation and establish family support could be verified via modification of working division in the family and avoiding traditional working division among family members , and emphasis on the role participation in family and also dividing the home duties between wife , husband and children . by doing this , the working mothers could have more time in spending for their children . regarding the social supports , the following activities could be the executing approaches and state supports for the working mothers that could satisfy and empower them in performing their maternal roles , in which case the children 's health could be guaranteed and hence the concerns of mothers regarding the role conflicts could be reduced : modifying the rules for maternal leave , changing the weekly hours of work and providing part - time and flexible jobs ( for all the women and not only the ones working for the government ) , constructing kindergartens in working areas , providing a suitable place for pumping the milk and storing it for the infants during breast feeding period and paying costs for taking care of children in institutions and kindergartens by government .

introduction : regarding the social and economic changes and developments , the increasing presence of working women in the present society and their important role in the family , paying more attentions to the importance of maternal role among working women who face multiple roles seems to be necessary . hence , the process of maternal role among working women has been investigated in this study.methods:the grounded theory approach is used in this qualitative research . in - depth and unstructured interviews were the main way in collecting the data . initially , the targeted sampling was started and continued gradually to the data saturation , in the form of theoretical sampling based on the obtained classes . the data was analyzed using strauss and corbin analysis . accuracy and validity by four criteria in this study included : credibility , dependability , confirmable potential , transferability or fittingness.results:data analysis led to the identification of the core variable of role conflict . the main classes of occupied mothers experiences included : different pregnancy experience , returning concerns , supportive umbrella , role assignation , role overlap , role strain , gradual acceptance , satisfaction and erosion.conclusions:the acceptance of numerous roles such as maternal and marital roles by working women creates various role expectations of them from their children , spouses , family and the society which in turn forces them to meet both family and job requirements and expectations ; this causes role conflict in working mothers and endure a lot of pressure and stress , that can influences of mental and physical health of the mothers .

INTRODUCTION METHODS RESULTS Returning concerns Role assignation Role overlap Role strain Satisfaction Erosion DISCUSSION Discussion about the core variable CONCLUSIONS

promoting the level of education , entering jobs and employment and increasing the social participation are among the social changes resulting from the transition from old to new society that a significant portion of the iranian women have experienced in recent years and could achieve their own interests and aspirations along their other traditional roles and attain social positions . two important and main roles , that is , spousal and maternal have been defined for women regarding the mental and physical characteristics of a woman , she will have to bear a special responsibility for them changes are formed in the traditional gender division of labor that had previously been defined for women to add a new role on their previous roles . in other words , occupation as an added role could involve obligations and responsibilities for working women , because they have an obligation to their housework duties and tasks in the field of employment outside the home as well . since becoming a mother and the maternal role are complex processes affected by personal and social factors such as supporting systems , economic conditions , and cultural situations ; it seems that analyzing the experiences and elaborations of working mothers is a step towards promoting proper life of women , children , and family as the most important pillar in the society as a whole , to understand the needs , specific problems , and even the probable strong points . this research seeks to answer this important question as , what is the process of maternal role among women ? the aim of this study was to clarify the process of maternal role among working women . in cases where the researcher has to consider the process of formation or exposure to certain social phenomena in question , the basic theory and method of grounded theory as a qualitative research method based on symbolic interaction are used . since the maternal role is a dynamic process of interaction formed by the relation with others in particular fields , and the working women make themselves compatible with the changes in life , the grounded theory methodology was chosen for this study . participants in this study were among the women working in one of the islamic azad university branches , including faculty and staff members and all the participants who had an experience of being a mother , and had at least one child . initially , the targeted sampling was started and gradually continued by stabilization of conceptual classes during the data analysis . participants were selected based on theoretical sampling according to the codes and classes continued until data saturation . after the saturation level of the data was achieved , interviews were conducted with nine participants . participants based on text written consent , freely participated in the study . data collection and data analysis in this study were done simultaneously and the data were analyzed according to strauss and corbin approach and divided into three stages including open coding , axial coding , and selective coding ; which led to identifying the variable core as the main class relating with other classes to describe them . the four criteria ; including credibility , dependability , conformability , and transferability or fittingness were considered for the validity and credibility of the study . for the credibility and validation of the findings , the method regarding the participants check was used that indicated whether the participants recognize the data and the results are in conformity with their experiences in terms of accuracy , completeness , and interpretation ; or not . scaling ideas were taken and the amount realized from the data was compared with the participants . the question that defines this criterion is about the dependency of the results to each otherto fulfill this criterion , the study 's findings were given to three working mother who did not participate in the study and they verified the relevance of them with their own experiences . conformability in qualitative research and is equivalent to reliability in quantitative research and represents stability and reliability of the data over time and under identical conditions . to develop the ability to approve or object to all of the survey data , researchers tried to fully explain all the stages of the research to ensure the integrity of the open and axial coding , the data and findings were given to other researchers familiar with qualitative studies to verify their accuracies . transferability or fitness means fitness and mobility of the outcomes to be used in similar conditions . discussing and comparing our findings with other studies is a method to evaluate the appropriateness of the data that has been used in this study . data obtained from analysis of the interviews included , 414 codes from the open coding phase , 42 category of the axial coding stage and 9 main themes of working mothers experiences which consisted of different pregnancy experience , returning concerns , supportive umbrella , role assignation , role overlap , role strain , gradual acceptance , satisfaction , and erosion that led to the identification of the core variable of role conflict that resulted from selective coding considered as the basic social psychological process , which is faced by working mothers in the study and developed over time , that is shown in figure 1 . the process of maternal role the following are some of the components of maternal role in the process described in this study : one of the major problems of working mothers in the study with their maternal role was their return to work after maternity leave . mental concerns and worries about finding the right person and place to care for a child , how to breast feed the infant , obligations in full - time cooperation in the organization , and inadequate supports of the organization are the concerns for most mothers participating in the study . this theme reflected maternal duties in the absence of mother , including relatives of mother , supportive resources in the community such as kindergartens , private institutions , and service or using family members or neighbors due to necessity because of the particular circumstances . in other words , working mothers in the study had to assign their maternal roles to other when they were at work in order to meet the diverse needs of their child with the help of others . in this i asked one of the relatives of one of my neighbors to take care of my children and i used to pay her monthly wages . it indicates reduction of mental and physical potentials of mother in passage of time , due to being inconsiderate to her own health , tolerating extensive forces including stress , anxiety , and depression that are due to unpleasant experiences during the growth of her children and because of the existing occupations . the data in this study shows that worries and mainly anxiety that are common in all the participants includes multiple stresses for family duties , work and existence of numerous roles , and also coordination of traditional and modern roles ; among which the maternal role and relevant duties are having priority and importance . on the other hand , accepting various roles increases expectations for different roles by the children , spouse , family , and the community . this obligated the mothers within this study to respond to the expectation and needs of family and working roles . certainly this affects the maternal role of the mothers either intentionally or unintentionally , causing negligence in doing their important duties or other roles , or causes a balance between their roles by the support of the spouse , family and the relatives , or to tolerate the stresses all by her own . the participants have experienced various approaches as stated in their interviews and have had proposal in these regards , which are to be pointed in the conclusion section . one of the major problems of working mothers in the study with their maternal role was their return to work after maternity leave . in this regard , the participant number ( 5 ) states : i was very worried from the beginning to what i should do about my child after my maternity leave , where should i put him , what i should do for breast feeding him . this theme reflected maternal duties in the absence of mother , including relatives of mother , supportive resources in the community such as kindergartens , private institutions , and service or using family members or neighbors due to necessity because of the particular circumstances . in other words , working mothers in the study had to assign their maternal roles to other when they were at work in order to meet the diverse needs of their child with the help of others . in this i asked one of the relatives of one of my neighbors to take care of my children and i used to pay her monthly wages . it indicates reduction of mental and physical potentials of mother in passage of time , due to being inconsiderate to her own health , tolerating extensive forces including stress , anxiety , and depression that are due to unpleasant experiences during the growth of her children and because of the existing occupations . the data in this study shows that worries and mainly anxiety that are common in all the participants includes multiple stresses for family duties , work and existence of numerous roles , and also coordination of traditional and modern roles ; among which the maternal role and relevant duties are having priority and importance . on the other hand , accepting various roles increases expectations for different roles by the children , spouse , family , and the community . this obligated the mothers within this study to respond to the expectation and needs of family and working roles . certainly this affects the maternal role of the mothers either intentionally or unintentionally , causing negligence in doing their important duties or other roles , or causes a balance between their roles by the support of the spouse , family and the relatives , or to tolerate the stresses all by her own . the participants have experienced various approaches as stated in their interviews and have had proposal in these regards , which are to be pointed in the conclusion section . considering the data origins and nature of the classes show that the working mothers have to bear extensive hardship in their experience from the starting point of their maternal role due to obligation in executing their work and providing the expected needs in social roles by the community without considering the special conditions of that period . , showed that 67.6% out of the total working mothers had moderate to high fatigue during pregnancies ; 90% used to stand on their feet for more than 3 h a day ; 58.2% expressed that despite fatigue , they continued their work ; 93% confronted tiring and fatigue resources such as long distance to work , having shifts , long working hours per week and standing for long periods ; 10% had early labor ; and 18.5% had children with less weight . the thought of going back to work after the end of maternal leave and continuing occupation role as in the past is a worrying concern that mothers have about taking care of their children during their absence from home . the result of the investigation by ukwuani and suchindran in nigeria , on the work by women and its effects on the feeding situations of the infants , showed that existence of very limited facilities and lack of official policies in that country regarding child care and working women , especially by the time of returning to work , have confronted the women with their child care , depriving the children from breast feeding and infections . with regard to the present study , inadequate supports by the organizations from the working women during pregnancy have caused the mothers to be worried about their child care after the maternal leave . the working women in the study noticed that they should provide the various needs of their children under the supports by other to continue their role in working position ; hence they had to temporarily assign replacements for themselves such as grandmothers , aunts , or kindergarten nurses or private companies instructors . the study by gibson and mace , shows that mothers relatives are more helpful to the girls than the fathers relatives in child care , in ethiopia . , from their research aiming in analysis of social supports from the working mothers in europe show that norwegian mother had the highest supports that needed less help by grand parents , in comparison to the working women in italy and spain.the findings are in conformity with the findings in this research . despite getting available aids from supporting sources and assigning the maternal role during the absence of the mother , it is expected that mothers immediately start their roles such as maternal , spousal and housing roles and provide the needs of the family members after their working and social roles . hence , what the mothers under study experienced was multiple roles and confrontation with different expected roles by others . the research result of rastgar khaled , shows that working women face numerous roles that are considered as the creating source of stress in women . according to the investigation by maghsodi and bostan , 46% of the working women stated that keeping up the child care affairs is their duty and 90% had difficulties in providing cleanliness and tidiness at home . in the present research , all the mothers stated that they do their traditional roles in the house and being occupied has not caused those roles to fade , but they regarded the maternal role to have great importance , among others . it has been stated by the participants that by passage of time for the maternal and occupation roles , more efforts and required for the conformity and compatibility with different life conditions in this case , parker and arthur , believe that one important thing about working and the family is balancing the relevant role also becoming compatible with different conditions . in the final stages of maternal role , the mothers having the feelings of neglecting maternal duties due to variety and contradicting of the roles are not satisfied with their maternal role , although they stated that they did best for their occupation . also the mothers , who had supports , were satisfied with their social role as well as their maternal role . according to the qualitative study by mc carthy , all the women had intensive emotions regarding their maternal roles , while the professional role was also important for them and they regarded this role to be satisfactory . moreover , the finding showed that working women were capable of providing the working / family balance in their mid - professional age rather than the beginning of their work and they had better feelings in that age . not considering the physical and mental requirements such as related requirements to health due to shortage of time or giving priority to the children 's needs and also unpleasant stresses and experiences from maternal and working roles simultaneously and the conflicts due to them cause reduction of energy and emergence of mental and physical fatigue for the mothers and hence they are confronted with a phenomenon called erosion . most of the participants in this study had no proper feelings for being healthy and involved with light to moderate physical illnesses . but , the distance , number of children and family supports were also among the effective factors on the physical health of the anthers that should not be neglected . the final result regarding the expressing of the process of maternal role in working women indicates that maternal roles is a concept that is rather exclusive , multilateral , gradual and affected by different factors . role conflict is a social conflict and occurs due to stresses on the person for her to have different roles simultaneously . the note to point is that the working participants who had more working hours during a week had to bear higher pressure as compared to the participants who were the members of the scientific board , in doing their assigned roles . according to tavassoli , 60% of women emphasized on the importance of the traditional role in house keeping and considered it to be prior to working outside the house . in this study , there is a contradiction between working as an economic activity in the community and maintaining the family and child care . this task should be done by the aid and support of the family members and the working place authorities . in this regard , the result of the study by emanuel that was done in australia shows that 81% of the women believed that social supports are related to developing the maternal role . accordingly , the social support is the most important factor in developing maternal role , affecting successful compatibilities with maternal role , having self confidence and satisfaction in the ability for growing and taking care of the children . according to this study , the cooperation and supports from the working place with mothers were in executing official statements for executing maternal leaves and using hourly leaves for breast feeding and better collaboration was done with scientific board members in affairs requiring cooperation and supports of senior authorities about the participant . according to the performed studies , one way to reduce conflicts and pressure for the role of working mothers is full supports and cooperation with the working mother . this cooperation causes the mothers to have more time for the affairs regarding child care and performing their maternal role . in the present study , supports and cooperation with mothers have had direct relations with reducing role conflicts that they are facing . the final result regarding the expressing of the process of maternal role in working women indicates that maternal roles is a concept that is rather exclusive , multilateral , gradual and affected by different factors . according to maghsodi and bostan , 90% of women had difficulties in cleaning and tiding responsibilities , 66% of them had to prepare food the day before and 39% of them were alone in their child care and had no aids from their spouses . the note to point is that the working participants who had more working hours during a week had to bear higher pressure as compared to the participants who were the members of the scientific board , in doing their assigned roles . according to tavassoli , 60% of women emphasized on the importance of the traditional role in house keeping and considered it to be prior to working outside the house . in this study , there is a contradiction between working as an economic activity in the community and maintaining the family and child care . this task should be done by the aid and support of the family members and the working place authorities . in this regard , the result of the study by emanuel that was done in australia shows that 81% of the women believed that social supports are related to developing the maternal role . accordingly , the social support is the most important factor in developing maternal role , affecting successful compatibilities with maternal role , having self confidence and satisfaction in the ability for growing and taking care of the children . according to this study , the cooperation and supports from the working place with mothers were in executing official statements for executing maternal leaves and using hourly leaves for breast feeding and better collaboration was done with scientific board members in affairs requiring cooperation and supports of senior authorities about the participant . according to the performed studies , one way to reduce conflicts and pressure for the role of working mothers is full supports and cooperation with the working mother . this cooperation causes the mothers to have more time for the affairs regarding child care and performing their maternal role . in the present study , supports and cooperation with mothers have had direct relations with reducing role conflicts that they are facing . emergence of social and economic evolution has increased the trend of women 's presence in the present community , from one side , and on the other side it has caused their confrontation with various family and social roles . since maternal role is one of the most important roles for women , this could intentionally or unintentionally affect the execution of the role . but the inevitable fact is that wherever in the world are the working women , they have similar concerns and their most important challenge is their maternal role and the quality in their child care . but as the finding of this research showed , the thing that is contributed to the working mother from the social and family combining roles is the role conflict due to simultaneity of their various roles . this will affect the process of their maternal role and it has caused the execution of this important role to face difficulties , to have a completely different process for the non - working women . what is certain is that the level and amount of responsibilities and the expectation due to family roles are more than the duties in our community of the working roles , such that women should spend more energy and power on them . it is specially observed about maternal duties of working women , since iranian women are mainly emotional and dependent on their children and the mental involvement for proper execution of the duties are always accompanied by their feelings . hence , according to the finding of the present research and other relevant investigations , it seems that the most important practical approach for reducing the conflict in the maternal roles of the working women and better execution of these roles is having family and social supports . creation and establish family support could be verified via modification of working division in the family and avoiding traditional working division among family members , and emphasis on the role participation in family and also dividing the home duties between wife , husband and children . by doing this , the working mothers could have more time in spending for their children . regarding the social supports , the following activities could be the executing approaches and state supports for the working mothers that could satisfy and empower them in performing their maternal roles , in which case the children 's health could be guaranteed and hence the concerns of mothers regarding the role conflicts could be reduced : modifying the rules for maternal leave , changing the weekly hours of work and providing part - time and flexible jobs ( for all the women and not only the ones working for the government ) , constructing kindergartens in working areas , providing a suitable place for pumping the milk and storing it for the infants during breast feeding period and paying costs for taking care of children in institutions and kindergartens by government .

specialized neuronal circuits integrate metabolic signals related to energy stores and needs and consequently adjust energy intake , utilization and expenditure . the melanocortin system is one of the most well described circuits which control body weight and food intake . the two main antagonistic components of this system are the anorexigenic pomc and orexigenic npy neurons located in the arcuate nucleus of the hypothalamus . it has been recently shown that density and input type on pomc and npy neurons vary in adult mice , depending on the metabolic state and the associated fluctuation of circulating hormones [ 46 ] . such synaptic plasticity modifies the activity of these neurons and thereby the responsiveness of the melanocortin system . it has been proposed that the synaptic plasticity of this system in response to acute changes in fuel availability was essential to the accurate control of food intake and the maintenance of body weight in adulthood [ 79 ] . moreover , altered synaptic plasticity of pomc and npy neurons was found in diet - induced obese rodents suggesting that inability to dynamically rewire feeding circuits could contribute to obesity . polysialic acid ( psa ) is a cell - surface glycan that modulates cell - to - cell interactions . polysialylation of cell adhesion proteins is involved in various synaptic plasticity - dependent processes in the central nervous system including learning , pain modulation and the control of neuroendocrine functions . we previously reported that polysialylation was also required for the adaptive synaptic plasticity of feeding circuits during acute positive energy balance . precisely , high - fat diet ( hfd ) given for three days caused a psa - dependent increase in the excitatory inputs connecting pomc neurons in mice . the polysialyltransferase pst-1 , encoded by the st8sia4 gene was identified as the enzyme involved in the psa - dependent control of energy intake . however the biological mediators and the intracellular cascade of events leading to the dietary fat - induced psa - dependent synaptic plasticity in the hypothalamus have not been characterized yet . it is now clearly established that modifications in gene expression patterns are required to elicit structural and functional synaptic plasticity in response to experience and environmental cues [ 1820 ] . the molecular mechanisms supporting these changes in gene expression were explored in several synaptic plasticity - dependent processes such as learning , memory formation , addiction and stress [ 2123 ] . in these models , alterations in expression , localization or activity of transcription regulatory factors and in chromatin structure are instrumental in the signaling pathway leading to synapse remodeling . in particular , changes in the acetylation of histone tails and in the activity of histone acetyltransferases or histone deacetylases are associated with changes in plasticity - related gene expression in the brain areas supporting these synaptic plasticity - dependent processes , i.e. the hippocampus , the prefrontal cortex and the nucleus accumbens [ 2528 ] . here we explored the role of hypothalamic polysialylation in the long - term maintenance of body weight . we also deciphered the molecular sequence underlying the activation of polysialylation in the hypothalamus upon overfeeding . hypothalamic psa removal and st8sia4 silencing increased body weight gain on hfd , indicating that st8sia4 is essential to maintain energy homeostasis . we showed that dietary fat rapidly incited post - translational histone modifications on the st8sia4 gene in the mediobasal hypothalamus ( mbh ) . the histone acetyltransferase mof was required for the hfd - induced acetylation of histone h4 on lysine 16 and the subsequent activation of st8sia4 gene transcription . moreover , mof silencing in the mbh of adult mice fed a hfd reduced hypothalamic psa levels , caused overfeeding and exacerbated diet - induced obesity . experiments were carried out on 8-week - old male c57bl/6jola mice ( harlan laboratories ) . animal care and experimental procedures were performed with approval from the animal care and use committee of the university of burgundy . mice were housed in individual cages and maintained on a standard light / dark cycle . they were fed either a standard diet ( a04 ; safe laboratories ) or a high fat diet ( hfd ) ( safe laboratories as previously described ) . mice were anesthetized with isoflurane ( abbott ) and placed in a stereotaxic apparatus ( david kopf instruments ) in flat skull position . after dermal disinfection with betadine solution , the skin and cranial muscles were incised and the skull was exposed . small holes were drilled and a double cannula with a cap ( c235g-0.8/sp 4.6 mm ; plastic one ) was inserted to target above the mediobasal hypothalamus ( mbh ) using the following coordinates : 1.4 mm posterior to the bregma , 0.4 mm lateral to the sagittal suture , and 4.6 mm below the skull surface ( supplementary figure 1a ) . after cannulation , animals were kept under controlled temperature and rehydrated with intraperitoneal injections of physiological fluid . mice were then housed individually and were allowed 1 week for recovery before experiment . for injection mice were injected with either vehicle or endoneuraminidase n ( endon , 0.28 units / side , 400 nl ; eurobio ) at a rate of 100 nl / min , weekly during one month , with a 5.6 mm injector ( c235i / sp 5.6 mm ; plastic one ) inserted into the cannula targetting the mbh . mice were anesthetized with isoflurane ( abbott ) and placed in a stereotactic apparatus ( david kopf instruments ) . mice received a bilateral injection of lentiviral transduction particles ( 400 nl per side , 23 10 tu / ml ) , using the following stereotactic coordinates : 1.4 mm posterior to the bregma , 0.4 mm lateral to the sagittal suture , and 5.6 mm below the skull surface ( supplementary figure 1b ) . the lentiviral particles contained either a non - target control shrna ( non - mammalian control ; sigma aldrich ) , or a shrna targeting mof ( trcn0000039323 ; sigma aldrich ) or st8sia4 ( trcn0000093936 ) . the viral suspension was delivered at a rate of 100 nl / min through a 34 g blunt needle mounted on a 10 l syringe ( nanofil device from wpi ) controlled by a micropump ( ump2 from wpi ) . mice were also injected with analgesic ( buprenorphine ; 0.1 mg / kg i.p . ; buprecare , animalcare ) and ibuprofen ( 20 mg / kg ; advil ) was added to the drinking water for 3 days . nuclear magnetic resonance was used to determine the body composition of the mice ( qnmr system echomri-500 t , echo medical systems ) . explants were immediately cross - linked in 1.5% formaldehyde for 10 min at room temperature and quenched by adding glycine to a final concentration of 0.125 m. explants were then washed 3 times in ice - cold pbs . tissues were first homogenized in a cell lysis buffer containing 50 mm hepes ph 7.5 , 140 mm nacl , 1 mm edta , 10% glycerol , 0.5% np-40 and 0.25% triton x-100 , 1 complete protease inhibitors cocktail ( roche ) . the pellets were finally resuspended in a nuclear lysis buffer ( tris hcl 10 mm , nacl 140 mm , edta 1 mm , egta 0.5 mm , 0.1% sds , 0.1% na deoxycholate ) , 1 complete protease inhibitors cocktail ( roche ) and sonicated for 20 min in a bioruptor ( diagenode ; settings : high ; 30 s on/30 s off ) . this chromatin - shearing protocol produces fragments ranging from 150 to 400 bp in length . for chip , antibodies ( listed in table 1 ) were conjugated to magnetic dynabeads ( invitrogen ) . chromatin was incubated with beads - bound antibodies overnight at 4 c in a buffer containing tris hcl 10 mm , nacl 140 mm , edta 1 mm , egta 0.5 mm , 0.1% sds , 0.5% na deoxycholate , 1% np-40 and 1 complete protease inhibitors cocktail ( roche ) and then washed 6 times in ripa buffer and once in tris elution buffer ( tris 50 mm , edta 10 mm , sds 1% ) was added and samples were incubated at 65 c , 1400 rpm , in a thermomixer for 30 min to remove beads . chromatin in the supernatant was then reverse cross - linked by incubating 10 h at 65 c . quantitative pcr analysis was performed using the fast sybr green master mix ( applied biosystems ) . the following primers were used for pcr amplification : were as follow : 5-gccagtaacgcaaggcaaca-3 and 5-cgtagcagggaaacgataag-3 for st8sia4 and 5-tctggctcagctcagtctcc-3 and 5-gcttttcctgcccgaga-3 for st8sia2 . quantitative pcr analysis was performed using inventoried taqman gene expression assays and the taqman fast universal pcr master mix ( applied biosystems ) . taqman assay ids for the mrna analyzed were as follow : mm00458911_m1 for mof , mm01292231_m1 for st8sia4 , mm00456815_m1 for ncam1 , mm00456289_m1 for st8sia2 , mm00450174_m1 for gne , mm02342429_g1 for ppia . for each reaction , 1 l of a 1:10 dilution of the rt product was used in a final volume of 10 l and each sample was assayed in triplicate . pcr amplification was run on a stepone plus realtime pcr system ( applied biosystems ) and the stepone gene expression software was used for gene expression analysis . tissue lysis was performed in ripa lysis buffer using the tissuelyser system ( qiagen ) and 5 mm stainless steel beads ( qiagen ) . homogenates were then centrifuged 5 min at 5000 g and supernatants were collected for psa assay using an enzyme - linked immunosorbent assay kit ( psa ncam elisa kit ; eurobio ) . total protein concentration was determined using the protein assay kit ll ( bio - rad ) . data sets with two independent groups with similar variances were analyzed for statistical significance using an unpaired two - tailed student 's t test . data sets with more than two groups were analyzed using one - way analysis of variance ( anova ) followed by a dunnett 's post - hoc test . for statistical analyses in figure 6c and e , significant differences were indicated as follow : * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 . to assess the role of hypothalamic polysialylation in the long - term maintenance of body weight of mice on hfd , we generated psa - depleted mice . we used endoneuraminidase n ( endon ) , a bacterial enzyme that specifically removes psa residues from cell adhesion molecules . endon remains active at least eight days after a single injection in the brain parenchyma . we chronically depleted psa in this area by bilateral intraparenchymal injections of endon once a week during one month in mice fed with hfd ( figure 1a ) . we observed that the repeated injections of endon reduced by 8090% the psa level in the mbh ( figure 1b ) . the repeated injections of endon did not modify body weight in mice fed a standard diet for one month ( figure 1c e , and supplementary figure 2 ) . however , the persistent loss of hypothalamic psa caused by endon dramatically increased the weight gain of mice on hfd , in comparison to vehicle - treated mice ( figure 1c ) . as a result , body weight gain and fat mass measured after one month in mice on hfd were higher in endon - treated mice relative to vehicle - treated mice ( figure 1d and e ) . pst-1 is the major polysialyltransferase in adult hypothalamus . to test whether hypothalamic pst-1 is involved in the long - term maintenance of body weight , we examined the metabolic consequences of st8sia4 knock - down in the hypothalamus of mice fed a hfd . we used lentiviral vectors - mediated rna interference for specifically knocking down st8sia4 expression in the hypothalamus of adult mice . control mice received lentiviral vectors carrying a shrna sequence directed against a non - mammalian target . three weeks after surgery , st8sia4 mrna level was reduced by 21% in st8sia4 knock - down ( kd ) animals ( pst-1 kd ) mice compared to control mice ( figure 2a ) . the expression of st8sia2 , ncam , and gne was not altered ( figure 2a ) , indicating that the rnai - mediated knockdown of st8sia4 was specific and did not modify expression of other genes involved in the signaling pathway of psa . body weight gain and energy intake of control and pst-1 kd mice fed a standard diet for 2 months were similar ( supplementary figure 3 ) . to investigate whether a reduction of polysialylation in the hypothalamus increased the vulnerability to dio , pst-1 kd mice were fed a hfd for two months . in these mice , knock - down efficiency was determined by post - mortem analysis of psa level in mbh biopsies . only mice with more than 5% reduction of hypothalamic psa after screening , we detected a significant reduction of the psa level by 25.7% in the mbh of pst-1 kd mice , in comparison to control mice ( figure 2b ) . a retrospective analysis showed that pst-1 kd mice gained progressively more weight during the 2-month hfd than control mice ( figure 2c ) . body weight gain and adiposity of pst-1 kd mice on hfd were therefore significantly increased ( figure 2d and e ) . we next investigated the regulation of st8sia4 expression in the hypothalamus of hfd - fed mice . we determined if hfd exposure triggered chromatin remodeling and transcriptional activation of the st8sia4 gene in the mbh . chromatin modifications were analyzed on the promoter of st8sia4 by chip with chromatin extracts obtained from the mbh of mice fed a hfd for 24 h72 h ( figure 3 and supplementary figure 4 ) . after 24 h on hfd , an increase in h3 and h4 acetylation and a decrease in h4 methylation were observed ( figure 3a ; 2.25 , 1.86 and 1.83 fold for h3k14ac , h4k16ac and h4k20me respectively ) . the levels of h4k16ac , h4k20me and h3k14ac returned to basal values after 72 h on hfd , indicating that these post - translational modifications were transient ( supplementary figure 4b ) . hfd feeding for 48 h triggered a dissociation of histones h3 and h4 from the promoter of this gene ( figure 3b ; 1.66 fold and 1.74 fold decrease respectively ) . the decrease in histone interaction with st8sia4 promoter preceded an increase in pol2-s5p level -the activated form of rna polymerase ii- indicating an activation of st8sia4 transcription ( figure 3b ) . st8sia4 mrna levels were also significantly higher after 3 days on hfd ( figure 3c ) . additional analysis revealed no post - translational histone modifications , no histone dissociation and no change in rna polymerase ii phosphorylation on the promoter of the st8sia2 gene , encoding the pst-2 polysialyltransferase ( supplementary figure 4c and 4d ) . the chromatin remodeling events detected in the mbh after hfd introduction were thus specific to the st8sia4 polysialyltransferase gene . in order to identify the histone acetyltransferases ( hat ) that trigger the increase in histone acetylation and the subsequent activation of st8sia4 transcription in the mbh of hfd - fed mice , the recruitment of cbp , gcn5 , mof and tip60 was analyzed by chip . these hats are representative members of the 3 different families of histone lysine acetyltransferases and are known to acetylate either h3k14 or h4k16 or both [ 3234 ] . their interaction with st8sia4 was analyzed in mbh chromatin extracts from mice fed a hfd for 6 h or 10 h ( figure 4 ) . after 6 h on hfd , a 2.20 fold increase in mof was detected on st8sia4 promoter . this increase was persistent after 10 h of hfd exposure . at this time point , gcn5 was also recruited . the early recruitment of mof suggested that this hat plays a critical role in mediating the effects of hfd on the activation of st8sia4 transcription in the mbh . to determine the role of mof in hfd - induced hypothalamic polysialylation , we knocked down mof in the hypothalamus and tested whether st8sia4 transcription and psa levels could still be increased upon hfd exposure . lentiviral particles containing a mof - targeting shrna sequence were stereotactically injected in the mbh of adult mice . after 3 weeks of recovery , a 26.73% decrease in mof mrna level was detected in the hypothalamus of silenced mice ( mof kd mice ) ( figure 5a ) . since h4k16ac has been identified as the main substrate of mof in many cell types and organisms [ 32,3537 ] , we tested whether mof knock - down altered the hfd - induced increase in h4k16ac level on st8sia4 in the mbh ( figure 5b ) . after 24 h of hfd feeding , the level of h4k16ac on st8sia4 was reduced by 25.39% in mof kd mice in comparison to control mice . the level of h4k16ac in hfd - fed mof - kd mice was similar to the level in standard - diet fed mice ( figure 3a ) , indicating that mof knock - down abolished the hfd - induced acetylation of h4k16 . after 3 days on hfd , the level of pol2-s5p detected on st8sia4 was strongly reduced in mof - kd mice in comparison to the control group ( 38.33% reduction , figure 5c ) . again , pol2-s5p level in mof - kd mice was similar to the level in standard - diet fed mice ( figure 3b ) . we finally assessed polysialylation capacities in mof - kd mice by measuring both st8sia4 mrna level and psa level in the mbh after exposure to hfd for 3 days ( figure 5c and d ) . st8sia4 mrna level was reduced by 38.73% and psa level was reduced by 14.10% in mof - kd mice . altogether , these results indicated that mof is crucial for activating the polysialylation pathway in the mbh in response to hfd . we finally examined the consequences of mof knock - down on the psa - dependent short - term feeding response to hfd and the long - term regulation of body weight on hfd . the short - term feeding response to hfd was assessed over 8 days of hfd exposure . when fed a standard diet , control and mof kd mice displayed similar body weight and daily energy intake ( figure 6a and b ) . in both control and mof kd mice , hfd caused a transient increase in energy intake ( figure 6c ) . however the normalization of energy intake was slightly but significantly altered in mof kd mice . consequently , the cumulative energy intake of mof kd mice during the first week of hfd was higher in this group ( figure 6d ) . hfd feeding revealed an alteration of the homeostatic control of food intake in mof kd mice . to assess the role of mof in the long - term regulation of energy homeostasis , control and mof kd mice food intake and body weight were recorded daily during this period . only mice with more than 5% reduction of hypothalamic mof mrna level as a reference , body weight of control and mof kd mice fed a standard diet was measured over 6 weeks after surgery . no significant differences were observed in body weight gain between control mice and mof kd mice ( + 2.49 0.28 g vs 2.96 0.52 g ) . however , upon hfd , a dramatic increase in body weight was observed in mof kd mice ( figure 6e ) . the body weight gain of mof kd mice over the 8 weeks of hfd was 31.85% higher than the one of the control group ( figure 6f ) . this increase in body weight gain was due to an increase in fat mass in mof kd mice ( figure 6 g ) . the higher cumulative energy intake of mof kd mice in comparison to control mice over the 8-week period likely explained this phenotype ( figure 6h , mof kd mice : 786.83 14.06 kcal and control mice : 708.41 21.79 obesity is on the rise in both developed and developing countries , and it is now considered as a worldwide epidemic . it has been proposed that obesity could be a brain disease , and recently , several molecular brain targets of therapeutic interest have been identified . investigation of central regulatory pathways involved in the control of appetite and body weight could probably help with the fight against obesity . we previously identified polysialylation as an essential process in the nutritionally regulated synaptic plasticity of the melanocortin system . we now show that polysialylation is essential to the long - term regulation of body weight . furthermore , the experimental data presented here unveil an unexpected role for the histone acetyltransferase mof in the brain control of energy balance . developmental malformation of the melanocortin system , i.e. disruption of neural projection pathways within the hypothalamus , could contribute to the obese phenotype and related metabolic disorders . moreover , the wiring of arcuate pomc neurons of rats kept on standard diet predicts vulnerability to weight gain on hfd , suggesting that accurate connectivity in this neuronal system is crucial to maintain energy homeostasis . besides , an abnormal synaptic organization of feeding circuits that can be acutely reversed by hormonal or pharmacological treatments has been found in obese mice and rats . thus , it has been suggested that the altered synaptic plasticity in feeding circuits , in addition to steady neuroanatomical defects , might promote dio . nevertheless , whether synaptic inadequacy is cause or consequence of the metabolic imbalance is still not clear . here , we report that the lack of hypothalamic psa strongly accelerated the onset of dio in mice . given the role of psa in synaptic plasticity , our results suggest that the inflexibility of feeding circuits is a risk factor for obesity . this is in line with the well - documented adaptive function of the plasticity in feeding circuits in response to metabolic fluctuations . the removal of hypothalamic psa was sufficient to cause severe overweight in mice fed a hfd for 12 months , suggesting that the inability to properly rewire feeding circuits depending on the nutritional conditions , makes mice prone to obesity . since the human hypothalamus is able to undergo hormone - dependent structural plasticity , our findings could be relevant to the etiology of obesity in humans as well . indeed , large - scale studies on the genetic determinants of body mass index have revealed the strong influence of some genes involved in neuronal plasticity . interestingly , the polymorphism in negr1 , the gene coding the neuronal growth regulator 1 , a cell adhesion molecule involved in the brain remodeling , seems to be causal in high body weight . moreover , genetic defects reducing the levels of plasticity - related molecules such as the brain - derived neurotrophic factor ( bdnf ) or its receptor trkb cause severe early - onset obesity . therefore , it is conceivable that an altered synaptic plasticity could predispose humans to obesity . to gain insight into the molecular sequence underlying the synaptic plasticity - dependent control of energy balance , we explored the chromatin remodeling events underlying the activation of the st8sia4 gene expression upon high fat diet ( hfd ) . we found that the hfd induced a decrease in the level of monomethylated h4k20 and an increase in acetylation of histones h3k14 and h4k16 on the st8sia4 gene . in mammals , histones localized in silent heterochromatic regions are usually characterized by high levels of methylation at certain specific sites and low levels of acetylation . h4k20me1 marks are mostly found in regions of facultative heterochromatin and h4k20 demethylation has been associated with transcription activation in different models [ 5153 ] . both h3k14 and h4k16 acetylation impact chromatin structure and allow switching from a repressive state to a transcriptionally active state [ 5457 ] . in our study , the decrease in h4k20me1 and the increase in h3 and h4 acetylation upon hfd the level of rna polymerase ii phosphorylated on the serine 5 of the carboxy - terminal domain increased subsequently . moreover , the phosphorylation of the serine 5 of the rna polymerase ii is acquired during the transcription initiation phase . tracing back the signaling cascade leading to the activation of st8sia4 transcription , we identified two histone acetyltransferases , gcn5 and mof , recruited on st8sia4 within the few hours following the introduction of hfd . transcription activation relies on cross - regulation of histone post - translational modifications through the stepwise or concomitant recruitment of histone modifying enzymes . of note , h3k14 and h4k16 are major acetylation targets for gcn5 and mof , respectively . additionally , mof depletion in drosophila or human cells resulted in a genome - wide loss of h4k16ac . in our study however , phf8 is the only histone h4k20me1 demethylase identified up to now and its activity is associated with a positive regulation of gene expression . phf8 could thus contribute , in cooperation with gcn5 and mof to the activation of st8sia4 transcription in the hypothalamus upon high fat feeding . in our study , since mof recruitment was already detected after 6 h of hfd feeding , we focused on the function of this hat in the activation of st8sia4 expression upon hfd exposure . the depletion of mof in the mbh completely abolished the hfd - induced increase in h4k16 acetylation on st8sia4 and the subsequent recruitment of the rna polymerase ii on this gene . the early recruitment of mof on st8sia4 upon hfd exposure , the absence of activation of st8sia4 transcription and the decrease in psa level after mof knock - down strongly suggested a crucial role for mof in mediating the effect of hfd exposure on the establishment of psa - dependent synaptic plasticity in the hypothalamus . mof kd mice displayed an impairment of the short - term feeding response to hfd , which is actually a psa - dependent process . in addition , we show that mof kd mice displayed a higher propensity to develop dio . reducing polysialylation by st8sia4 silencing or psa removal from the hypothalamus accelerated the onset of a dio phenotype as well . afterwards , studies performed on mammalian cells and tumor biopsies indicated that mof is crucial to several cell functions such as cell proliferation and differentiation , dna damage repair , autophagy and tumorigenesis [ 6567 ] . in vivo , a purkinje cells - specific deletion of mof in the cerebellum produced neurological abnormalities including impaired motor coordination , ataxia and a backward - walking phenotype . the present work demonstrates that mediobasal hypothalamic mof is involved in the regulation of energy homeostasis through an activation of polysialylation . whereas some studies suggest that mof is globally involved in the activation of transcription , others indicate that this hat rather activates defined subset of genes and has a function in transcription regulation in specific cell types only . though we can not rule out that a larger mof - dependent transcriptomic program contributes to the accelerated onset of diet - induced obesity in mof - depleted mice , the subsequent deficit in hypothalamus polysialylation capacities of these mice might largely contribute to their increased vulnerability to dio . in few recent studies , mof has been detected in enhancer regions on mammalian genes . for instance , mof is recruited to foxp3-target genes by directly interacting with foxp3 and the transcription factor p53 is an acetylation target of mof . this suggests that mof could be recruited to the st8sia4 gene by interacting directly with a transcription factor activating st8sia4 transcription upon high fat feeding . otherwise , its recruitment could involve mediator complex which is known to convey regulatory information to the basal rna polymerase ii transcription machinery by interacting both with gene - specific transcription factors and with histone modifying enzymes . the mechanism by which mof is targeted to st8sia4 upon hfd feeding remains to be determined . in conclusion , the present work clearly indicated the causal role of altered psa signaling in accelerating the development of obesity . given the role of psa in the modulation of cell - to - cell interactions these findings strengthen the concept that individual capacity to synaptic plasticity is a key factor in the development of overweight with obesogenic foods .

overfeeding causes rapid synaptic remodeling in hypothalamus feeding circuits . polysialylation of cell surface molecules is a key step in this neuronal rewiring and allows normalization of food intake . here we examined the role of hypothalamic polysialylation in the long - term maintenance of body weight , and deciphered the molecular sequence underlying its nutritional regulation . we found that upon high fat diet ( hfd ) , reduced hypothalamic polysialylation exacerbated the diet - induced obese phenotype in mice . upon hfd , the histone acetyltransferase mof was rapidly recruited on the st8sia4 polysialyltransferase - encoding gene . mof silencing in the mediobasal hypothalamus of adult mice prevented activation of the st8sia4 gene transcription , reduced polysialylation , altered the acute homeostatic feeding response to hfd and increased the body weight gain . these findings indicate that impaired hypothalamic polysialylation contribute to the development of obesity , and establish a role for mof in the brain control of energy balance .

Introduction Materials and methods Results Discussion Author contributions

the melanocortin system is one of the most well described circuits which control body weight and food intake . the two main antagonistic components of this system are the anorexigenic pomc and orexigenic npy neurons located in the arcuate nucleus of the hypothalamus . it has been recently shown that density and input type on pomc and npy neurons vary in adult mice , depending on the metabolic state and the associated fluctuation of circulating hormones [ 46 ] . such synaptic plasticity modifies the activity of these neurons and thereby the responsiveness of the melanocortin system . it has been proposed that the synaptic plasticity of this system in response to acute changes in fuel availability was essential to the accurate control of food intake and the maintenance of body weight in adulthood [ 79 ] . moreover , altered synaptic plasticity of pomc and npy neurons was found in diet - induced obese rodents suggesting that inability to dynamically rewire feeding circuits could contribute to obesity . polysialylation of cell adhesion proteins is involved in various synaptic plasticity - dependent processes in the central nervous system including learning , pain modulation and the control of neuroendocrine functions . we previously reported that polysialylation was also required for the adaptive synaptic plasticity of feeding circuits during acute positive energy balance . precisely , high - fat diet ( hfd ) given for three days caused a psa - dependent increase in the excitatory inputs connecting pomc neurons in mice . the polysialyltransferase pst-1 , encoded by the st8sia4 gene was identified as the enzyme involved in the psa - dependent control of energy intake . however the biological mediators and the intracellular cascade of events leading to the dietary fat - induced psa - dependent synaptic plasticity in the hypothalamus have not been characterized yet . it is now clearly established that modifications in gene expression patterns are required to elicit structural and functional synaptic plasticity in response to experience and environmental cues [ 1820 ] . in particular , changes in the acetylation of histone tails and in the activity of histone acetyltransferases or histone deacetylases are associated with changes in plasticity - related gene expression in the brain areas supporting these synaptic plasticity - dependent processes , i.e. the hippocampus , the prefrontal cortex and the nucleus accumbens [ 2528 ] . here we explored the role of hypothalamic polysialylation in the long - term maintenance of body weight . we also deciphered the molecular sequence underlying the activation of polysialylation in the hypothalamus upon overfeeding . hypothalamic psa removal and st8sia4 silencing increased body weight gain on hfd , indicating that st8sia4 is essential to maintain energy homeostasis . we showed that dietary fat rapidly incited post - translational histone modifications on the st8sia4 gene in the mediobasal hypothalamus ( mbh ) . the histone acetyltransferase mof was required for the hfd - induced acetylation of histone h4 on lysine 16 and the subsequent activation of st8sia4 gene transcription . moreover , mof silencing in the mbh of adult mice fed a hfd reduced hypothalamic psa levels , caused overfeeding and exacerbated diet - induced obesity . animal care and experimental procedures were performed with approval from the animal care and use committee of the university of burgundy . they were fed either a standard diet ( a04 ; safe laboratories ) or a high fat diet ( hfd ) ( safe laboratories as previously described ) . small holes were drilled and a double cannula with a cap ( c235g-0.8/sp 4.6 mm ; plastic one ) was inserted to target above the mediobasal hypothalamus ( mbh ) using the following coordinates : 1.4 mm posterior to the bregma , 0.4 mm lateral to the sagittal suture , and 4.6 mm below the skull surface ( supplementary figure 1a ) . mice received a bilateral injection of lentiviral transduction particles ( 400 nl per side , 23 10 tu / ml ) , using the following stereotactic coordinates : 1.4 mm posterior to the bregma , 0.4 mm lateral to the sagittal suture , and 5.6 mm below the skull surface ( supplementary figure 1b ) . the lentiviral particles contained either a non - target control shrna ( non - mammalian control ; sigma aldrich ) , or a shrna targeting mof ( trcn0000039323 ; sigma aldrich ) or st8sia4 ( trcn0000093936 ) . nuclear magnetic resonance was used to determine the body composition of the mice ( qnmr system echomri-500 t , echo medical systems ) . chromatin in the supernatant was then reverse cross - linked by incubating 10 h at 65 c . taqman assay ids for the mrna analyzed were as follow : mm00458911_m1 for mof , mm01292231_m1 for st8sia4 , mm00456815_m1 for ncam1 , mm00456289_m1 for st8sia2 , mm00450174_m1 for gne , mm02342429_g1 for ppia . to assess the role of hypothalamic polysialylation in the long - term maintenance of body weight of mice on hfd , we generated psa - depleted mice . endon remains active at least eight days after a single injection in the brain parenchyma . we chronically depleted psa in this area by bilateral intraparenchymal injections of endon once a week during one month in mice fed with hfd ( figure 1a ) . we observed that the repeated injections of endon reduced by 8090% the psa level in the mbh ( figure 1b ) . the repeated injections of endon did not modify body weight in mice fed a standard diet for one month ( figure 1c e , and supplementary figure 2 ) . however , the persistent loss of hypothalamic psa caused by endon dramatically increased the weight gain of mice on hfd , in comparison to vehicle - treated mice ( figure 1c ) . as a result , body weight gain and fat mass measured after one month in mice on hfd were higher in endon - treated mice relative to vehicle - treated mice ( figure 1d and e ) . to test whether hypothalamic pst-1 is involved in the long - term maintenance of body weight , we examined the metabolic consequences of st8sia4 knock - down in the hypothalamus of mice fed a hfd . we used lentiviral vectors - mediated rna interference for specifically knocking down st8sia4 expression in the hypothalamus of adult mice . the expression of st8sia2 , ncam , and gne was not altered ( figure 2a ) , indicating that the rnai - mediated knockdown of st8sia4 was specific and did not modify expression of other genes involved in the signaling pathway of psa . body weight gain and energy intake of control and pst-1 kd mice fed a standard diet for 2 months were similar ( supplementary figure 3 ) . to investigate whether a reduction of polysialylation in the hypothalamus increased the vulnerability to dio , pst-1 kd mice were fed a hfd for two months . only mice with more than 5% reduction of hypothalamic psa after screening , we detected a significant reduction of the psa level by 25.7% in the mbh of pst-1 kd mice , in comparison to control mice ( figure 2b ) . body weight gain and adiposity of pst-1 kd mice on hfd were therefore significantly increased ( figure 2d and e ) . we next investigated the regulation of st8sia4 expression in the hypothalamus of hfd - fed mice . we determined if hfd exposure triggered chromatin remodeling and transcriptional activation of the st8sia4 gene in the mbh . chromatin modifications were analyzed on the promoter of st8sia4 by chip with chromatin extracts obtained from the mbh of mice fed a hfd for 24 h72 h ( figure 3 and supplementary figure 4 ) . the levels of h4k16ac , h4k20me and h3k14ac returned to basal values after 72 h on hfd , indicating that these post - translational modifications were transient ( supplementary figure 4b ) . the decrease in histone interaction with st8sia4 promoter preceded an increase in pol2-s5p level -the activated form of rna polymerase ii- indicating an activation of st8sia4 transcription ( figure 3b ) . additional analysis revealed no post - translational histone modifications , no histone dissociation and no change in rna polymerase ii phosphorylation on the promoter of the st8sia2 gene , encoding the pst-2 polysialyltransferase ( supplementary figure 4c and 4d ) . the chromatin remodeling events detected in the mbh after hfd introduction were thus specific to the st8sia4 polysialyltransferase gene . in order to identify the histone acetyltransferases ( hat ) that trigger the increase in histone acetylation and the subsequent activation of st8sia4 transcription in the mbh of hfd - fed mice , the recruitment of cbp , gcn5 , mof and tip60 was analyzed by chip . after 6 h on hfd , a 2.20 fold increase in mof was detected on st8sia4 promoter . the early recruitment of mof suggested that this hat plays a critical role in mediating the effects of hfd on the activation of st8sia4 transcription in the mbh . to determine the role of mof in hfd - induced hypothalamic polysialylation , we knocked down mof in the hypothalamus and tested whether st8sia4 transcription and psa levels could still be increased upon hfd exposure . lentiviral particles containing a mof - targeting shrna sequence were stereotactically injected in the mbh of adult mice . after 3 weeks of recovery , a 26.73% decrease in mof mrna level was detected in the hypothalamus of silenced mice ( mof kd mice ) ( figure 5a ) . since h4k16ac has been identified as the main substrate of mof in many cell types and organisms [ 32,3537 ] , we tested whether mof knock - down altered the hfd - induced increase in h4k16ac level on st8sia4 in the mbh ( figure 5b ) . the level of h4k16ac in hfd - fed mof - kd mice was similar to the level in standard - diet fed mice ( figure 3a ) , indicating that mof knock - down abolished the hfd - induced acetylation of h4k16 . after 3 days on hfd , the level of pol2-s5p detected on st8sia4 was strongly reduced in mof - kd mice in comparison to the control group ( 38.33% reduction , figure 5c ) . we finally assessed polysialylation capacities in mof - kd mice by measuring both st8sia4 mrna level and psa level in the mbh after exposure to hfd for 3 days ( figure 5c and d ) . altogether , these results indicated that mof is crucial for activating the polysialylation pathway in the mbh in response to hfd . we finally examined the consequences of mof knock - down on the psa - dependent short - term feeding response to hfd and the long - term regulation of body weight on hfd . the short - term feeding response to hfd was assessed over 8 days of hfd exposure . however the normalization of energy intake was slightly but significantly altered in mof kd mice . consequently , the cumulative energy intake of mof kd mice during the first week of hfd was higher in this group ( figure 6d ) . hfd feeding revealed an alteration of the homeostatic control of food intake in mof kd mice . to assess the role of mof in the long - term regulation of energy homeostasis , control and mof kd mice food intake and body weight were recorded daily during this period . only mice with more than 5% reduction of hypothalamic mof mrna level as a reference , body weight of control and mof kd mice fed a standard diet was measured over 6 weeks after surgery . no significant differences were observed in body weight gain between control mice and mof kd mice ( + 2.49 0.28 g vs 2.96 0.52 g ) . however , upon hfd , a dramatic increase in body weight was observed in mof kd mice ( figure 6e ) . the body weight gain of mof kd mice over the 8 weeks of hfd was 31.85% higher than the one of the control group ( figure 6f ) . this increase in body weight gain was due to an increase in fat mass in mof kd mice ( figure 6 g ) . the higher cumulative energy intake of mof kd mice in comparison to control mice over the 8-week period likely explained this phenotype ( figure 6h , mof kd mice : 786.83 14.06 kcal and control mice : 708.41 21.79 obesity is on the rise in both developed and developing countries , and it is now considered as a worldwide epidemic . investigation of central regulatory pathways involved in the control of appetite and body weight could probably help with the fight against obesity . we previously identified polysialylation as an essential process in the nutritionally regulated synaptic plasticity of the melanocortin system . we now show that polysialylation is essential to the long - term regulation of body weight . furthermore , the experimental data presented here unveil an unexpected role for the histone acetyltransferase mof in the brain control of energy balance . developmental malformation of the melanocortin system , i.e. disruption of neural projection pathways within the hypothalamus , could contribute to the obese phenotype and related metabolic disorders . moreover , the wiring of arcuate pomc neurons of rats kept on standard diet predicts vulnerability to weight gain on hfd , suggesting that accurate connectivity in this neuronal system is crucial to maintain energy homeostasis . thus , it has been suggested that the altered synaptic plasticity in feeding circuits , in addition to steady neuroanatomical defects , might promote dio . here , we report that the lack of hypothalamic psa strongly accelerated the onset of dio in mice . given the role of psa in synaptic plasticity , our results suggest that the inflexibility of feeding circuits is a risk factor for obesity . this is in line with the well - documented adaptive function of the plasticity in feeding circuits in response to metabolic fluctuations . the removal of hypothalamic psa was sufficient to cause severe overweight in mice fed a hfd for 12 months , suggesting that the inability to properly rewire feeding circuits depending on the nutritional conditions , makes mice prone to obesity . since the human hypothalamus is able to undergo hormone - dependent structural plasticity , our findings could be relevant to the etiology of obesity in humans as well . indeed , large - scale studies on the genetic determinants of body mass index have revealed the strong influence of some genes involved in neuronal plasticity . interestingly , the polymorphism in negr1 , the gene coding the neuronal growth regulator 1 , a cell adhesion molecule involved in the brain remodeling , seems to be causal in high body weight . to gain insight into the molecular sequence underlying the synaptic plasticity - dependent control of energy balance , we explored the chromatin remodeling events underlying the activation of the st8sia4 gene expression upon high fat diet ( hfd ) . we found that the hfd induced a decrease in the level of monomethylated h4k20 and an increase in acetylation of histones h3k14 and h4k16 on the st8sia4 gene . in our study , the decrease in h4k20me1 and the increase in h3 and h4 acetylation upon hfd the level of rna polymerase ii phosphorylated on the serine 5 of the carboxy - terminal domain increased subsequently . moreover , the phosphorylation of the serine 5 of the rna polymerase ii is acquired during the transcription initiation phase . tracing back the signaling cascade leading to the activation of st8sia4 transcription , we identified two histone acetyltransferases , gcn5 and mof , recruited on st8sia4 within the few hours following the introduction of hfd . phf8 could thus contribute , in cooperation with gcn5 and mof to the activation of st8sia4 transcription in the hypothalamus upon high fat feeding . in our study , since mof recruitment was already detected after 6 h of hfd feeding , we focused on the function of this hat in the activation of st8sia4 expression upon hfd exposure . the depletion of mof in the mbh completely abolished the hfd - induced increase in h4k16 acetylation on st8sia4 and the subsequent recruitment of the rna polymerase ii on this gene . the early recruitment of mof on st8sia4 upon hfd exposure , the absence of activation of st8sia4 transcription and the decrease in psa level after mof knock - down strongly suggested a crucial role for mof in mediating the effect of hfd exposure on the establishment of psa - dependent synaptic plasticity in the hypothalamus . mof kd mice displayed an impairment of the short - term feeding response to hfd , which is actually a psa - dependent process . in vivo , a purkinje cells - specific deletion of mof in the cerebellum produced neurological abnormalities including impaired motor coordination , ataxia and a backward - walking phenotype . the present work demonstrates that mediobasal hypothalamic mof is involved in the regulation of energy homeostasis through an activation of polysialylation . whereas some studies suggest that mof is globally involved in the activation of transcription , others indicate that this hat rather activates defined subset of genes and has a function in transcription regulation in specific cell types only . though we can not rule out that a larger mof - dependent transcriptomic program contributes to the accelerated onset of diet - induced obesity in mof - depleted mice , the subsequent deficit in hypothalamus polysialylation capacities of these mice might largely contribute to their increased vulnerability to dio . this suggests that mof could be recruited to the st8sia4 gene by interacting directly with a transcription factor activating st8sia4 transcription upon high fat feeding . in conclusion , the present work clearly indicated the causal role of altered psa signaling in accelerating the development of obesity . given the role of psa in the modulation of cell - to - cell interactions these findings strengthen the concept that individual capacity to synaptic plasticity is a key factor in the development of overweight with obesogenic foods .

when engineering proteins , an important problem to be considered is to which extent a mutation will affect the stability of the new protein with respect to the wild type . they are mainly based on the development of different energy functions , suited to compute the stability free energy changes ( 15 ) . recently , an approach based on a neural network system was described ( 6 ) . in this application , instead of directly estimating the relative stability changes upon protein mutation [ the g value , ( 15 ) ] , a neural network predicts the direction towards which the mutation shifts the stability of the protein ( namely the g sign ) . all the methods developed so far , are however limited in that prediction can be carried out only when the protein 3d structure is available in the pdb database . in the post - genomic era , however , mutagenesis experiments may start from the proteome . therefore , the development of predictors that help to design mutated proteins from the only sequence is urgent . here , we present a new server i - mutant ( 2.0 ) . i - mutant2.0 can predict the stability change of the mutated protein structure , and , for the first time , it can predict to which extent a mutation in a protein sequence will or will not affect the stability of the folded protein . i - mutant2.0 is based on support vector machines ( svms ) and it has been trained to predict both the direction ( the g sign ) of the protein stability changes and the g associated values . thanks to the availability of a large database of thermodynamic data of mutated proteins ( 7 ) , we show that for the specific task of predicting the g sign , i - mutant2.0 correctly predicts ( with a cross - validation procedure ) 80 or 77% of the data set , depending on the input of structural or sequence information , respectively . when predicting g values associated with the mutation , the correlation of predicted with expected values , as taken from the experimental database , is 0.71 and 0.62 , depending on the structure- and sequence - base prediction , respectively . i - mutant2.0 was trained to accomplish four different tasks : prediction of the direction of the protein stability changes upon single point mutation from the protein tertiary structure ( a classification task);prediction of the g value of the protein stability changes upon single point mutation from the protein tertiary structure ( a function approximation task);prediction of the direction of the protein stability changes upon single point mutation only from the protein sequence ( a classification task);prediction of the g value of the protein stability changes upon single point mutation only from the protein sequence ( a function approximation task ) . prediction of the direction of the protein stability changes upon single point mutation from the protein tertiary structure ( a classification task ) ; prediction of the g value of the protein stability changes upon single point mutation from the protein tertiary structure ( a function approximation task ) ; prediction of the direction of the protein stability changes upon single point mutation only from the protein sequence ( a classification task ) ; prediction of the g value of the protein stability changes upon single point mutation only from the protein sequence ( a function approximation task ) . we tested several kernels and we found that the most convenient for the problems at hand is the one based on radial basis functions ( rbf , kernel = exp [ gxixj2 ] ) . the results described here are therefore relative only to the rbf kernel . for the classification task and for assigning the g values , we basically adopt the same input code by identifying two labels : one represents the increased protein stability ( g > 0 , label is + ) , the other is associated with the destabilizing mutation ( g < 0 , label is ) . the first two input values account , respectively , for the temperature and the ph at which the stability of the mutated protein was experimentally determined . the next 20 values ( for 20 residue types ) explicitly define the mutation ( we set 1 to the element corresponding to the deleted residue and 1 to the new residue , all the remaining elements are kept equal to 0 ) . spatial environment when the protein structure is available or the nearest sequence neighbors , when only the protein sequence is available . when the protein structure is known ( and the prediction is performed on the protein structure ) each of the 20 values is the number of the encoded residue type , to be found inside a sphere of a 0.9 nm radius and centered on the coordinates of the residue that undergoes mutation . alternatively , when the prediction is performed starting from the protein sequence , each of the 20 input values is again the number of the encoded residue type , to be found inside a symmetrical window , centered at the mutated residue , that spans the sequence towards the left ( n - terminus ) and the right ( c - terminus ) for a total length of 19 residues . the rsa value ( relative solvent accessible area ) can be calculated with the dssp program ( 8) only when prediction is structure based , dividing the accessible surface area value of the mutated residue by the free residue surface . in this case , a further input value ( for a total sum of 43 numbers ) includes the relative solvent accessible area of the mutated residue only when the protein structure is considered . this strategy is similar to what was previously done with a neural network - based predictor for predicting stability changes upon mutation starting from the protein structure ( 6 ) . the ri value ( reliability index ) can be computed only when the sign of the stability change is predicted and is evaluated from the output of the svm o as ri = 20*abs(o 0.5 ) . i - mutant2.0 was trained and tested with a cross - validation procedure on a data set derived from the current release ( december 2004 ) of the thermodynamic database for proteins and mutants [ protherm , ( 7 ) ] . the data set of proteins was extracted from protherm with the following constraints : only single point mutations were considered for each protein ( no multiple mutations were taken into account).the correspondent free energy change of protein stability ( g value ) had been experimentally detected and experimental conditions ( temperature and ph ) were also listed in the database . only single point mutations were considered for each protein ( no multiple mutations were taken into account ) . the correspondent free energy change of protein stability ( g value ) had been experimentally detected and experimental conditions ( temperature and ph ) were also listed in the database . after this filtering procedure , our data set comprised 2087 different single mutations in 65 different proteins , 58 out of which were also known with atomic resolution . for each mutation , the corresponding free energy change was collected . the subset of structures known with atomic resolution ( with a total of 1948 different single mutations ) contains proteins uniformly distributed in the four predominant structural classes according to the scop classification ( ) . , we list the accuracy obtained when i - mutant2.0 is adopted as a classifier , and discriminates whether a given mutation increases ( label + ) or decreases ( label ) the protein stability . the accuracy of the structure - based prediction is 0.80 and it is higher than that obtained when the prediction is sequence based . a similar value was previously obtained with a neural network - based predictor , trained / tested on a data set smaller than that adopted in this study ( 6 ) . however , a remarkable result of this paper is that the overall accuracy is 0.77 even when the predictor is sequence based . i - mutant2.0 was also trained / tested to predict the value of the free energy stability change upon single point mutation , starting from the protein structure or sequence . in this case , the accuracy was evaluated by measuring the correlation between the predicted ( adopting a cross - validation procedure ) and the observed g values . the correlation of the predicted and experimental data is 0.71 , with a standard error of 1.3 kcal / mol , when the method is structure based ( figure 1 ) . when it is sequence based , the correlation between the observed and the predicted data is 0.62 with a standard error of 1.45 kcal / mol . as a final observation , it must be considered that 89% of the proteins are contributing to the training / testing set adopted to implement both the sequence- and structure - based svm method . with this in mind , the small scoring difference in accuracy that i - mutant2.0 achieves starting from the protein structure or sequence ( 0.80 versus 0.77 ) suggests that to a large extent protein stability can also be correctly evaluated when specific interactions within the sequence neighbors are captured . i - mutant2.0 was trained to accomplish four different tasks : prediction of the direction of the protein stability changes upon single point mutation from the protein tertiary structure ( a classification task);prediction of the g value of the protein stability changes upon single point mutation from the protein tertiary structure ( a function approximation task);prediction of the direction of the protein stability changes upon single point mutation only from the protein sequence ( a classification task);prediction of the g value of the protein stability changes upon single point mutation only from the protein sequence ( a function approximation task ) . prediction of the direction of the protein stability changes upon single point mutation from the protein tertiary structure ( a classification task ) ; prediction of the g value of the protein stability changes upon single point mutation from the protein tertiary structure ( a function approximation task ) ; prediction of the direction of the protein stability changes upon single point mutation only from the protein sequence ( a classification task ) ; prediction of the g value of the protein stability changes upon single point mutation only from the protein sequence ( a function approximation task ) . we tested several kernels and we found that the most convenient for the problems at hand is the one based on radial basis functions ( rbf , kernel = exp [ gxixj2 ] ) . the results described here are therefore relative only to the rbf kernel . for the classification task and for assigning the g values , we basically adopt the same input code by identifying two labels : one represents the increased protein stability ( g > 0 , label is + ) , the other is associated with the destabilizing mutation ( g < 0 , label is ) . the first two input values account , respectively , for the temperature and the ph at which the stability of the mutated protein was experimentally determined . the next 20 values ( for 20 residue types ) explicitly define the mutation ( we set 1 to the element corresponding to the deleted residue and 1 to the new residue , all the remaining elements are kept equal to 0 ) . spatial environment when the protein structure is available or the nearest sequence neighbors , when only the protein sequence is available . when the protein structure is known ( and the prediction is performed on the protein structure ) each of the 20 values is the number of the encoded residue type , to be found inside a sphere of a 0.9 nm radius and centered on the coordinates of the residue that undergoes mutation . alternatively , when the prediction is performed starting from the protein sequence , each of the 20 input values is again the number of the encoded residue type , to be found inside a symmetrical window , centered at the mutated residue , that spans the sequence towards the left ( n - terminus ) and the right ( c - terminus ) for a total length of 19 residues . the rsa value ( relative solvent accessible area ) can be calculated with the dssp program ( 8) only when prediction is structure based , dividing the accessible surface area value of the mutated residue by the free residue surface . in this case , a further input value ( for a total sum of 43 numbers ) includes the relative solvent accessible area of the mutated residue only when the protein structure is considered . this strategy is similar to what was previously done with a neural network - based predictor for predicting stability changes upon mutation starting from the protein structure ( 6 ) . the ri value ( reliability index ) can be computed only when the sign of the stability change is predicted and is evaluated from the output of the svm o as ri = 20*abs(o 0.5 ) . i - mutant2.0 was trained and tested with a cross - validation procedure on a data set derived from the current release ( december 2004 ) of the thermodynamic database for proteins and mutants [ protherm , ( 7 ) ] . the data set of proteins was extracted from protherm with the following constraints : only single point mutations were considered for each protein ( no multiple mutations were taken into account).the correspondent free energy change of protein stability ( g value ) had been experimentally detected and experimental conditions ( temperature and ph ) were also listed in the database . only single point mutations were considered for each protein ( no multiple mutations were taken into account ) . the correspondent free energy change of protein stability ( g value ) had been experimentally detected and experimental conditions ( temperature and ph ) were also listed in the database . after this filtering procedure , our data set comprised 2087 different single mutations in 65 different proteins , 58 out of which were also known with atomic resolution . for each mutation , the subset of structures known with atomic resolution ( with a total of 1948 different single mutations ) contains proteins uniformly distributed in the four predominant structural classes according to the scop classification ( ) . , we list the accuracy obtained when i - mutant2.0 is adopted as a classifier , and discriminates whether a given mutation increases ( label + ) or decreases ( label ) the protein stability . the accuracy of the structure - based prediction is 0.80 and it is higher than that obtained when the prediction is sequence based . a similar value was previously obtained with a neural network - based predictor , trained / tested on a data set smaller than that adopted in this study ( 6 ) . however , a remarkable result of this paper is that the overall accuracy is 0.77 even when the predictor is sequence based . i - mutant2.0 was also trained / tested to predict the value of the free energy stability change upon single point mutation , starting from the protein structure or sequence . in this case , the accuracy was evaluated by measuring the correlation between the predicted ( adopting a cross - validation procedure ) and the observed g values . the correlation of the predicted and experimental data is 0.71 , with a standard error of 1.3 kcal / mol , when the method is structure based ( figure 1 ) . when it is sequence based , the correlation between the observed and the predicted data is 0.62 with a standard error of 1.45 kcal / mol . as a final observation , it must be considered that 89% of the proteins are contributing to the training / testing set adopted to implement both the sequence- and structure - based svm method . with this in mind , the small scoring difference in accuracy that i - mutant2.0 achieves starting from the protein structure or sequence ( 0.80 versus 0.77 ) suggests that to a large extent protein stability can also be correctly evaluated when specific interactions within the sequence neighbors are captured . on the i - mutant2.0 web page , two alternatives are available : predicting the protein stability changes upon single point mutation starting from the protein structure or sequence ( see figure 2 ) . in the first case , the pdb code of the protein is required ; in the latter , the protein sequence needs to be pasted in the appropriate box . as discussed above and listed in table 1 , the accuracy is higher when the prediction is structure based . nevertheless , even when it is sequence based the score is remarkable . following the first option , two choices are available : ( i ) i - mutant2.0 acts as a classifier , and the sign of the free energy stability change upon mutation is predicted ; ( ii ) i - mutant2.0 acts as a regression estimator and the value of free energy stability change ( g ) upon mutation is predicted . in both cases , i - mutant2.0 allows the selection of the prediction of protein stability changes at different ranges of temperature and ph ( see figure 2 ) . the user can start from the protein structure or sequence . in either case , the prediction of the value of free energy change or only its sign can be obtained . in all cases , a table of 19 rows ( the 19 residues that are different from the one present in the sequence at a selected position ) is returned . a further option , when activated , allows to highlight only the row corresponding to a given mutation . the four different possible predictive options correspond to a different number of columns returned in the output table . the common number is six columns , listing respectively : the sequence position under consideration , the original residue name ( one letter code ) , the mutated residue ( one letter code ) , the predicted free energy change value ( ddg ) or the sign of the prediction ( increase / decrease ) , the temperature and the ph at which the prediction has been carried out . one more column may be present in the output table that lists in turn the reliability index value of the prediction ( ri ) or the solvent accessible surface area [ rsa , computed with the dssp program ( 8) ] . this occurs when the sign of the stability change is predicted starting from the protein sequence or the ddg value of the free energy change is predicted starting from the protein structure , respectively . both rsa and ri are computed and present in the output table only when the sign of the protein stability change is predicted starting from the structure . in this case , the number of columns in the output table is 8 . from the above description , it is evident that both the structure - based and the sequence - based predictions of the sign of the protein stability change upon mutation are those endowed with the reliability scoring index ( ri ) , and this allows the sorting out of the subset of more reliable predictions [ see ( 6 ) ] . when the ddg values are predicted from the protein structure or sequence , an estimate of the standard error can be evaluated from the linear regression between the predicted and the expected values and can be associated to the predicted value . results obtained for the structure - based case are shown in figure 1 . with this procedure , the standard error value is 1.30 and 1.45 kcal / mol , when the prediction is structure or sequence based , respectively . on the i - mutant2.0 web page , two alternatives are available : predicting the protein stability changes upon single point mutation starting from the protein structure or sequence ( see figure 2 ) . in the first case , the pdb code of the protein is required ; in the latter , the protein sequence needs to be pasted in the appropriate box . as discussed above and listed in table 1 , the accuracy is higher when the prediction is structure based . nevertheless , even when it is sequence based the score is remarkable . following the first option , two choices are available : ( i ) i - mutant2.0 acts as a classifier , and the sign of the free energy stability change upon mutation is predicted ; ( ii ) i - mutant2.0 acts as a regression estimator and the value of free energy stability change ( g ) upon mutation is predicted . in both cases , i - mutant2.0 allows the selection of the prediction of protein stability changes at different ranges of temperature and ph ( see figure 2 ) . the user can start from the protein structure or sequence . in either case , the prediction of the value of free energy change or only its sign can be obtained . in all cases , a table of 19 rows ( the 19 residues that are different from the one present in the sequence at a selected position ) is returned . a further option , when activated , allows to highlight only the row corresponding to a given mutation . the four different possible predictive options correspond to a different number of columns returned in the output table . the common number is six columns , listing respectively : the sequence position under consideration , the original residue name ( one letter code ) , the mutated residue ( one letter code ) , the predicted free energy change value ( ddg ) or the sign of the prediction ( increase / decrease ) , the temperature and the ph at which the prediction has been carried out . one more column may be present in the output table that lists in turn the reliability index value of the prediction ( ri ) or the solvent accessible surface area [ rsa , computed with the dssp program ( 8) ] . this occurs when the sign of the stability change is predicted starting from the protein sequence or the ddg value of the free energy change is predicted starting from the protein structure , respectively . both rsa and ri are computed and present in the output table only when the sign of the protein stability change is predicted starting from the structure . in this case , the number of columns in the output table is 8 . from the above description , it is evident that both the structure - based and the sequence - based predictions of the sign of the protein stability change upon mutation are those endowed with the reliability scoring index ( ri ) , and this allows the sorting out of the subset of more reliable predictions [ see ( 6 ) ] . when the ddg values are predicted from the protein structure or sequence , an estimate of the standard error can be evaluated from the linear regression between the predicted and the expected values and can be associated to the predicted value . results obtained for the structure - based case are shown in figure 1 . with this procedure , the standard error value is 1.30 and 1.45 kcal / mol , when the prediction is structure or sequence based , respectively . correlation plot between the experimentally observed and the predicted g values when the svm method is structure based . snapshot of the i - mutant2.0 input pages for the prediction of the protein stability change upon mutation with the protein structure through its pdb code ( top ) or starting from the sequence ( bottom ) . i - mutant2.0 cross - validation accuracy q2 = number of correct predictions / number of examples . p(s ) = number of correct prediction for class s / all prediction made for s. q(s ) = number of correct prediction for class s / observed in class s. c = matthews 's correlation coefficient [ compare with ( 6 ) ] .

i - mutant2.0 is a support vector machine ( svm)-based tool for the automatic prediction of protein stability changes upon single point mutations . i - mutant2.0 predictions are performed starting either from the protein structure or , more importantly , from the protein sequence . this latter task , to the best of our knowledge , is exploited for the first time . the method was trained and tested on a data set derived from protherm , which is presently the most comprehensive available database of thermodynamic experimental data of free energy changes of protein stability upon mutation under different conditions . i - mutant2.0 can be used both as a classifier for predicting the sign of the protein stability change upon mutation and as a regression estimator for predicting the related g values . acting as a classifier , i - mutant2.0 correctly predicts ( with a cross - validation procedure ) 80% or 77% of the data set , depending on the usage of structural or sequence information , respectively . when predicting g values associated with mutations , the correlation of predicted with expected / experimental values is 0.71 ( with a standard error of 1.30 kcal / mol ) and 0.62 ( with a standard error of 1.45 kcal / mol ) when structural or sequence information are respectively adopted . our web interface allows the selection of a predictive mode that depends on the availability of the protein structure and/or sequence . in this latter case , the web server requires only pasting of a protein sequence in a raw format . we therefore introduce i - mutant2.0 as a unique and valuable helper for protein design , even when the protein structure is not yet known with atomic resolution . availability : .

INTRODUCTION DESCRIPTION I-Mutant2.0 accuracy SERVER DETAILS Inputs Outputs Figures and Tables

when engineering proteins , an important problem to be considered is to which extent a mutation will affect the stability of the new protein with respect to the wild type . they are mainly based on the development of different energy functions , suited to compute the stability free energy changes ( 15 ) . in this application , instead of directly estimating the relative stability changes upon protein mutation [ the g value , ( 15 ) ] , a neural network predicts the direction towards which the mutation shifts the stability of the protein ( namely the g sign ) . all the methods developed so far , are however limited in that prediction can be carried out only when the protein 3d structure is available in the pdb database . therefore , the development of predictors that help to design mutated proteins from the only sequence is urgent . i - mutant2.0 can predict the stability change of the mutated protein structure , and , for the first time , it can predict to which extent a mutation in a protein sequence will or will not affect the stability of the folded protein . i - mutant2.0 is based on support vector machines ( svms ) and it has been trained to predict both the direction ( the g sign ) of the protein stability changes and the g associated values . thanks to the availability of a large database of thermodynamic data of mutated proteins ( 7 ) , we show that for the specific task of predicting the g sign , i - mutant2.0 correctly predicts ( with a cross - validation procedure ) 80 or 77% of the data set , depending on the input of structural or sequence information , respectively . when predicting g values associated with the mutation , the correlation of predicted with expected values , as taken from the experimental database , is 0.71 and 0.62 , depending on the structure- and sequence - base prediction , respectively . i - mutant2.0 was trained to accomplish four different tasks : prediction of the direction of the protein stability changes upon single point mutation from the protein tertiary structure ( a classification task);prediction of the g value of the protein stability changes upon single point mutation from the protein tertiary structure ( a function approximation task);prediction of the direction of the protein stability changes upon single point mutation only from the protein sequence ( a classification task);prediction of the g value of the protein stability changes upon single point mutation only from the protein sequence ( a function approximation task ) . prediction of the direction of the protein stability changes upon single point mutation from the protein tertiary structure ( a classification task ) ; prediction of the g value of the protein stability changes upon single point mutation from the protein tertiary structure ( a function approximation task ) ; prediction of the direction of the protein stability changes upon single point mutation only from the protein sequence ( a classification task ) ; prediction of the g value of the protein stability changes upon single point mutation only from the protein sequence ( a function approximation task ) . we tested several kernels and we found that the most convenient for the problems at hand is the one based on radial basis functions ( rbf , kernel = exp [ gxixj2 ] ) . for the classification task and for assigning the g values , we basically adopt the same input code by identifying two labels : one represents the increased protein stability ( g > 0 , label is + ) , the other is associated with the destabilizing mutation ( g < 0 , label is ) . the first two input values account , respectively , for the temperature and the ph at which the stability of the mutated protein was experimentally determined . spatial environment when the protein structure is available or the nearest sequence neighbors , when only the protein sequence is available . when the protein structure is known ( and the prediction is performed on the protein structure ) each of the 20 values is the number of the encoded residue type , to be found inside a sphere of a 0.9 nm radius and centered on the coordinates of the residue that undergoes mutation . alternatively , when the prediction is performed starting from the protein sequence , each of the 20 input values is again the number of the encoded residue type , to be found inside a symmetrical window , centered at the mutated residue , that spans the sequence towards the left ( n - terminus ) and the right ( c - terminus ) for a total length of 19 residues . the rsa value ( relative solvent accessible area ) can be calculated with the dssp program ( 8) only when prediction is structure based , dividing the accessible surface area value of the mutated residue by the free residue surface . in this case , a further input value ( for a total sum of 43 numbers ) includes the relative solvent accessible area of the mutated residue only when the protein structure is considered . this strategy is similar to what was previously done with a neural network - based predictor for predicting stability changes upon mutation starting from the protein structure ( 6 ) . the ri value ( reliability index ) can be computed only when the sign of the stability change is predicted and is evaluated from the output of the svm o as ri = 20*abs(o 0.5 ) . i - mutant2.0 was trained and tested with a cross - validation procedure on a data set derived from the current release ( december 2004 ) of the thermodynamic database for proteins and mutants [ protherm , ( 7 ) ] . the data set of proteins was extracted from protherm with the following constraints : only single point mutations were considered for each protein ( no multiple mutations were taken into account).the correspondent free energy change of protein stability ( g value ) had been experimentally detected and experimental conditions ( temperature and ph ) were also listed in the database . only single point mutations were considered for each protein ( no multiple mutations were taken into account ) . the correspondent free energy change of protein stability ( g value ) had been experimentally detected and experimental conditions ( temperature and ph ) were also listed in the database . after this filtering procedure , our data set comprised 2087 different single mutations in 65 different proteins , 58 out of which were also known with atomic resolution . for each mutation , the corresponding free energy change was collected . the subset of structures known with atomic resolution ( with a total of 1948 different single mutations ) contains proteins uniformly distributed in the four predominant structural classes according to the scop classification ( ) . , we list the accuracy obtained when i - mutant2.0 is adopted as a classifier , and discriminates whether a given mutation increases ( label + ) or decreases ( label ) the protein stability . the accuracy of the structure - based prediction is 0.80 and it is higher than that obtained when the prediction is sequence based . a similar value was previously obtained with a neural network - based predictor , trained / tested on a data set smaller than that adopted in this study ( 6 ) . however , a remarkable result of this paper is that the overall accuracy is 0.77 even when the predictor is sequence based . i - mutant2.0 was also trained / tested to predict the value of the free energy stability change upon single point mutation , starting from the protein structure or sequence . in this case , the accuracy was evaluated by measuring the correlation between the predicted ( adopting a cross - validation procedure ) and the observed g values . the correlation of the predicted and experimental data is 0.71 , with a standard error of 1.3 kcal / mol , when the method is structure based ( figure 1 ) . when it is sequence based , the correlation between the observed and the predicted data is 0.62 with a standard error of 1.45 kcal / mol . as a final observation , it must be considered that 89% of the proteins are contributing to the training / testing set adopted to implement both the sequence- and structure - based svm method . with this in mind , the small scoring difference in accuracy that i - mutant2.0 achieves starting from the protein structure or sequence ( 0.80 versus 0.77 ) suggests that to a large extent protein stability can also be correctly evaluated when specific interactions within the sequence neighbors are captured . i - mutant2.0 was trained to accomplish four different tasks : prediction of the direction of the protein stability changes upon single point mutation from the protein tertiary structure ( a classification task);prediction of the g value of the protein stability changes upon single point mutation from the protein tertiary structure ( a function approximation task);prediction of the direction of the protein stability changes upon single point mutation only from the protein sequence ( a classification task);prediction of the g value of the protein stability changes upon single point mutation only from the protein sequence ( a function approximation task ) . prediction of the direction of the protein stability changes upon single point mutation from the protein tertiary structure ( a classification task ) ; prediction of the g value of the protein stability changes upon single point mutation from the protein tertiary structure ( a function approximation task ) ; prediction of the direction of the protein stability changes upon single point mutation only from the protein sequence ( a classification task ) ; prediction of the g value of the protein stability changes upon single point mutation only from the protein sequence ( a function approximation task ) . we tested several kernels and we found that the most convenient for the problems at hand is the one based on radial basis functions ( rbf , kernel = exp [ gxixj2 ] ) . for the classification task and for assigning the g values , we basically adopt the same input code by identifying two labels : one represents the increased protein stability ( g > 0 , label is + ) , the other is associated with the destabilizing mutation ( g < 0 , label is ) . the first two input values account , respectively , for the temperature and the ph at which the stability of the mutated protein was experimentally determined . spatial environment when the protein structure is available or the nearest sequence neighbors , when only the protein sequence is available . when the protein structure is known ( and the prediction is performed on the protein structure ) each of the 20 values is the number of the encoded residue type , to be found inside a sphere of a 0.9 nm radius and centered on the coordinates of the residue that undergoes mutation . alternatively , when the prediction is performed starting from the protein sequence , each of the 20 input values is again the number of the encoded residue type , to be found inside a symmetrical window , centered at the mutated residue , that spans the sequence towards the left ( n - terminus ) and the right ( c - terminus ) for a total length of 19 residues . the rsa value ( relative solvent accessible area ) can be calculated with the dssp program ( 8) only when prediction is structure based , dividing the accessible surface area value of the mutated residue by the free residue surface . in this case , a further input value ( for a total sum of 43 numbers ) includes the relative solvent accessible area of the mutated residue only when the protein structure is considered . this strategy is similar to what was previously done with a neural network - based predictor for predicting stability changes upon mutation starting from the protein structure ( 6 ) . the ri value ( reliability index ) can be computed only when the sign of the stability change is predicted and is evaluated from the output of the svm o as ri = 20*abs(o 0.5 ) . i - mutant2.0 was trained and tested with a cross - validation procedure on a data set derived from the current release ( december 2004 ) of the thermodynamic database for proteins and mutants [ protherm , ( 7 ) ] . the data set of proteins was extracted from protherm with the following constraints : only single point mutations were considered for each protein ( no multiple mutations were taken into account).the correspondent free energy change of protein stability ( g value ) had been experimentally detected and experimental conditions ( temperature and ph ) were also listed in the database . only single point mutations were considered for each protein ( no multiple mutations were taken into account ) . the correspondent free energy change of protein stability ( g value ) had been experimentally detected and experimental conditions ( temperature and ph ) were also listed in the database . after this filtering procedure , our data set comprised 2087 different single mutations in 65 different proteins , 58 out of which were also known with atomic resolution . for each mutation , the subset of structures known with atomic resolution ( with a total of 1948 different single mutations ) contains proteins uniformly distributed in the four predominant structural classes according to the scop classification ( ) . , we list the accuracy obtained when i - mutant2.0 is adopted as a classifier , and discriminates whether a given mutation increases ( label + ) or decreases ( label ) the protein stability . the accuracy of the structure - based prediction is 0.80 and it is higher than that obtained when the prediction is sequence based . a similar value was previously obtained with a neural network - based predictor , trained / tested on a data set smaller than that adopted in this study ( 6 ) . however , a remarkable result of this paper is that the overall accuracy is 0.77 even when the predictor is sequence based . i - mutant2.0 was also trained / tested to predict the value of the free energy stability change upon single point mutation , starting from the protein structure or sequence . in this case , the accuracy was evaluated by measuring the correlation between the predicted ( adopting a cross - validation procedure ) and the observed g values . the correlation of the predicted and experimental data is 0.71 , with a standard error of 1.3 kcal / mol , when the method is structure based ( figure 1 ) . when it is sequence based , the correlation between the observed and the predicted data is 0.62 with a standard error of 1.45 kcal / mol . as a final observation , it must be considered that 89% of the proteins are contributing to the training / testing set adopted to implement both the sequence- and structure - based svm method . with this in mind , the small scoring difference in accuracy that i - mutant2.0 achieves starting from the protein structure or sequence ( 0.80 versus 0.77 ) suggests that to a large extent protein stability can also be correctly evaluated when specific interactions within the sequence neighbors are captured . on the i - mutant2.0 web page , two alternatives are available : predicting the protein stability changes upon single point mutation starting from the protein structure or sequence ( see figure 2 ) . in the first case , the pdb code of the protein is required ; in the latter , the protein sequence needs to be pasted in the appropriate box . as discussed above and listed in table 1 , the accuracy is higher when the prediction is structure based . nevertheless , even when it is sequence based the score is remarkable . following the first option , two choices are available : ( i ) i - mutant2.0 acts as a classifier , and the sign of the free energy stability change upon mutation is predicted ; ( ii ) i - mutant2.0 acts as a regression estimator and the value of free energy stability change ( g ) upon mutation is predicted . in both cases , i - mutant2.0 allows the selection of the prediction of protein stability changes at different ranges of temperature and ph ( see figure 2 ) . the user can start from the protein structure or sequence . in either case , the prediction of the value of free energy change or only its sign can be obtained . in all cases , a table of 19 rows ( the 19 residues that are different from the one present in the sequence at a selected position ) is returned . the common number is six columns , listing respectively : the sequence position under consideration , the original residue name ( one letter code ) , the mutated residue ( one letter code ) , the predicted free energy change value ( ddg ) or the sign of the prediction ( increase / decrease ) , the temperature and the ph at which the prediction has been carried out . this occurs when the sign of the stability change is predicted starting from the protein sequence or the ddg value of the free energy change is predicted starting from the protein structure , respectively . both rsa and ri are computed and present in the output table only when the sign of the protein stability change is predicted starting from the structure . in this case , the number of columns in the output table is 8 . from the above description , it is evident that both the structure - based and the sequence - based predictions of the sign of the protein stability change upon mutation are those endowed with the reliability scoring index ( ri ) , and this allows the sorting out of the subset of more reliable predictions [ see ( 6 ) ] . when the ddg values are predicted from the protein structure or sequence , an estimate of the standard error can be evaluated from the linear regression between the predicted and the expected values and can be associated to the predicted value . with this procedure , the standard error value is 1.30 and 1.45 kcal / mol , when the prediction is structure or sequence based , respectively . on the i - mutant2.0 web page , two alternatives are available : predicting the protein stability changes upon single point mutation starting from the protein structure or sequence ( see figure 2 ) . in the first case , the pdb code of the protein is required ; in the latter , the protein sequence needs to be pasted in the appropriate box . as discussed above and listed in table 1 , the accuracy is higher when the prediction is structure based . nevertheless , even when it is sequence based the score is remarkable . following the first option , two choices are available : ( i ) i - mutant2.0 acts as a classifier , and the sign of the free energy stability change upon mutation is predicted ; ( ii ) i - mutant2.0 acts as a regression estimator and the value of free energy stability change ( g ) upon mutation is predicted . in both cases , i - mutant2.0 allows the selection of the prediction of protein stability changes at different ranges of temperature and ph ( see figure 2 ) . the user can start from the protein structure or sequence . in either case , the prediction of the value of free energy change or only its sign can be obtained . the common number is six columns , listing respectively : the sequence position under consideration , the original residue name ( one letter code ) , the mutated residue ( one letter code ) , the predicted free energy change value ( ddg ) or the sign of the prediction ( increase / decrease ) , the temperature and the ph at which the prediction has been carried out . one more column may be present in the output table that lists in turn the reliability index value of the prediction ( ri ) or the solvent accessible surface area [ rsa , computed with the dssp program ( 8) ] . this occurs when the sign of the stability change is predicted starting from the protein sequence or the ddg value of the free energy change is predicted starting from the protein structure , respectively . both rsa and ri are computed and present in the output table only when the sign of the protein stability change is predicted starting from the structure . in this case , the number of columns in the output table is 8 . from the above description , it is evident that both the structure - based and the sequence - based predictions of the sign of the protein stability change upon mutation are those endowed with the reliability scoring index ( ri ) , and this allows the sorting out of the subset of more reliable predictions [ see ( 6 ) ] . when the ddg values are predicted from the protein structure or sequence , an estimate of the standard error can be evaluated from the linear regression between the predicted and the expected values and can be associated to the predicted value . with this procedure , the standard error value is 1.30 and 1.45 kcal / mol , when the prediction is structure or sequence based , respectively . correlation plot between the experimentally observed and the predicted g values when the svm method is structure based . snapshot of the i - mutant2.0 input pages for the prediction of the protein stability change upon mutation with the protein structure through its pdb code ( top ) or starting from the sequence ( bottom ) . i - mutant2.0 cross - validation accuracy q2 = number of correct predictions / number of examples .

oligonucleotides ( oligos ) have re - emerged as a major pharmaceutical target due to the unprecedented opportunity for controlling protein expression mediated by short rna oligomers ( ca . 20 nucleotides long ) through rna interference ( rnai ) with small interfering rna ( sirna ) or micro - rna ( mirna ) , and these have in turn re - invigorated research in the field of anti - sense oligonucleotides ( aso / as - on).[13 ] excitement rose with the recent demonstration of safe and effective delivery of oligos in humans . this imperative has underlined the need for scalable methods of rna synthesis . today the overwhelming majority of oligos are prepared using solid - phase oligo synthesis ( spos ) , but this is very challenging to scale up . we are developing a liquid - phase oligo synthesis ( lpos ) synthetic strategy that will be more amenable to standard batch production techniques than spos , using organic - solvent nanofiltration ( osn ) as the critical scalable technology for separating the growing oligo from all other reagents . we now report the lpos - osn preparation of a 2-methyl rna phosphorothioate 9-mer , monitoring all reactions by hplc , p nmr spectroscopy and ms . the defining characteristic of spos is the ease of separation of the growing oligo from excess reagents : the solid synthesis support bed / column is simply washed with solvent to remove any molecular species not covalently attached to it . spos has been scaled up to 12 kg per batch , and the largest trial of the new generation of rnai therapies required a few kg of oligo . thus it is expected that 100s of kilograms of oligo might be required annually to treat rare diseases , and possibly tons for major ones . the leading companies in the field have claimed that spos can be extended to yet larger scales . however , the specialized equipment is demanding and expensive to use in an industrial setting , and we believe that spos , even with major advances , is incapable of approaching the 100 kg scale per batch , because of the challenge of completely and reproducibly washing large beds of synthesis support.[9a ] therefore a very serious gap is expected to open between oligo supply and demand that will restrict this otherwise promising new mode of therapy . consequently , a new method of oligo production is urgently required . early on scalability was identified as the achilles heel of spos , and lpos has long been proposed to overcome this problem . however , the critical question that must be addressed in any lpos strategy is how to separate the growing oligo from excess reagents and byproducts . so far , amongst the alternative strategies reported for the synthesis of oligos , chromatography has been dismissed as too time - consuming , solvent intensive , and inefficient . whilst approaches including size - exclusion chromatography and extraction have been proposed to overcome this separation problem , precipitation of polymer - supported oligo has been explored much more widely . initially dna oligos supported on poly(styrene ) ( the same solid - phase support as had recently been used by merryfield for peptide synthesis ) were assembled by means of khorana s phosphodiester approach.[10 , 15 ] subsequent authors explored poly(vinyl alcohol ) ( pva)[16 , 17 ] and cellulose as supports , but poly(ethylene glycol ) came to dominate this strategy wherein the oligonucleotidyl - peg was usually precipitated with diethyl ether.[17 , 1921 ] recently a discrete , non - polymeric synthesis support was developed in which four oligo chains were grown simultaneously around a pentaerythritol core , and the products were then precipitated from methanol.[22 , 23 ] whilst approaches to oligo synthesis based upon precipitation or crystallisation are in theory scalable , it is questionable whether this would be truly practical . process development of industrial precipitation is often time - consuming and labour - intensive , with the conditions being unique to each compound.[9b ] during bonora s preparation of 10 mg dna 20-mer , 79 separate precipitations and crystallisations were required . furthermore , it is inevitable that material will always be lost to incomplete separation : on the peg support it was found that losses , starting around 1 % per cycle , became more significant as the increasing solubility of the growing oligo began to overwhelm the polymer - driven phase separation ; during the preparation of an rna 5-mer on the small pentaerythritol support , the coupling cycle yield only averaged 85 % for a 54 % overall yield , which would be unacceptable for commercial production . the use of membrane - based technologies for the separation of synthetic biopolymers has been little explored , despite their evident potential to realise scalable liquid - phase approaches to valuable targets . in the first such synthesis , bayer and mutter bound peptides to mono - methyl peg-10 000 ( mpeg ) that was purified by ultrafiltration . this approach was repeated for oligos by the same laboratory , using the now obsolete phosphodiester coupling strategy and either pva or peg-10 000 polymeric supports . alternation between chain extension in organic solvent and diafiltration in water after each chain extension cycle probably makes this strategy impractical . we postulated that organic - solvent nanofiltration ( osn ) could fulfil the critical separation role in an lpos strategy conducted entirely in organic solution , figure 1 . furthermore , since organic - solvent stable nanofiltration ( as opposed to ultrafiltration ) membranes are now available , we proposed that a smaller , discrete synthesis support could be used . during osn , solutes are separated by size exclusion and geometric selection as they pass through a membrane possessing nanometer - scale permeation pathways . solutes that can not pass through the membrane are said to be rejected and remain in the upstream retentate . we further postulated that lpos - osn would provide an excellent platform for monitoring the ongoing oligo synthesis , by means of sampling using a simple liquid draw - off . although in principle it is also possible to monitor chain extension progress with spos , we are unaware of any report of such a procedure . this is most likely due to the difficulty of engineering repeated access to beds of solid support in large diameter , pressurized steel columns ( columns for preparing 12 kg oligo by spos have diameters 50100 cm and pressure ratings of 1520 bar ) . ready access to samples , although providing the opportunity to optimize reactions and to rescue failed steps , is of marginal value on the small synthetic scales regularly produced today . however , in the future , during the preparation of tens of kilograms to tons of oligos , it would be economically unacceptable to risk the complete loss of such large batches of very expensive building blocks without critical quality control . the lpos - osn concept : a ) chain extension reaction ; b ) diafiltration by osn to remove excess reagents ; c ) 5-o deprotection ; d ) diafiltration by osn to remove excess reagents , then repeat cycle to the desired length . building on our earlier experience with membrane - enhanced peptide synthesis ( meps ) in organic solvent ( dmf ) , we initially explored osn separation of mpeg-5000-supported dinucleotides . however , even ,-bis(dinucleotidyl)-peg-10 000 had too low a rejection for practical lpos - osn . therefore we instead adopted monodisperse tris(octagol ) homostar 1 ( a homostar is a star polymer in which all the arms are identical ) as a branched lpos support , see scheme 1 . we hypothesised that this would have three advantages : 1 ) branching should inhibit threading of the supported oligo into the membrane permeation pathways , and therefore increase membrane rejection of the construct ; 2 ) with three oligos growing around one hub , the molecular weight will rise by three nucleotides per cycle , rapidly increasing the overall size , and hence the rejection of the tris(oligonucleotidyl ) support with oligo length ; and 3 ) since the tris(oligonucleotidyl ) support is a discrete species , hplc and mass spectral ( ms ) analyses of real - time synthetic quality should be feasible . we next required an osn membrane compatible with acetonitrile , the solvent in which phosphoramidite couplings are typically conducted . the membrane should also be compatible with feed mixtures containing typical oligo coupling , oxidation / thioylation , capping and 5-o - unblocking reagents . furthermore , the membrane must be able to permeate nucleotide monomer debris after oligo chain extension ; these species are the largest molecular weight debris generated during the synthesis cycle . to meet these challenges we developed a new class of osn membrane ( pbi-17dbx ) , prepared from poly(benzimidazole ) and cross - linked with para - dibromoxylene . pbi-17dbx is very resistant to chemical degradation and gave highly reproducible performance in ch3cn , whilst being open enough to allow species of similar size to nucleotide monomers to permeate . second- and third - generation therapeutic oligos most commonly contain either 2-deoxy or 2-modified nucleosides ( e.g. ; ch3o- , ch3och2ch2o- , f- ) , as well as more complex locked / bridged ribose analogues.[3032 ] for this reason we elected to focus on nucleic acid analogues for our lpos - osn test sequence , instead of native rna . adoption of lpos - osn by other groups would be encouraged if this technology was compatible with commercial building blocks and common protective group combinations . therefore we selected readily available 2-methoxy nucleosides , activated as their 2-cyanoethyl ( cne ) n , n - diisopropylphosphoramidites , carrying 5-o-(4,4-dimethoxytriphenylmethyl ) ( dmtr ) temporary protection and with various amides blocking the exocyclic amino groups of the nucleobases . we also selected the widely used , first - generation phosphorothioate modification as a target for this pilot project because the debris from thioylation reagents is likely to be a more severe test of lpos - osn purification than common oxidants ( e.g. iodine pyridine water , or tert - butyl hydroperoxide ) . all previous lpos studies , except for that of lonnberg , have concerned the synthesis of dna oligos , which are easier to prepare than rna . furthermore , with the exception of bonora s dna 20-mer prepared using numerous precipitations and crystallisations , the largest oligo prepared by lpos to date using iterative synthesis is a dna 10-mer , through h - phosphonate coupling ; a dna phosphorothioate 15-mer has also been reported , but this was constructed using dimer building blocks . as a challenging target for this new lpos - osn technology , we set out to synthesise a 2-methyl rna phosphorothioate 9-mer section of the m23d aso . to assess the performance of the support and phosphoramidite chemistry in this new environment , we planned to undertake global deprotection at both the 5-mer ( four chain extensions ) and 9-mer ( eight chain extensions ) stages . building on our earlier experience with membrane - enhanced peptide synthesis ( meps ) in organic solvent ( dmf ) , we initially explored osn separation of mpeg-5000-supported dinucleotides . however , even ,-bis(dinucleotidyl)-peg-10 000 had too low a rejection for practical lpos - osn . therefore we instead adopted monodisperse tris(octagol ) homostar 1 ( a homostar is a star polymer in which all the arms are identical ) as a branched lpos support , see scheme 1 . we hypothesised that this would have three advantages : 1 ) branching should inhibit threading of the supported oligo into the membrane permeation pathways , and therefore increase membrane rejection of the construct ; 2 ) with three oligos growing around one hub , the molecular weight will rise by three nucleotides per cycle , rapidly increasing the overall size , and hence the rejection of the tris(oligonucleotidyl ) support with oligo length ; and 3 ) since the tris(oligonucleotidyl ) support is a discrete species , hplc and mass spectral ( ms ) analyses of real - time synthetic quality should be feasible . we next required an osn membrane compatible with acetonitrile , the solvent in which phosphoramidite couplings are typically conducted . the membrane should also be compatible with feed mixtures containing typical oligo coupling , oxidation / thioylation , capping and 5-o - unblocking reagents . furthermore , the membrane must be able to permeate nucleotide monomer debris after oligo chain extension ; these species are the largest molecular weight debris generated during the synthesis cycle . to meet these challenges we developed a new class of osn membrane ( pbi-17dbx ) , prepared from poly(benzimidazole ) and cross - linked with para - dibromoxylene . pbi-17dbx is very resistant to chemical degradation and gave highly reproducible performance in ch3cn , whilst being open enough to allow species of similar size to nucleotide monomers to permeate . second- and third - generation therapeutic oligos most commonly contain either 2-deoxy or 2-modified nucleosides ( e.g. ; ch3o- , ch3och2ch2o- , f- ) , as well as more complex locked / bridged ribose analogues.[3032 ] for this reason we elected to focus on nucleic acid analogues for our lpos - osn test sequence , instead of native rna . adoption of lpos - osn by other groups would be encouraged if this technology was compatible with commercial building blocks and common protective group combinations . therefore we selected readily available 2-methoxy nucleosides , activated as their 2-cyanoethyl ( cne ) n , n - diisopropylphosphoramidites , carrying 5-o-(4,4-dimethoxytriphenylmethyl ) ( dmtr ) temporary protection and with various amides blocking the exocyclic amino groups of the nucleobases . we also selected the widely used , first - generation phosphorothioate modification as a target for this pilot project because the debris from thioylation reagents is likely to be a more severe test of lpos - osn purification than common oxidants ( e.g. iodine pyridine water , or tert - butyl hydroperoxide ) . all previous lpos studies , except for that of lonnberg , have concerned the synthesis of dna oligos , which are easier to prepare than rna . furthermore , with the exception of bonora s dna 20-mer prepared using numerous precipitations and crystallisations , the largest oligo prepared by lpos to date using iterative synthesis is a dna 10-mer , through h - phosphonate coupling ; a dna phosphorothioate 15-mer has also been reported , but this was constructed using dimer building blocks . as a challenging target for this new lpos - osn technology , we set out to synthesise a 2-methyl rna phosphorothioate 9-mer section of the m23d aso . to assess the performance of the support and phosphoramidite chemistry in this new environment , we planned to undertake global deprotection at both the 5-mer ( four chain extensions ) and 9-mer ( eight chain extensions ) stages . homostar 1 was first condensed with 4.5 equivalents 5-dmtr-2-methyl-3-succinyl uridine ( 2 , dmtr - mu - suc - oh ) , see scheme 1 . classical activation with 8 equivalents n , n-diisopropyl carbodiimide , in addition to catalytic 4-(dimethylamino)pyridine ( dmap , 0.2 equiv ) in thf , was incomplete with excess uridine succinate being consumed as the acyl urea . to maximize the analytical potential of lpos - osn it is highly desirable to drive loading of the synthesis support to completion to give a homogeneous product , as well as to avoid waste of expensive excess nucleoside on a large scale . thus , condensation of 4.5 equivalents uridine succinate ( 2 ) with homostar 1 was initiated with more reactive 2,6-dichlorobenzoyl chloride ( dcbcl ) and n - methyl imidazole ( nmi ) , after which no peg - terminal hydroxyls remained . the resultant tris - dmtr - ether ( 3 ) was then detritylated with dichloroacetic acid ( dca ) , using pyrrole as a cation scavenger , to provide fully loaded homostar 4 in 80 % yield over the two steps , ready to commence the chain extension cycle . at this stage a small amount of dcb - ester ( 5 ) was separated chromatographically from 4 ; although this contaminant would not affect oligo synthesis at all , in this study it was removed to simplify hplc analysis of chain extension . the loaded synthesis support 4 ( 1.24 g ) was next chain extended with 5-dmtr-2-methyl n - acetylcytidine ( dmtr - mc ) phosphoramidite 6 ( 1.5 equiv per oh ) to mumc homostar 7 under typical conditions , see scheme 1 : ethylthiotetrazole ( ett , 3 equiv per oh ) in ch3cn , 35 min , then phenylacetyl disulfide ( pads ) in pyridine , 30 min , monitoring by hplc ( see scheme 1 and supporting information ) . for this pilot study unusually long times were used for both coupling and thioylation so that the reactions could be sampled and monitored in real time before moving on to the next process . for this reason , the widely used ett ( pka 4.3 , 0.25 m in ch3cn ) was selected as the activator , firstly because it is a compromise that provides higher activity than classical tetrazole ( pka 4.8 ) , but less than 4-nitrophenyl tetrazole ( npt , pka 3.7 ) . secondly , in larger scale couplings with 2-methyl phosphoramidites , 0.5 m ett has proved effective over 515 min reaction times , so 0.25 m ett is commensurate with our longer reactions . furthermore , although npt has been reported to give very high coupling yields with 2-methyl phosphoramidites , we were concerned that the greater acidity of this activator than ett would exacerbate contamination from double coupling during the long reaction times used here . during spos mass transfer for fast reactions , such as phosphoramidite coupling , mass transfer is the rate - limiting step.[9a , 24 ] thus , if the same chemistry is used in both cases , yields in lpos are expected to be higher than in spos . consequently , in an lpos strategy capping should not be as critical as in spos , and this step was omitted simplifying the process during pilot study development . indeed , it is hoped that assay of the chain extension reaction will in future permit identification of otherwise economically catastrophic failed couplings on very large scales , and provide the opportunity to repeat the reaction . however , if capping were implemented before the assay , it would then be impossible to recover a failed coupling . once chain extension and thioylation were complete , the crude mixture was then diluted with ch3cn , and poured directly into the osn apparatus ( see supporting information ) . the rig was pressurized with nitrogen to force solvent and solutes through the pbi-17dbx membrane , a process termed diafiltration . thus , the efficiency of osn can be related to how many retentate system volumes , or diavolumes , must be permeated to achieve a given degree of purification of the retentate . after 12 diavolumes , all small molecules had been removed from the retentate . however , along with the desired dmtr - dinucleotidyl homostar ( 7 ) , most of the building block related species , consisting of a mixture of amidates ( 8) and thioate salts ( 9 ) , were retained ( corresponding to n - op in figure 1 ) . notably , the proportion of phosphoryl species 8 b and 9 b compared to thioyl derivatives 8 a and 9 a ( p = o vs. p = s ) , determined by p nmr spectroscopy of the mixture ( see figure 2 a ) , increased substantially when the amount of pads was reduced from 10 to 3 equivalents per 5-oh . indeed , thioamidate 8 a was almost undetectable when the intermediate phosphite was thioylated with 3 equivalents pads , although amidate 8 b rose to between 15 and 25 % of the p nmr signal integral intensity of product 7 . osn of dinucleotidyl homostar monitored by p nmr spectroscopy : a ) tris(mumc - dmtr ) homostar 7 after permeating 12 diavolumes of ch3cn apart from the homostar , amidates 8 a and 8 b , and thioate salts 9 a and 9 b are present ; b ) tris(mumc - oh ) homostar 10 after permeating 5 diavolumes 1 % dca - ch3cn and 10 diavolumes ch3cn ; c ) expansion of b ) exhibiting the two diastereoisomers of the internucleotide linkage , plus a low level of possible n - deacetylation of cytosine ( see supporting information ) . the crude tris(mumc - dmtr ) homostar 7 was washed from the osn rig and re - dissolved in ch2cl2 . to this were added pyrrole then dca , and after 30 min the detritylation was complete by hplc . unlike in spos , in which the detritylation equilibrium is driven to completion by flushing the dmtr cation away from 5-oh oligo bound to the solid support , in solution phase a scavenger ( here pyrrole ) is necessary to ensure total unblocking . otherwise even small amounts of mono - tritylated homostar would be carried through to the next cycle where ( even after capping ) subsequent detritylation would lead to n1 short - mers . it had been anticipated that at this stage the smaller fragments from the excess building block ( dmtr - pyrrole , and 5-oh amidates 11 and thioates 12 , now corresponding to p and n - oh in figure 1 ) would then permeate , but they did not . suspecting that ion exchange could occur between the protonatable pbi membrane surface and thioate salts 12 , 1 vol % dca was added to the first five diavolumes . after a total of 15 diavolumes had permeated the product tris(mumc - oh ) homostar 10 was then of a similar purity to that achieved by flash chromatography . however , the detritylated amidates ( 11 ) were slower to permeate than the thioate salts 12 , and thioamidate 11 a exhibited substantially greater rejection than amidate 11 b. thus by reducing the excess of pads from 10 to 3 equivalents , when very little or no thioamidate 11 a formed , the purity of dinucleotidyl homostar 10 was maximised ( figure 2 b ) . dmtr - pyrrole was the only contaminant significantly rejected by pbi 17dbx ( see hplc in supporting information ) . thus , although dmtr - pyrrole probably does not interfere with subsequent couplings , this was removed by precipitation of tris(mumc - oh ) homostar 10 in diethyl ether so that an accurate mass recovery could be determined ; apart from dmtr - pyrrole no other species could be detected in the supernatant by h or p nmr spectroscopy . after the first chain extension cycle a moderate 75 % yield of tris(mumc - oh ) homostar 10 was isolated . the only detectable impurity was a low level of cytosine n - deacetylation , identified by lc - ms ( see supporting information ) and believed to be the minor peaks in figure 2 c. despite the need to remove dmtr - pyrrole by precipitation , the above cycle was repeated on homostar 10 , see scheme 2 ; from this point on , all chain extension cycles start with 1.21.4 g tris(5-ho - oligo ) homostar . thus , after chain extension , dmtr-3-mer homostar 13 was partially purified by osn ( 12 diavolumes ) then detritylated , after which all the nucleotidyl debris was separated by osn , and precipitation was again used to remove residual dmtr - pyrrole . this time during the second diafiltration , the first five diavolumes contained only 0.1 % dca to minimize n - deacetylation . the 85 % yield of tris(mumcmc - oh ) homostar 14 was significantly higher than that of dinucleotidyl homostar 10 at the same stage ( see scheme 2 , inset graph ) , indicating that as expected the homostar rejection had risen with oligo length . it should be noted that chain extension cycle yields are calculated assuming 100 % purity of the product homostar . however , as low levels of side - reactions accumulate on the growing oligo , the purity can not be 100 % , so the molecular weight can not be precisely defined , and the yields are more correctly referred to as apparent yields . inset : change in apparent yield of isolated 5-oh tris(oligonucleotidyl ) homostar with oligo length . both the tritylated ( 13 ) and detritylated ( 14 ) tris(trinucleotidyl ) homostars were less soluble in ch3cn than the shorter species 4 , 7 and 10a trend that continued with increasing length . noting that all the oligonucleotidyl homostars ( 1418 and 2027 ) were highly soluble in dmf , all subsequent phosphoramidite couplings were conducted in ch3cn dmf ( ca . 9:1 ) ; this solubility of a branched 2-me - rna 24-mer oligonucleotidyl homostar ( 18 ) may be favorably contrasted with the previously reported poor solubility of 5-oh dna 8-mers in ch3cn . the solvent was also changed during osn from neat ch3cn , in which tris(tetranucleotidyl ) homostar 16 is almost insoluble , to ch3oh ch3cn ( 1:4 or 1:3 v / v ) in which all the oligonucleotidyl homostars are soluble up to at least tris(9-mer ) homostar 27 ( 0.4 wt % 27 during final diafiltration ; the saturation conc . finally , the dca in the second diafiltration was replaced by 1 % pyridinium dichloroacetate ( pydca ) which promoted permeation of thioate salts 12 just as effectively as un - buffered dca . this protocol was used on 4-mer 16 and for all later chain extension cycles , following each reaction by hplc , and assaying the products by p nmr spectroscopy and maldi ms , both before and after detritylation ( see supporting information ) . two further rounds of chain extension were conducted , with the apparent yield continuing to rise ( 82 % 16 , 94 % 18 , see inset graph , scheme 2 ) . although hplc usefully exhibited retention times lengthening in relation to the number of 5-dmtr ethers per homostar , both during chain extension and detritylation , by 5-mers 17 and 18 the peaks were too broad to be of further analytical use ( see supporting information ) , presumably due to the exponentially growing number of diastereoisomers at the p - centers of the oligo backbone . however , p nmr spectroscopy continued to demonstrate acceptably low levels of amidate contamination after each cycle . furthermore , ms confirmed that full - length tris(mumcmcmamu - oh ) homostar 18 was the principal product . by contrast , ms of an oligo conjugated to a polydisperse support would be spread over too many polymeric homologues to provide sufficiently intense peaks for analysis . pentanucleotidyl homostar 18 was deprotected first with diethylamine , then overnight in aqueous ammonia at 55 c . the following day , trituration with ch3cn removed protective group debris to give crude 2-methyl rna phosphorothioate 5-mer 19 . hplc assay of this material ( figure 3 a ) exhibited a moderate purity of 74 % , with both short - mer and long - mer contaminants . although these short - mers could be explained by lack of capping , we believe that they actually derive from chain extension of residual amidate building block 11 after osn . examination of the mass spectra of the 5-oh tris(oligonucleotidyl ) homostars 10 , 14 , 16 and 18 ( from 2-mer to 5-mer ) exhibit no detectable ions corresponding to incomplete chain extension . since our synthesis support possesses three arms , if 1 % incomplete chain extension had occurred , this would afford approximately 3 % homostar having one arm bearing the n1 short - mer . thus , assuming that maldi ionization of full - length oligohomostars and their singly truncated oligohomostar contaminants are similar , mass spectral analysis of homostars supported oligonucleotides should usefully amplify sequence errors to detectable amounts . the long - mers probably arise from two sources : 1 ) relatively long coupling times compared to spos ( 35 min vs. 612 min ) were used here to allow time for hplc confirmation of complete coupling . 2 ) n - deacetylation of cytosine residues ( as observed at the dimer stage ) could provide sites for branching , although we suspect that the switching from dca to pydca in the second diafiltration of each cycle largely suppressed this . hplc of deprotected oligos : a ) crude 5-mer 19 , 75 % purity ; b ) crude 9-mer 28 , 49 % , containing 8-mer 29 , 18 % ; c ) purified 9-mer 28 , 94 % , from lpos - osn ; d ) 9-mer 28 , 95 % , from spos . chain extension was continued from tris(pentanucleotidyl ) homostar 18 with the same protocol for another four cycles , and p nmr spectroscopy and ms now served as the principal methods of product characterization , to obtain 1.36 g of the desired tris(nonanucleotidyl ) homostar 27 . apparent yields of detritylated 6- to 9-mer homostars 21 , 23 , 25 and 27 now plateaued around an average of 95 % ( see inset graph , scheme 2 ) . finally , at the 9-mer stage ms indicated incomplete coupling with approximately 90 % conversion per chain ( 2526 ) . this material was detritylated and deprotected as before to characterize the crude 9-mer by hplc ( figure 3 b ) ; as implied by the homostar ms , hplc indicated 49 % of the desired 9-mer 28 , plus 17 % of the expected 8-mer impurity 29 ( confirmed by lc - ms , see supporting information ) . the 9-mer was then fractionated for confirmatory analysis through two ion - exchange columns to 94 % purity ( figure 3 c ) , and desalted providing a 16 % ( 29 mg ) yield of pure 9-mer 28 from 5-oh homostar 27 . this material displayed identical hplc ( figure 3 d ) and ms to 9-mer 28 prepared by spos . in this report we have demonstrated for the first time a new liquid - phase synthesis and separation paradigm for oligonucleotides : liquid - phase oligonucleotide synthesis / organic - solvent nanofiltration , lpos - osn . this promising technology has yet to equal the speed and purity of spos , requiring around two days per chain extension cycle with the limited area available from current laboratory - scale flat membrane cells . however , the fact that we were able to perform eight chain extension and detritylation cycles , with intermediate purifications , all in the liquid phase demonstrates that it has high potential . compared to competing precipitation strategies , lpos - osn is more amenable to industrial exploitation because liquid - phase handling is intrinsically scalable . lpos - osn also has the major advantage over spos that it is straightforward to sample and monitor every step of the process . apart from p nmr spectroscopy and hplc , the choice of a monodisperse support also allowed characterization of the growing oligonucleotidyl homostars by ms . from the above experience , several modifications can be suggested to improve future protocols : shortening the coupling time , and analysing only after thioylation , will reduce long - mer formation . minimising 5-unblocking time , and therefore acid exposure , will minimise cytosine deacetylation , and again possible long - mer formation . biasing building block debris away from amidates ( 8/11 ) into thioates ( 9/12 ) that are more easily removed by diafiltration would suppress short - mer formation . the overall yield of fully protected 5-oh tris(oligonucleotidyl ) homostar 27 from loaded uridine homostar 4 is only about 39 % , mainly due to poor recovery from the early cycles of osn ; the first three couplings ( 44-mer 14 ) give a cumulative yield of only 52 % , but the next five couplings ( 4-mer 189-mer 27 ) have a combined yield of about 76 % . this will be much improved using our recently developed 2-stage diafiltration ; we would expect early stage recovery to be > 95 % and from 5-mer onwards > 99 % . as with spos , we have recently demonstrated that an additional stage of diafiltration with a low - molecular - weight cut - off membrane can be used to recycle the permeate solvent , greatly reducing the potential cost on an industrial scale . as the scale of lpos - osn increases , an alternative analysis to direct hplc of the retentate will be required ; we believe that rapid ammonia - methylamine ( ama ) global deprotection , followed by hplc of the crude unblocked oligo will provide a suitable method to assay for complete chain extension . finally , identifying a membrane that permeates dmtr derivatives , or using a smaller 5-protecting group , such as the methoxyisopropylidene acetal , would make the process even more efficient . chemical shifts in ppm are referenced with respect to residual solvent signals : h ( chcl3 ) 7.25 ppm , h ( chd2od ) 3.31 ppm , h ( cd3cochd2 ) 2.05 ppm ; c ( cdcl3 ) 77.50 ppm , c ( cd3od ) 49.15 ppm , c ( cd3cochd2 ) 29.92 ppm . the splitting patterns for h nmr spectra are denoted as follows ; s ( singlet ) , d ( doublet ) , t ( triplet ) , q ( quartet ) , quin ( quintet ) , m ( multiplet ) , br ( broad ) and combinations thereof . c nmr assignments ( c , ch , ch2 and ch3 ) and h nmr assignments were established with the aid of dept-135 , hsqc and cosy experiments . molecular fragments not abbreviated in main text are denoted as follows : u , uracil ; c , cytosine ; ri , ribose ; suc , succinate ; hub , c6h3(ch2or)3 . nmr spectrscopy of small dmtr derivatives was conducted in the presence of either a small amount of et3n or of pyridine . mass spectra were recorded on micromass maldi micro mx , or micromass lct premier ( esi ) mass spectrometers . other reagents were purchased from sigma - aldrich ltd . and used as supplied , except where specified . dichloromethane , acetonitrile , thf and dmf were dried and stored over baked 4 molecular sieves . triethylamine , diethyl ether , methanol , isopropanol and n - methyl imidazole were used as supplied . flash chromatography was conducted in a 9 cm diameter , porosity 3 glass sinter funnel : geduran ( si 60 ) from merck was used for normal phase columns , and merck silanised silica for reverse phase columns . thin - layer chromatography was carried out using merck silica gel 60 f254 aluminium - backed plates ; compounds were visualised using uv light or kmno4 stain . solid phase oligonucleotide synthesis ( spos ) was carried out on a ge akta oligopilot 10 , using preloaded 2ome u primer support 200 and manufacturer s standard protocols on a 30 mol scale . cleavage from solid support and deprotection of nucleobases was carried out in 0.88 aqueous ammonia at 55 c for 16 h. tris-1,3,5-{-[2-o - methyl-5-o-(4,4-dimethoxytriphenylmethyl)uridine-3-o - succinyloxy]octa(ethylene glycol)--oxymethyl}benzene ( 3 ) : compound 1 ( 1.125 g , 1.00 mmol ) was co - evaporated from ch3cn ( 315 ml ) , re - dissolved in ch2cl2 ( 7 ml ) and a dropping funnel was fitted to the flask . 2-o - methyl-3-o - succinyl-5-o-(4,4-dimethoxytriphenylmethyl)uridine , triethylammonium salt ( 2 , 3.047 g , 4.00 mmol ) was co - evaporated from thf ( 350 ml ) , then re - dissolved in ch2cl2 ( 14 ml ) and n - methylimidazole ( 0.63 ml , 7.9 mmol ) then 2,6-dichlorobenzoyl chloride ( dcbcl , 0.54 ml , 3.77 mmol ) were added . after stirring for 20 min , the activated succinate solution was transferred to the dropping funnel , rinsing out the flask with further ch2cl2 ( 5 ml ) , and this solution slowly added to homostar 1 over 45 min . after stirring over night the aqueous layer was back - extracted with small portions of ch2cl2 ( 50 ml3 ) , the combined organic layers dried over na2so4 , and the solvent stripped off in vacuo . the residual foam may be purified chromatographically by fractionation through a column of silanised silica , eluting with a gradient of ch3cn water ( 3:7 to 8:2 v / v containing 0.5 % sat . chcl3 1:9 + trace et3n ) 0.56 ; h nmr ( 400 mhz , cdcl3 ) : =7.88 ( d , j=8.2 hz , 3 h ; u ch ) , 7.387.36 ( m , 6 h ; dmtr ch ) , 7.32 ( t , j=7.5 hz , 6 h ; dmtr ch ) , 7.297.24 ( m , 18 h ; hub ch + dmtr ch ) , 6.86 ( d , j=8.9 hz , 12 h ; dmtr ch ) , 6.04 ( d , j=3.7 hz , 3 h ; 1-ch ) , 5.33 ( d , j=8.2 hz , 3 h ; u ch ) , 5.29 ( t , j=5.6 hz , 3 h ; 3-ch ) , 4.55 ( s , 6 h ; hub - ch2o ) , 4.264.24 ( m , 9 h ; 4-ch + suc - och2 ) , 4.10 ( dd , j=5.1 , 3.7 hz , 3 h ; 2-ch ) , 3.81 ( s , 18 h ; och3 ) , 3.713.58 ( m , 93 h ; ch2o + 5-chh ) , 3.483.44 ( m , 12 h ; 2-och3 + 5-chh ) , 2.732.66 ppm ( m , 12 h ; suc ch2 ) ; c nmr ( 101 mhz , cdcl3 ) : =171.98 ( 3 c ; suc c = o ) , 171.53 ( 3 c ; suc c = o ) , 163.29 ( 3 c ; u c ) , 158.73 ( 6 c ; dmtr c ) , 150.30 ( 3 c ; u c ) , 144.13 ( 3 c ; dmtr c ) , 139.78 ( 3 c ; u 6-ch ) , 138.57 ( 3 c ; hub c ) , 134.98 ( 3 c ; dmtr c ) , 134.86 ( 3 c ; dmtr c ) , 130.17 ( 6 c ; dmtr ch ) , 130.09 ( 6 c ; dmtr ch ) , 128.08 ( 12 c ; dmtr ch ) , 127.24 ( 3 c ; dmtr ch ) , 126.32 ( 3 c ; hub ch ) , 113.33 ( 12 c ; dmtr ch ) , 102.51 ( 3 c ; u 5-ch ) , 87.39 ( 3 c ; dmtr c ) , 87.13 ( 3 c ; 1-ch ) , 82.18 ( 3 c ; ri ch ) , 81.03 ( 3 c ; ri ch ) , 73.06 ( 3 c ; hub - ch2o ) , 70.54 ( 39 c ; ch2o ) , 70.36 ( 3 c ; ri ch ) , 69.51 ( 3 c ; ch2o ) , 69.02 ( 3 c ; ch2o ) , 63.94 ( 3 c ; ch2o ) , 61.56 ( 3 c ; 5-ch2 ) , 59.03 ( 3 c ; 2-och3 ) , 55.26 ( 6 c ; dmtr och3 ) , 28.88 ( 3 c ; suc ch2 ) , 28.83 ppm ( 3 c ; suc ch2 ) ; ms ( maldi - tof+ ) : m / z calcd for [ c162h210n6nao57 ] : 3175.4 ; found : 3176 [ 3+na ] . tris-1,3,5-[-(2-o - methyluridine-3-o - succinyloxy)octa(ethylene glycol)--oxymethyl]benzene ( 4 ) : the crude tris(dmtr - mu ) homostar 3 ( 4.438 g from the above procedure ) was dissolved in ch2cl2 ( 40 ml ) and pyrrole ( 1.39 ml , 19.6 mmol ) was added . dichloroacetic acid was then added in aliquots ( 0.40 ml , 4.8 mmol ) until a strong orange colour remained , and then dissipated over 20 min ; four aliquots were required to overwhelm buffering of the crude dmtr ether and tlc confirmed complete unblocking of intermediate 3 . the aqueous layer was back - extracted with ch2cl2 ( 50 ml4 ) , organic layers combined , dried over na2so4 and the solvent stripped off under reduced pressure . the residue was fractionated through a column of silica gel ( 180 ml ) in a large sinter funnel , eluting with a gradient of ch3oh bands containing dcb - ester 5 ( 409 mg ) , mixed with some product , and compound 4 ( 1.644 g , 80 % ) were isolated as colourless gums . rf ( ch3oh chcl3 1:9 ) 4 0.35 ; 5 0.51 ; h nmr ( 400 mhz , cdcl3 ) : =9.53 ( br d , j=2.3 , 3 h ; u 4-nh ) , 7.85 ( d , j=8.1 hz , 3 h ; u 6-ch ) , 7.23 ( s , 3 h ; hub ch ) , 5.85 ( d , j=5.1 hz , 3 h ; 1-ch ) , 5.76 ( dd , j=8.0 , 2.0 hz , 3 h ; u 5-ch ) , 5.32 ( t , j=4.6 hz , 3 h ; 3-ch ) 4.55 ( s , 6 h ; hub - ch2o ) , 4.26 ( dt , j=5.4 , 2.0 hz , 6 h ; suc - och2 ) , 4.224.19 ( m , 6 h ; 4-ch + 2-ch ) , 3.94 ( br d , j=12.0 hz , 3 h ; 5-chh ) , 3.79 ( br dd , j=12.3 , 4.0 hz , 3 h ; 5-chh ) , 3.723.61 ( m , 90 h ; ch2o ) , 3.43 ( s , 9 h ; 2-och3 ) , 2.762.68 ppm ( m , 12 h ; suc ch2ch2 ) ; c nmr ( 101 mhz , cdcl3 ) : =172.08 ( 3 c ; suc c = o ) , 171.68 ( 3 c ; suc c = o ) , 163.92 ( 3 c ; u c ) , 150.75 ( 3 c ; u c ) , 141.22 ( 3 c ; u 6-ch ) , 138.51 ( 3 c ; hub c ) , 126.29 ( 3 c ; hub ch ) , 102.64 ( 3 c ; u 5-ch ) , 88.69 ( 3 c ; 1-ch ) , 83.18 ( 3 c ; 2/4-ch ) , 81.43 ( 3 c ; 2/4-ch ) , 73.00 ( 3 c ; hub - ch2o ) , 70.96 ( 3 c ; 3-ch ) , 70.48 ( 39 c ; ch2o ) , 69.46 ( 3 c ; ch2o ) , 68.96 ( 3 c ; ch2o ) , 63.90 ( 3 c ; suco - ch2 ) , 61.15 ( 3 c ; 5-ch2 ) , 58.90 ( 3 c ; 2-och3 ) , 28.95 ppm ( 6 c ; suc ch2ch2 ) ; ms ( maldi - tof+ ) : m / z calcd for [ c99h156n6nao51 ] : 2268.97 ; found : 2269.0 [ 4+na ] . tris-1,3,5-{-(2-o - me-5-o-{[2-o - me-5-o-(dmtr)-4-n - acetylcytosin-3-yl](2-cyanoethyloxy)thiophosphoryl}uridinyl-3-o - succinyloxy)octa(ethylene glycol)--oxymethyl}benzene , tris(mupsmc - odmtr ) homostar ( 7 ) : tris(2-methyluridine ) homostar 4 ( 422 mg , 0.188 mmol ) and compound 6 ( 678 mg , 0.846 mmol , 4.5 equiv ) were co - evaporated from ch3cn ( 310 ml ) in vacuo . to the residue was added 0.25 m ett in ch3cn ( 6.77 ml , 1.69 mmol , 9 equiv ) , and after 40 min , pads ( 1.71 g , 5.65 mmol , 30 equiv ) and pyridine ( 6.8 ml ) were added . after a further 60 min the solvent was stripped off under reduced pressure and the residue fractionated through a column of silanised silica , eluting with a gradient of water the resultant emulsion was extracted with ch2cl2 ( 100 ml4 ) , the organic layer dried over na2so4 , and then evaporated to dryness . the appropriate fractions were extracted as before to afford compound 7 ( 538 mg , 64 % ) . rf ( ch3oh ch2cl2 1:9 ) 0.41 ; intermediate tris(phosphite triester ) 0.54 ; h nmr ( 400 mhz , d6-acetone ) : =10.38 ( br s , 1.5 h ; nh ) , 10.25 ( s , 1.5 h ; nh ) , 10.19 ( s , 1.5 h ; nh ) , 10.15 ( br s , 1.5 h ; nh ) , 8.49 ( d , j=7.4 hz , 3 h ; c ch ) , 7.75 ( d , j=8.2 hz , 1.5 h ; u ch ) , 7.64 ( d , j=8.2 hz , 1.5 h ; u ch ) , 7.547.51 ( m , 6 h ; dmtr ch ) , 7.427.36 ( m , 18 h ; dmtr ch ) , 7.337.30 ( m , 3 h ; dmtr ch ) , 7.28 ( s , 3 h ; hub ch ) , 7.13 ( d , j=7.2 hz , 1.5 h ; c ch ) , 7.12 ( d , j=7.4 hz , 1.5 h ; c ch ) , 6.976.93 ( m , 12 h ; dmtr ch ) , 6.076.04 ( m , 3 h ; 1-ch ) , 5.995.96 ( m , 3 h ; 1-ch ) , 5.71 ( d , j=7.7 hz , 1.5 h ; u ch ) , 5.70 ( d , j=7.8 hz , 1.5 h ; u ch ) , 5.375.26 ( m , 2 h ; 23-ch ) , 4.56 ( s , 6 h ; hub - ch2o ) , 4.534.37 ( m , 9 h ; u 4-ch + c 4-ch + pochhch2cn ) , 4.364.15 ( m , 21 h ; u 2-ch + c 2-ch + 5-ch2 + pochhch2cn + suc - och2 ) , 3.84 ( s , 9 h ; dmtr och3 ) , 3.83 ( s , 9 h ; dmtr och3 ) , 3.693.53 ( m , 105 h ; ch2o + 5-ch2 + 2-och3 ) , 3.41 ( s , 4.5 h ; 2-och3 ) , 3.40 ( s , 4.5 h ; 2-och3 ) , 3.01 ( t , j=7.2 hz , 3 h ; ch2cn ) , 2.92.87 ( m , 3 h ; ch2cn ) , 2.762.73 ( m , 6 h ; suc ch2 ) , 2.702.67 ( m , 6 h ; suc ch2 ) , 2.25 ( s , 4.5 h ; ac ch3 ) , 2.24 ppm ( s , 4.5 h ; ac ch3 ) ; c nmr ( 101 mhz , d6-acetone ) : =171.89 ( 3 c ; c = o ) , 171.45 ( 3 c ; c = o ) , 170.66 ( 3 c ; c = o ) , 163.09 ( 3 c ; u / c c ) , 162.68 ( 1.5 c ; u / c c ) , 162.64 ( 1.5 c ; u / c c ) , 158.92 ( 6 c ; dmtr c ) , 154.71 ( 1.5 c ; c c ) , 154.60 ( 1.5 c ; c c ) , 150.47 ( 1.5 c ; u c ) , 150.44 ( 1.5 c ; u c ) , 144.44 ( 1.5 c ; c ch ) , 144.30 ( 1.5 c ; c ch ) , 144.27 ( 1.5 c ; dmtr c ) , 140.02 ( 3 c ; u 6-ch ) , 139.00 ( 3 c ; hub c ) , 135.43 ( 1.5 c ; dmtr c ) , 135.33 ( 1.5 c ; dmtr c ) , 130.27 ( 6 c ; dmtr ch ) , 130.18 ( 6 c ; dmtr ch ) , 128.48 ( 3 c ; dmtr ch ) , 128.40 ( 3 c ; dmtr ch ) , 128.02 ( 6 c ; dmtr ch ) , 127.18 ( 3 c ; dmtr ch ) , 125.70 ( 3 c ; hub ch ) , 117.59 ( 1.5 c ; cn ) , 117.31 ( 1.5 c ; cn ) , 113.27 ( 12 c ; dmtr ch ) , 102.65 ( 1.5 c ; u 5-ch ) , 102.52 ( 1.5 c ; u 5-ch ) , 96.15 ( 3 c ; c ch ) , 88.80 ( 1.5 c ; 1-ch ) , 88.51 ( 1.5 c ; 1-ch ) , 88.04 ( 1.5 c ; 1-ch ) , 87.99 ( 1.5 c ; 1-ch ) , 87.08 ( 1.5 c ; dmtr c ) , 87.06 ( 1.5 c ; dmtr c ) , 82.09 ( 1.5 c ; ri ch ) , 81.78 ( 1.5 c ; ri ch ) , 80.91 ( 3 c ; ri ch ) , 80.79 ( 3 c ; ri ch ) , 80.1580.00 ( m , 3 c ; ri ch ) , 73.51 ( 1.5 c ; ri ch ) , 73.28 ( 1.5 c ; ri ch ) , 72.54 ( 3 c ; hub - ch2o ) , 70.58 ( 3 c ; ri ch ) , 70.35 ( 39 c ; ch2 ) , 69.64 ( 3 c ; ch2o ) , 68.72 ( 3 c ; ch2o ) , 67.4267.10 ( 3 c ; u 5-ch2 ) , 63.7563.48 ( m , 6 c ; ch2o + poch2ch2cn ) , 60.57 ( 1.5 c ; c 5-ch2 ) , 60.49 ( 1.5 c ; c 5-ch2 ) , 58.40 ( 1.5 c ; 2-och3 ) , 58.33 ( 1.5 c ; 2-och3 ) , 58.13 ( 1.5 c ; 2-och3 ) , 57.83 ( 1.5 c ; 2-och3 ) , 54.81 ( 3 c ; dmtr och3 ) , 54.79 ( 3 c ; dmtr och3 ) , 28.67 ( 6 c ; suc ch2 ) , 24.13 ( 1.5 c ; ac ch3 ) , 24.05 ( 1.5 c ; ac ch3 ) , 19.0718.90 ppm ( m , 3 c ; ch2cn ) ; p nmr ( 162 mhz , d6-acetone ) =67.33 ( 0.39 p ) , 67.11 ppm ( 0.61 p ) ; ms ( maldi - tof+ ) : m / z calcd for [ c207h269n18nao79p3s3 ] : 4484.59 ; found : 4484.1 [ 7+na+h2o ] . tris(mupsmc - oh ) homostar ( 10 ) : tris(dmtro-2-mer ) homostar 7 ( 192 mg , 43 mol ) was placed in ch2cl2 ( 1 ml ) , to which pyrrole ( 35 l ) then dca ( 35 l ) were added . after 60 min the reaction was complete by tlc and 1 m triethylammonium bicarbonate ( teab , 0.5 ml ) was added , followed by sufficient ch3cn to give a clear solution . the solution was concentrated at reduced pressure until all ch2cl2 had evaporated , then silanised silica ( 10 ml ) was added to the remaining solution followed by the slow addition of water ( 90 ml ) with gentle swirling , plus 1 m teab ( 1 ml ) . the silica was collected in a glass sinter funnel , and the pad was washed with water ( 50 ml ) , then ch3cn water ( 1:3 v / v , 160 ml plus 1.5 ml teab ) and the filtrate was discarded . next the silica was washed with ch3cn ( 150 ml ) and the filtrate evaporated to dryness . the residue ( 156 mg ) was taken up in chcl3 ( 20 ml ) and to the swirled solution was added normal phase silica ( 10 ml ) . the silica was collected in a glass sinter funnel , the pad was washed with further chcl3 ( 130 ml ) and the filtrate discarded . finally the silica was washed with ch3oh chcl3 ( 1:9 v / v , 150 ml ) and the filtrate evaporated to dryness to afford compound 10 ( 118 mg , 77 % ) . chcl3 1:9 ) 0.19 ; h nmr ( 500 mhz , cdcl3cd3od 2:1 v / v ) : =8.42 ( d , j=7.5 hz , 1.5 h ; c ch ) , 8.41 ( d , j=7.6 hz , 1.5 h ; c ch ) , 7.63 ( d , j=8.1 hz , 1.5 h ; u ch ) , 7.402 ( d , j=7.5 hz , 1.5 h ; c ch ) , 7.397 ( d , j=7.6 hz , 1.5 h ; c ch ) , 7.21 ( s , 3 h ; hub ch ) , 5.98 ( d , j=3.7 hz , 3 h ; ri 1-ch ) , 5.91 ( d , j=4.7 hz , 1.5 h ; ri 1-ch ) , 5.90 ( d , j=4.9 hz , 1.5 h ; ri 1-ch ) , 5.75 ( d , j=8.1 hz , 1.5 h ; u ch ) , 5.72 ( d , j=8.1 hz , 1.5 h ; u ch ) , 5.21 ( t , j=5.2 hz , 3 h ; u 3-ch ) 5.034.97 ( m , 3 h ; c 3-ch ) , 4.52 ( s , 6 h ; hub - ch2o ) , 4.454.23 ( m , 18 h ; poch2ch2cn + u 5- ch2 + c 4-ch + u 4-ch ) , 4.214.19 ( m , 6 h ; suc - och2 ) , 4.15 ( br t , j=4.3 hz , 1.5 h ; u 2-ch ) , 4.14 ( br t , j=4.4 hz , 1.5 h ; u 2-ch ) , 4.04 ( dd , j=4.9 , 1.3 hz , 1.5 h ; c 2-ch ) , 4.03 ( dd , j=5.3 , 1.3 hz , 1.5 h ; c 2-ch ) , 3.953.91 ( m , 3 h ; 5-chh ) , 3.783.73 ( m , 3 h ; 5-chh ) , 3.67 ( t , j=4.8 hz , 6 h ; ch2o ) , 3.653.59 ( m , 84 h ; ch2o ) , 3.53 ( s , 4.5 h ; 2-och3 ) , 3.50 ( s , 4.5 h ; 2-och3 ) , 3.39 ( s , 9 h ; 2-och3 ) , 2.852.82 ( m , 6 h ; ch2cn ) , 2.722.69 ( m , 6 h ; suc ch2ch2 ) , 2.672.64 ( m , 6 h ; suc ch2ch2 ) , 2.17 ppm ( s , 9 h ; ac ch3 ) ; c nmr ( 126 mhz , cdcl3cd3od 2:1 v / v ) : =172.40 ( 3 c ; c = o ) , 171.75 ( 1.5 c ; c = o ) , 171.71 ( 1.5 c ; c = o ) , 171.56 ( 3 c ; c = o ) , 164.15 ( 3 c ; u / c c ) , 162.83 ( 3 c ; u / c c ) , 156.09 ( 3 c ; c c ) , 150.52 ( 3 c ; u c ) , 145.39 ( 3 c ; c ch ) , 140.21 ( 3 c ; u 6-ch ) , 138.47 ( 3 c ; hub c ) , 126.40 ( 3 c ; hub ch ) , 117.03 ( 1.5 c ; cn ) , 116.96 ( 1.5 c ; cn ) , 102.79 ( 1.5 c ; u 5-ch ) , 102.73 ( 1.5 c ; u 5-ch ) , 97.40 ( 1.5 c ; c ch ) , 97.34 ( 1.5 c ; c ch ) , 89.22 ( 1.5 c ; c 1-ch ) , 89.10 ( 1.5 c ; c 1-ch ) , 88.08 ( 1.5 c ; u 1-ch ) , 88.02 ( 1.5 c ; u 1-ch ) , 83.2983.19 ( m , 3 c ; u 4-ch ) , 82.17 ( 3 c ; c 2-ch ) , 81.09 ( 1.5 c ; u 2-ch ) , 81.04 ( 1.5 c ; u 2-ch ) , 80.0879.97 ( m , 3 c ; c 4-ch ) , 74.5074.42 ( m , 3 c ; c 3-ch ) , 72.93 ( 3 c ; hub - ch2o ) , 70.35 ( 39 c ; ch2o ) , 70.18 ( 3 c ; u 3-ch ) , 69.43 ( 3 c ; ch2o ) , 68.87 ( 3 c ; ch2o ) , 66.70 ( br , 3 c ; u 5-ch2 ) , 63.91 ( 3 c ; sucoch2 ) , 63.1463.07 ( m , 3 c ; poch2ch2cn ) , 59.65 ( 3 c ; c 5-ch2 ) , 58.81 ( 3 c ; 2-och3 ) , 58.57 ( 1.5 c ; 2-och3 ) , 58.54 ( 1.5 c ; 2-och3 ) , 28.76 ( 6 c ; suc ch2 ) , 24.24 ( 3 c ; ac ch3 ) , 19.2219.10 ppm ( m , 3 c , ch2cn ) ; p nmr ( 202 mhz , cdcl3cd3od 2:1 v / v ) : =67.81 ( 0.44 p ) , 67.59 ppm ( 0.56 p ) ; ms ( maldi - tof+ ) : m / z calcd for [ c144h214n18nao72p3s3]=3537.20 ; found : 3538.3 [ 10+h ] . tris(mupsmcpsmcpsmapsmupsmu - oh ) homostar ( 21)typical chain extension cycle : tris(ho-5-mer ) homostar 18 ( 1.286 g , 0.171 mmol ) was dissolved in dmf ( 4 ml ) to which was added ch3cn ( 20 ml ) and the solution evaporated in vacuo ; this was repeated with dmf ( 2 ml ) plus ch3cn ( 20 ml ) , and finally with neat ch3cn ( 20 ml ) . phosphoramidite 6 ( 585 mg , 0.769 mmol , 4.5 equiv ) was then added to the residue followed by 0.25 m ett in ch3cn ( 6.2 ml , 1.55 mmol , 9 equiv ) . after 35 min pads ( 465 mg , 1.55 mmol , 9 equiv ) and pyridine ( 6.2 ml ) were added , and after a further 35 min the reaction was diluted into ch3oh ch3cn ( 3:17 v / v ) and poured into the osn rig . once 12 diavolumes had permeated , the retentate was evaporated to give crude 5-dmtr homostar 20 ( 1.779 g ) as a brown glass . partially purified tris(dmtro-6-mer ) homostar 20 ( 1.708 g ) was placed in ch2cl2 ( 28 ml ) , to which was added pyrrole ( 0.48 ml ) then dca ( 0.28 ml ) . after 45 min the mixture was diluted with ch3cn ( 100 ml ) and the liquid concentrated until all the ch2cl2 had evaporated . to this solution was then added ch3oh ( 20 ml ) , the solution was diluted with further ch3oh ch3cn ( 3:17 v / v ) containing pyridinium.dca ( 0.5 vol % ) and 5 diavolumes were permeated . the flux was observed to drop significantly , so this was followed by 10 diavolumes ch3oh ch3cn ( 1:4 v / v ) when the flux improved . the retentate was evaporated to dryness , and the residual glass was re - dissolved in ch2cl2ch3oh ( 10 ml ) . the solution was added dropwise to briskly stirred diethyl ether ( 300 ml ) , and the precipitate collected to afford tris(ho-6-mer ) homostar 21 ( 1.394 g , 98 % ) as a brown powder . p nmr ( 202 mhz , cdcl3-cd3od 2:1 v / v ) : =67.867.1 ppm ( m , 15 p ) ; ms ( maldi - tof+ ) : m / z calcd for [ c330h429n66nao153p15s15 ] : 8734.0 ; found : 8728 [ 21+na ] . chemical shifts in ppm are referenced with respect to residual solvent signals : h ( chcl3 ) 7.25 ppm , h ( chd2od ) 3.31 ppm , h ( cd3cochd2 ) 2.05 ppm ; c ( cdcl3 ) 77.50 ppm , c ( cd3od ) 49.15 ppm , c ( cd3cochd2 ) 29.92 ppm . the splitting patterns for h nmr spectra are denoted as follows ; s ( singlet ) , d ( doublet ) , t ( triplet ) , q ( quartet ) , quin ( quintet ) , m ( multiplet ) , br ( broad ) and combinations thereof . c nmr assignments ( c , ch , ch2 and ch3 ) and h nmr assignments were established with the aid of dept-135 , hsqc and cosy experiments . molecular fragments not abbreviated in main text are denoted as follows : u , uracil ; c , cytosine ; ri , ribose ; suc , succinate ; hub , c6h3(ch2or)3 . nmr spectrscopy of small dmtr derivatives was conducted in the presence of either a small amount of et3n or of pyridine . mass spectra were recorded on micromass maldi micro mx , or micromass lct premier ( esi ) mass spectrometers . other reagents were purchased from sigma - aldrich ltd . and used as supplied , except where specified . dichloromethane , acetonitrile , thf and dmf were dried and stored over baked 4 molecular sieves . triethylamine , diethyl ether , methanol , isopropanol and n - methyl imidazole were used as supplied . flash chromatography was conducted in a 9 cm diameter , porosity 3 glass sinter funnel : geduran ( si 60 ) from merck was used for normal phase columns , and merck silanised silica for reverse phase columns . thin - layer chromatography was carried out using merck silica gel 60 f254 aluminium - backed plates ; compounds were visualised using uv light or kmno4 stain . solid phase oligonucleotide synthesis ( spos ) was carried out on a ge akta oligopilot 10 , using preloaded 2ome u primer support 200 and manufacturer s standard protocols on a 30 mol scale . cleavage from solid support and deprotection of nucleobases was carried out in 0.88 aqueous ammonia at 55 c for 16 h. tris-1,3,5-{-[2-o - methyl-5-o-(4,4-dimethoxytriphenylmethyl)uridine-3-o - succinyloxy]octa(ethylene glycol)--oxymethyl}benzene ( 3 ) : compound 1 ( 1.125 g , 1.00 mmol ) was co - evaporated from ch3cn ( 315 ml ) , re - dissolved in ch2cl2 ( 7 ml ) and a dropping funnel was fitted to the flask . 2-o - methyl-3-o - succinyl-5-o-(4,4-dimethoxytriphenylmethyl)uridine , triethylammonium salt ( 2 , 3.047 g , 4.00 mmol ) was co - evaporated from thf ( 350 ml ) , then re - dissolved in ch2cl2 ( 14 ml ) and n - methylimidazole ( 0.63 ml , 7.9 mmol ) then 2,6-dichlorobenzoyl chloride ( dcbcl , 0.54 ml , 3.77 mmol ) were added . after stirring for 20 min , the activated succinate solution was transferred to the dropping funnel , rinsing out the flask with further ch2cl2 ( 5 ml ) , and this solution slowly added to homostar 1 over 45 min . after stirring over night the aqueous layer was back - extracted with small portions of ch2cl2 ( 50 ml3 ) , the combined organic layers dried over na2so4 , and the solvent stripped off in vacuo . the residual foam may be purified chromatographically by fractionation through a column of silanised silica , eluting with a gradient of ch3cn water ( 3:7 to 8:2 v / v containing 0.5 % sat . chcl3 1:9 + trace et3n ) 0.56 ; h nmr ( 400 mhz , cdcl3 ) : =7.88 ( d , j=8.2 hz , 3 h ; u ch ) , 7.387.36 ( m , 6 h ; dmtr ch ) , 7.32 ( t , j=7.5 hz , 6 h ; dmtr ch ) , 7.297.24 ( m , 18 h ; hub ch + dmtr ch ) , 6.86 ( d , j=8.9 hz , 12 h ; dmtr ch ) , 6.04 ( d , j=3.7 hz , 3 h ; 1-ch ) , 5.33 ( d , j=8.2 hz , 3 h ; u ch ) , 5.29 ( t , j=5.6 hz , 3 h ; 3-ch ) , 4.55 ( s , 6 h ; hub - ch2o ) , 4.264.24 ( m , 9 h ; 4-ch + suc - och2 ) , 4.10 ( dd , j=5.1 , 3.7 hz , 3 h ; 2-ch ) , 3.81 ( s , 18 h ; och3 ) , 3.713.58 ( m , 93 h ; ch2o + 5-chh ) , 3.483.44 ( m , 12 h ; 2-och3 + 5-chh ) , 2.732.66 ppm ( m , 12 h ; suc ch2 ) ; c nmr ( 101 mhz , cdcl3 ) : =171.98 ( 3 c ; suc c = o ) , 171.53 ( 3 c ; suc c = o ) , 163.29 ( 3 c ; u c ) , 139.78 ( 3 c ; u 6-ch ) , 138.57 ( 3 c ; hub c ) , 134.98 ( 3 c ; dmtr c ) , 134.86 ( 3 c ; dmtr c ) , 130.17 ( 6 c ; dmtr ch ) , 130.09 ( 6 c ; dmtr ch ) , 128.08 ( 12 c ; dmtr ch ) , 127.24 ( 3 c ; dmtr ch ) , 126.32 ( 3 c ; hub ch ) , 113.33 ( 12 c ; dmtr ch ) , 102.51 ( 3 c ; u 5-ch ) , 87.39 ( 3 c ; dmtr c ) , 87.13 ( 3 c ; 1-ch ) , 82.18 ( 3 c ; ri ch ) , 81.03 ( 3 c ; ri ch ) , 73.06 ( 3 c ; hub - ch2o ) , 70.54 ( 39 c ; ch2o ) , 70.36 ( 3 c ; ri ch ) , 69.51 ( 3 c ; ch2o ) , 69.02 ( 3 c ; ch2o ) , 63.94 ( 3 c ; ch2o ) , 61.56 ( 3 c ; 5-ch2 ) , 59.03 ( 3 c ; 2-och3 ) , 55.26 ( 6 c ; dmtr och3 ) , 28.88 ( 3 c ; suc ch2 ) , 28.83 ppm ( 3 c ; suc ch2 ) ; ms ( maldi - tof+ ) : m / z calcd for [ c162h210n6nao57 ] : 3175.4 ; found : 3176 [ 3+na ] . tris-1,3,5-[-(2-o - methyluridine-3-o - succinyloxy)octa(ethylene glycol)--oxymethyl]benzene ( 4 ) : the crude tris(dmtr - mu ) homostar 3 ( 4.438 g from the above procedure ) was dissolved in ch2cl2 ( 40 ml ) and pyrrole ( 1.39 ml , 19.6 mmol ) was added . dichloroacetic acid was then added in aliquots ( 0.40 ml , 4.8 mmol ) until a strong orange colour remained , and then dissipated over 20 min ; four aliquots were required to overwhelm buffering of the crude dmtr ether and tlc confirmed complete unblocking of intermediate 3 . the aqueous layer was back - extracted with ch2cl2 ( 50 ml4 ) , organic layers combined , dried over na2so4 and the solvent stripped off under reduced pressure . the residue was fractionated through a column of silica gel ( 180 ml ) in a large sinter funnel , eluting with a gradient of ch3oh bands containing dcb - ester 5 ( 409 mg ) , mixed with some product , and compound 4 ( 1.644 g , 80 % ) were isolated as colourless gums . rf ( ch3oh chcl3 1:9 ) 4 0.35 ; 5 0.51 ; h nmr ( 400 mhz , cdcl3 ) : =9.53 ( br d , j=2.3 , 3 h ; u 4-nh ) , 7.85 ( d , j=8.1 hz , 3 h ; u 6-ch ) , 7.23 ( s , 3 h ; hub ch ) , 5.85 ( d , j=5.1 hz , 3 h ; 1-ch ) , 5.76 ( dd , j=8.0 , 2.0 hz , 3 h ; u 5-ch ) , 5.32 ( t , j=4.6 hz , 3 h ; 3-ch ) 4.55 ( s , 6 h ; hub - ch2o ) , 4.26 ( dt , j=5.4 , 2.0 hz , 6 h ; suc - och2 ) , 4.224.19 ( m , 6 h ; 4-ch + 2-ch ) , 3.94 ( br d , j=12.0 hz , 3 h ; 5-chh ) , 3.79 ( br dd , j=12.3 , 4.0 hz , 3 h ; 5-chh ) , 3.723.61 ( m , 90 h ; ch2o ) , 3.43 ( s , 9 h ; 2-och3 ) , 2.762.68 ppm ( m , 12 h ; suc ch2ch2 ) ; c nmr ( 101 mhz , cdcl3 ) : =172.08 ( 3 c ; suc c = o ) , 171.68 ( 3 c ; suc c = o ) , 163.92 ( 3 c ; u c ) , 150.75 ( 3 c ; u c ) , 141.22 ( 3 c ; u 6-ch ) , 138.51 ( 3 c ; hub c ) , 126.29 ( 3 c ; hub ch ) , 102.64 ( 3 c ; u 5-ch ) , 88.69 ( 3 c ; 1-ch ) , 83.18 ( 3 c ; 2/4-ch ) , 81.43 ( 3 c ; 2/4-ch ) , 73.00 ( 3 c ; hub - ch2o ) , 70.96 ( 3 c ; 3-ch ) , 70.48 ( 39 c ; ch2o ) , 69.46 ( 3 c ; ch2o ) , 68.96 ( 3 c ; ch2o ) , 63.90 ( 3 c ; suco - ch2 ) , 61.15 ( 3 c ; 5-ch2 ) , 58.90 ( 3 c ; 2-och3 ) , 28.95 ppm ( 6 c ; suc ch2ch2 ) ; ms ( maldi - tof+ ) : m / z calcd for [ c99h156n6nao51 ] : 2268.97 ; found : 2269.0 [ 4+na ] . tris-1,3,5-{-(2-o - me-5-o-{[2-o - me-5-o-(dmtr)-4-n - acetylcytosin-3-yl](2-cyanoethyloxy)thiophosphoryl}uridinyl-3-o - succinyloxy)octa(ethylene glycol)--oxymethyl}benzene , tris(mupsmc - odmtr ) homostar ( 7 ) : tris(2-methyluridine ) homostar 4 ( 422 mg , 0.188 mmol ) and compound 6 ( 678 mg , 0.846 mmol , 4.5 equiv ) were co - evaporated from ch3cn ( 310 ml ) in vacuo . to the residue was added 0.25 m ett in ch3cn ( 6.77 ml , 1.69 mmol , 9 equiv ) , and after 40 min , pads ( 1.71 g , 5.65 mmol , 30 equiv ) and pyridine ( 6.8 ml ) were added . after a further 60 min the solvent was stripped off under reduced pressure and the residue fractionated through a column of silanised silica , eluting with a gradient of water the resultant emulsion was extracted with ch2cl2 ( 100 ml4 ) , the organic layer dried over na2so4 , and then evaporated to dryness . the appropriate fractions were extracted as before to afford compound 7 ( 538 mg , 64 % ) . rf ( ch3oh ch2cl2 1:9 ) 0.41 ; intermediate tris(phosphite triester ) 0.54 ; h nmr ( 400 mhz , d6-acetone ) : =10.38 ( br s , 1.5 h ; nh ) , 10.25 ( s , 1.5 h ; nh ) , 10.19 ( s , 1.5 h ; nh ) , 10.15 ( br s , 1.5 h ; nh ) , 8.49 ( d , j=7.4 hz , 3 h ; c ch ) , 7.75 ( d , j=8.2 hz , 1.5 h ; u ch ) , 7.64 ( d , j=8.2 hz , 1.5 h ; u ch ) , 7.547.51 ( m , 6 h ; dmtr ch ) , 7.427.36 ( m , 18 h ; dmtr ch ) , 7.337.30 ( m , 3 h ; dmtr ch ) , 7.28 ( s , 3 h ; hub ch ) , 7.13 ( d , j=7.2 hz , 1.5 h ; c ch ) , 7.12 ( d , j=7.4 hz , 1.5 h ; c ch ) , 6.976.93 ( m , 12 h ; dmtr ch ) , 6.076.04 ( m , 3 h ; 1-ch ) , 5.995.96 ( m , 3 h ; 1-ch ) , 5.71 ( d , j=7.7 hz , 1.5 h ; u ch ) , 5.70 ( d , j=7.8 hz , 1.5 h ; u ch ) , 5.375.26 ( m , 2 h ; 23-ch ) , 4.56 ( s , 6 h ; hub - ch2o ) , 4.534.37 ( m , 9 h ; u 4-ch + c 4-ch + pochhch2cn ) , 4.364.15 ( m , 21 h ; u 2-ch + c 2-ch + 5-ch2 + pochhch2cn + suc - och2 ) , 3.84 ( s , 9 h ; dmtr och3 ) , 3.83 ( s , 9 h ; dmtr och3 ) , 3.693.53 ( m , 105 h ; ch2o + 5-ch2 + 2-och3 ) , 3.41 ( s , 4.5 h ; 2-och3 ) , 3.40 ( s , 4.5 h ; 2-och3 ) , 3.01 ( t , j=7.2 hz , 3 h ; ch2cn ) , 2.92.87 ( m , 3 h ; ch2cn ) , 2.762.73 ( m , 6 h ; suc ch2 ) , 2.702.67 ( m , 6 h ; suc ch2 ) , 2.25 ( s , 4.5 h ; ac ch3 ) , 2.24 ppm ( s , 4.5 h ; ac ch3 ) ; c nmr ( 101 mhz , d6-acetone ) : =171.89 ( 3 c ; c = o ) , 171.45 ( 3 c ; c = o ) , 170.66 ( 3 c ; c = o ) , 163.09 ( 3 c ; u / c c ) , 162.68 ( 1.5 c ; u / c c ) , 162.64 ( 1.5 c ; u / c c ) , 158.92 ( 6 c ; dmtr c ) , 154.71 ( 1.5 c ; c c ) , 154.60 ( 1.5 c ; c c ) , 150.47 ( 1.5 c ; u c ) , 150.44 ( 1.5 c ; u c ) , 144.44 ( 1.5 c ; c ch ) , 144.30 ( 1.5 c ; c ch ) , 144.27 ( 1.5 c ; dmtr c ) , 140.02 ( 3 c ; u 6-ch ) , 139.00 ( 3 c ; hub c ) , 135.43 ( 1.5 c ; dmtr c ) , 135.33 ( 1.5 c ; dmtr c ) , 135.19 ( 1.5 c ; dmtr c ) , 130.27 ( 6 c ; dmtr ch ) , 130.18 ( 6 c ; dmtr ch ) , 128.48 ( 3 c ; dmtr ch ) , 128.40 ( 3 c ; dmtr ch ) , 128.02 ( 6 c ; dmtr ch ) , 127.18 ( 3 c ; dmtr ch ) , 125.70 ( 3 c ; hub ch ) , 117.59 ( 1.5 c ; cn ) , 117.31 ( 1.5 c ; cn ) , 113.27 ( 12 c ; dmtr ch ) , 102.65 ( 1.5 c ; u 5-ch ) , 102.52 ( 1.5 c ; u 5-ch ) , 96.15 ( 3 c ; c ch ) , 88.80 ( 1.5 c ; 1-ch ) , 88.51 ( 1.5 c ; 1-ch ) , 88.04 ( 1.5 c ; 1-ch ) , 87.99 ( 1.5 c ; 1-ch ) , 87.08 ( 1.5 c ; dmtr c ) , 87.06 ( 1.5 c ; dmtr c ) , 82.09 ( 1.5 c ; ri ch ) , 81.78 ( 1.5 c ; ri ch ) , 80.91 ( 3 c ; ri ch ) , 80.79 ( 3 c ; ri ch ) , 80.1580.00 ( m , 3 c ; ri ch ) , 73.51 ( 1.5 c ; ri ch ) , 73.28 ( 1.5 c ; ri ch ) , 72.54 ( 3 c ; hub - ch2o ) , 70.58 ( 3 c ; ri ch ) , 70.35 ( 39 c ; ch2 ) , 69.64 ( 3 c ; ch2o ) , 68.72 ( 3 c ; ch2o ) , 67.4267.10 ( 3 c ; u 5-ch2 ) , 63.7563.48 ( m , 6 c ; ch2o + poch2ch2cn ) , 60.57 ( 1.5 c ; c 5-ch2 ) , 60.49 ( 1.5 c ; c 5-ch2 ) , 58.40 ( 1.5 c ; 2-och3 ) , 58.33 ( 1.5 c ; 2-och3 ) , 58.13 ( 1.5 c ; 2-och3 ) , 57.83 ( 1.5 c ; 2-och3 ) , 54.81 ( 3 c ; dmtr och3 ) , 54.79 ( 3 c ; dmtr och3 ) , 28.67 ( 6 c ; suc ch2 ) , 24.13 ( 1.5 c ; ac ch3 ) , 24.05 ( 1.5 c ; ac ch3 ) , 19.0718.90 ppm ( m , 3 c ; ch2cn ) ; p nmr ( 162 mhz , d6-acetone ) =67.33 ( 0.39 p ) , 67.11 ppm ( 0.61 p ) ; ms ( maldi - tof+ ) : m / z calcd for [ c207h269n18nao79p3s3 ] : 4484.59 ; found : 4484.1 [ 7+na+h2o ] . tris(mupsmc - oh ) homostar ( 10 ) : tris(dmtro-2-mer ) homostar 7 ( 192 mg , 43 mol ) was placed in ch2cl2 ( 1 ml ) , to which pyrrole ( 35 l ) then dca ( 35 l ) were added . after 60 min the reaction was complete by tlc and 1 m triethylammonium bicarbonate ( teab , 0.5 ml ) was added , followed by sufficient ch3cn to give a clear solution . the solution was concentrated at reduced pressure until all ch2cl2 had evaporated , then silanised silica ( 10 ml ) was added to the remaining solution followed by the slow addition of water ( 90 ml ) with gentle swirling , plus 1 m teab ( 1 ml ) . the silica was collected in a glass sinter funnel , and the pad was washed with water ( 50 ml ) , then ch3cn water ( 1:3 v / v , 160 ml plus 1.5 ml teab ) and the filtrate was discarded . next the silica was washed with ch3cn ( 150 ml ) and the filtrate evaporated to dryness . the residue ( 156 mg ) was taken up in chcl3 ( 20 ml ) and to the swirled solution was added normal phase silica ( 10 ml ) . the silica was collected in a glass sinter funnel , the pad was washed with further chcl3 ( 130 ml ) and the filtrate discarded . finally the silica was washed with ch3oh chcl3 ( 1:9 v / v , 150 ml ) and the filtrate evaporated to dryness to afford compound 10 ( 118 mg , 77 % ) . chcl3 1:9 ) 0.19 ; h nmr ( 500 mhz , cdcl3cd3od 2:1 v / v ) : =8.42 ( d , j=7.5 hz , 1.5 h ; c ch ) , 8.41 ( d , j=7.6 hz , 1.5 h ; c ch ) , 7.63 ( d , j=8.1 hz , 1.5 h ; u ch ) , 7.402 ( d , j=7.5 hz , 1.5 h ; c ch ) , 7.397 ( d , j=7.6 hz , 1.5 h ; c ch ) , 7.21 ( s , 3 h ; hub ch ) , 5.98 ( d , j=3.7 hz , 3 h ; ri 1-ch ) , 5.91 ( d , j=4.7 hz , 1.5 h ; ri 1-ch ) , 5.90 ( d , j=4.9 hz , 1.5 h ; ri 1-ch ) , 5.75 ( d , j=8.1 hz , 1.5 h ; u ch ) , 5.72 ( d , j=8.1 hz , 1.5 h ; u ch ) , 5.21 ( t , j=5.2 hz , 3 h ; u 3-ch ) 5.034.97 ( m , 3 h ; c 3-ch ) , 4.52 ( s , 6 h ; hub - ch2o ) , 4.454.23 ( m , 18 h ; poch2ch2cn + u 5- ch2 + c 4-ch + u 4-ch ) , 4.214.19 ( m , 6 h ; suc - och2 ) , 4.15 ( br t , j=4.3 hz , 1.5 h ; u 2-ch ) , 4.14 ( br t , j=4.4 hz , 1.5 h ; u 2-ch ) , 4.04 ( dd , j=4.9 , 1.3 hz , 1.5 h ; c 2-ch ) , 4.03 ( dd , j=5.3 , 1.3 hz , 1.5 h ; c 2-ch ) , 3.953.91 ( m , 3 h ; 5-chh ) , 3.783.73 ( m , 3 h ; 5-chh ) , 3.67 ( t , j=4.8 hz , 6 h ; ch2o ) , 3.653.59 ( m , 84 h ; ch2o ) , 3.53 ( s , 4.5 h ; 2-och3 ) , 3.50 ( s , 4.5 h ; 2-och3 ) , 3.39 ( s , 9 h ; 2-och3 ) , 2.852.82 ( m , 6 h ; ch2cn ) , 2.722.69 ( m , 6 h ; suc ch2ch2 ) , 2.672.64 ( m , 6 h ; suc ch2ch2 ) , 2.17 ppm ( s , 9 h ; ac ch3 ) ; c nmr ( 126 mhz , cdcl3cd3od 2:1 v / v ) : =172.40 ( 3 c ; c = o ) , 171.75 ( 1.5 c ; c = o ) , 171.71 ( 1.5 c ; c = o ) , 171.56 ( 3 c ; c = o ) , 164.15 ( 3 c ; u / c c ) , 162.83 ( 3 c ; u / c c ) , 156.09 ( 3 c ; c c ) , 150.52 ( 3 c ; u c ) , 145.39 ( 3 c ; c ch ) , 140.21 ( 3 c ; u 6-ch ) , 138.47 ( 3 c ; hub c ) , 126.40 ( 3 c ; hub ch ) , 117.03 ( 1.5 c ; cn ) , 116.96 ( 1.5 c ; cn ) , 102.79 ( 1.5 c ; u 5-ch ) , 102.73 ( 1.5 c ; u 5-ch ) , 97.40 ( 1.5 c ; c ch ) , 97.34 ( 1.5 c ; c ch ) , 89.22 ( 1.5 c ; c 1-ch ) , 89.10 ( 1.5 c ; c 1-ch ) , 88.08 ( 1.5 c ; u 1-ch ) , 88.02 ( 1.5 c ; u 1-ch ) , 83.2983.19 ( m , 3 c ; u 4-ch ) , 82.17 ( 3 c ; c 2-ch ) , 81.09 ( 1.5 c ; u 2-ch ) , 81.04 ( 1.5 c ; u 2-ch ) , 80.0879.97 ( m , 3 c ; c 4-ch ) , 74.5074.42 ( m , 3 c ; c 3-ch ) , 72.93 ( 3 c ; hub - ch2o ) , 70.35 ( 39 c ; ch2o ) , 70.18 ( 3 c ; u 3-ch ) , 69.43 ( 3 c ; ch2o ) , 68.87 ( 3 c ; ch2o ) , 66.70 ( br , 3 c ; u 5-ch2 ) , 63.91 ( 3 c ; sucoch2 ) , 63.1463.07 ( m , 3 c ; poch2ch2cn ) , 59.65 ( 3 c ; c 5-ch2 ) , 58.81 ( 3 c ; 2-och3 ) , 58.57 ( 1.5 c ; 2-och3 ) , 58.54 ( 1.5 c ; 2-och3 ) , 28.76 ( 6 c ; suc ch2 ) , 24.24 ( 3 c ; ac ch3 ) , 19.2219.10 ppm ( m , 3 c , ch2cn ) ; p nmr ( 202 mhz , cdcl3cd3od 2:1 v / v ) : =67.81 ( 0.44 p ) , 67.59 ppm ( 0.56 p ) ; ms ( maldi - tof+ ) : m / z calcd for [ c144h214n18nao72p3s3]=3537.20 ; found : 3538.3 [ 10+h ] . tris(mupsmcpsmcpsmapsmupsmu - oh ) homostar ( 21)typical chain extension cycle : tris(ho-5-mer ) homostar 18 ( 1.286 g , 0.171 mmol ) was dissolved in dmf ( 4 ml ) to which was added ch3cn ( 20 ml ) and the solution evaporated in vacuo ; this was repeated with dmf ( 2 ml ) plus ch3cn ( 20 ml ) , and finally with neat ch3cn ( 20 ml ) . phosphoramidite 6 ( 585 mg , 0.769 mmol , 4.5 equiv ) was then added to the residue followed by 0.25 m ett in ch3cn ( 6.2 ml , 1.55 mmol , 9 equiv ) . after 35 min pads ( 465 mg , 1.55 mmol , 9 equiv ) and pyridine ( 6.2 ml ) were added , and after a further 35 min the reaction was diluted into ch3oh ch3cn ( 3:17 v / v ) and poured into the osn rig . once 12 diavolumes had permeated , the retentate was evaporated to give crude 5-dmtr homostar 20 ( 1.779 g ) as a brown glass . partially purified tris(dmtro-6-mer ) homostar 20 ( 1.708 g ) was placed in ch2cl2 ( 28 ml ) , to which was added pyrrole ( 0.48 ml ) then dca ( 0.28 ml ) . after 45 min the mixture was diluted with ch3cn ( 100 ml ) and the liquid concentrated until all the ch2cl2 had evaporated . to this solution was then added ch3oh ( 20 ml ) , the solution was diluted with further ch3oh ch3cn ( 3:17 v / v ) containing pyridinium.dca ( 0.5 vol % ) and 5 diavolumes were permeated . the flux was observed to drop significantly , so this was followed by 10 diavolumes ch3oh ch3cn ( 1:4 v / v ) when the flux improved . the retentate was evaporated to dryness , and the residual glass was re - dissolved in ch2cl2ch3oh ( 10 ml ) . the solution was added dropwise to briskly stirred diethyl ether ( 300 ml ) , and the precipitate collected to afford tris(ho-6-mer ) homostar 21 ( 1.394 g , 98 % ) as a brown powder . p nmr ( 202 mhz , cdcl3-cd3od 2:1 v / v ) : =67.867.1 ppm ( m , 15 p ) ; ms ( maldi - tof+ ) : m / z calcd for [ c330h429n66nao153p15s15 ] : 8734.0 ; found : 8728 [ 21+na ] . as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors

due to the discovery of rnai , oligonucleotides ( oligos ) have re - emerged as a major pharmaceutical target that may soon be required in ton quantities . however , it is questionable whether solid - phase oligo synthesis ( spos ) methods can provide a scalable synthesis . liquid - phase oligo synthesis ( lpos ) is intrinsically scalable and amenable to standard industrial batch synthesis techniques . however , most reported lpos strategies rely upon at least one precipitation per chain extension cycle to separate the growing oligonucleotide from reaction debris . precipitation can be difficult to develop and control on an industrial scale and , because many precipitations would be required to prepare a therapeutic oligonucleotide , we contend that this approach is not viable for large - scale industrial preparation . we are developing an lpos synthetic strategy for 2-methyl rna phosphorothioate that is more amenable to standard batch production techniques , using organic solvent nanofiltration ( osn ) as the critical scalable separation technology . we report the first lpos - osn preparation of a 2-me rna phosphorothioate 9-mer , using commercial phosphoramidite monomers , and monitoring all reactions by hplc , 31p nmr spectroscopy and ms .

Introduction Selection of LPOS-OSN materials Results and Discussion Conclusion Experimental Section General experimental details Synthetic procedures Supporting Information

oligonucleotides ( oligos ) have re - emerged as a major pharmaceutical target due to the unprecedented opportunity for controlling protein expression mediated by short rna oligomers ( ca . 20 nucleotides long ) through rna interference ( rnai ) with small interfering rna ( sirna ) or micro - rna ( mirna ) , and these have in turn re - invigorated research in the field of anti - sense oligonucleotides ( aso / as - on). today the overwhelming majority of oligos are prepared using solid - phase oligo synthesis ( spos ) , but this is very challenging to scale up . we are developing a liquid - phase oligo synthesis ( lpos ) synthetic strategy that will be more amenable to standard batch production techniques than spos , using organic - solvent nanofiltration ( osn ) as the critical scalable technology for separating the growing oligo from all other reagents . we now report the lpos - osn preparation of a 2-methyl rna phosphorothioate 9-mer , monitoring all reactions by hplc , p nmr spectroscopy and ms . spos has been scaled up to 12 kg per batch , and the largest trial of the new generation of rnai therapies required a few kg of oligo . thus it is expected that 100s of kilograms of oligo might be required annually to treat rare diseases , and possibly tons for major ones . however , the specialized equipment is demanding and expensive to use in an industrial setting , and we believe that spos , even with major advances , is incapable of approaching the 100 kg scale per batch , because of the challenge of completely and reproducibly washing large beds of synthesis support. however , the critical question that must be addressed in any lpos strategy is how to separate the growing oligo from excess reagents and byproducts . initially dna oligos supported on poly(styrene ) ( the same solid - phase support as had recently been used by merryfield for peptide synthesis ) were assembled by means of khorana s phosphodiester approach. [22 , 23 ] whilst approaches to oligo synthesis based upon precipitation or crystallisation are in theory scalable , it is questionable whether this would be truly practical . furthermore , it is inevitable that material will always be lost to incomplete separation : on the peg support it was found that losses , starting around 1 % per cycle , became more significant as the increasing solubility of the growing oligo began to overwhelm the polymer - driven phase separation ; during the preparation of an rna 5-mer on the small pentaerythritol support , the coupling cycle yield only averaged 85 % for a 54 % overall yield , which would be unacceptable for commercial production . this approach was repeated for oligos by the same laboratory , using the now obsolete phosphodiester coupling strategy and either pva or peg-10 000 polymeric supports . alternation between chain extension in organic solvent and diafiltration in water after each chain extension cycle probably makes this strategy impractical . we postulated that organic - solvent nanofiltration ( osn ) could fulfil the critical separation role in an lpos strategy conducted entirely in organic solution , figure 1 . furthermore , since organic - solvent stable nanofiltration ( as opposed to ultrafiltration ) membranes are now available , we proposed that a smaller , discrete synthesis support could be used . we further postulated that lpos - osn would provide an excellent platform for monitoring the ongoing oligo synthesis , by means of sampling using a simple liquid draw - off . although in principle it is also possible to monitor chain extension progress with spos , we are unaware of any report of such a procedure . this is most likely due to the difficulty of engineering repeated access to beds of solid support in large diameter , pressurized steel columns ( columns for preparing 12 kg oligo by spos have diameters 50100 cm and pressure ratings of 1520 bar ) . however , in the future , during the preparation of tens of kilograms to tons of oligos , it would be economically unacceptable to risk the complete loss of such large batches of very expensive building blocks without critical quality control . the lpos - osn concept : a ) chain extension reaction ; b ) diafiltration by osn to remove excess reagents ; c ) 5-o deprotection ; d ) diafiltration by osn to remove excess reagents , then repeat cycle to the desired length . building on our earlier experience with membrane - enhanced peptide synthesis ( meps ) in organic solvent ( dmf ) , we initially explored osn separation of mpeg-5000-supported dinucleotides . however , even ,-bis(dinucleotidyl)-peg-10 000 had too low a rejection for practical lpos - osn . we hypothesised that this would have three advantages : 1 ) branching should inhibit threading of the supported oligo into the membrane permeation pathways , and therefore increase membrane rejection of the construct ; 2 ) with three oligos growing around one hub , the molecular weight will rise by three nucleotides per cycle , rapidly increasing the overall size , and hence the rejection of the tris(oligonucleotidyl ) support with oligo length ; and 3 ) since the tris(oligonucleotidyl ) support is a discrete species , hplc and mass spectral ( ms ) analyses of real - time synthetic quality should be feasible . adoption of lpos - osn by other groups would be encouraged if this technology was compatible with commercial building blocks and common protective group combinations . we also selected the widely used , first - generation phosphorothioate modification as a target for this pilot project because the debris from thioylation reagents is likely to be a more severe test of lpos - osn purification than common oxidants ( e.g. as a challenging target for this new lpos - osn technology , we set out to synthesise a 2-methyl rna phosphorothioate 9-mer section of the m23d aso . building on our earlier experience with membrane - enhanced peptide synthesis ( meps ) in organic solvent ( dmf ) , we initially explored osn separation of mpeg-5000-supported dinucleotides . however , even ,-bis(dinucleotidyl)-peg-10 000 had too low a rejection for practical lpos - osn . therefore we instead adopted monodisperse tris(octagol ) homostar 1 ( a homostar is a star polymer in which all the arms are identical ) as a branched lpos support , see scheme 1 . we hypothesised that this would have three advantages : 1 ) branching should inhibit threading of the supported oligo into the membrane permeation pathways , and therefore increase membrane rejection of the construct ; 2 ) with three oligos growing around one hub , the molecular weight will rise by three nucleotides per cycle , rapidly increasing the overall size , and hence the rejection of the tris(oligonucleotidyl ) support with oligo length ; and 3 ) since the tris(oligonucleotidyl ) support is a discrete species , hplc and mass spectral ( ms ) analyses of real - time synthetic quality should be feasible . [3032 ] for this reason we elected to focus on nucleic acid analogues for our lpos - osn test sequence , instead of native rna . adoption of lpos - osn by other groups would be encouraged if this technology was compatible with commercial building blocks and common protective group combinations . we also selected the widely used , first - generation phosphorothioate modification as a target for this pilot project because the debris from thioylation reagents is likely to be a more severe test of lpos - osn purification than common oxidants ( e.g. as a challenging target for this new lpos - osn technology , we set out to synthesise a 2-methyl rna phosphorothioate 9-mer section of the m23d aso . to maximize the analytical potential of lpos - osn it is highly desirable to drive loading of the synthesis support to completion to give a homogeneous product , as well as to avoid waste of expensive excess nucleoside on a large scale . the resultant tris - dmtr - ether ( 3 ) was then detritylated with dichloroacetic acid ( dca ) , using pyrrole as a cation scavenger , to provide fully loaded homostar 4 in 80 % yield over the two steps , ready to commence the chain extension cycle . at this stage a small amount of dcb - ester ( 5 ) was separated chromatographically from 4 ; although this contaminant would not affect oligo synthesis at all , in this study it was removed to simplify hplc analysis of chain extension . for this reason , the widely used ett ( pka 4.3 , 0.25 m in ch3cn ) was selected as the activator , firstly because it is a compromise that provides higher activity than classical tetrazole ( pka 4.8 ) , but less than 4-nitrophenyl tetrazole ( npt , pka 3.7 ) . consequently , in an lpos strategy capping should not be as critical as in spos , and this step was omitted simplifying the process during pilot study development . indeed , it is hoped that assay of the chain extension reaction will in future permit identification of otherwise economically catastrophic failed couplings on very large scales , and provide the opportunity to repeat the reaction . however , if capping were implemented before the assay , it would then be impossible to recover a failed coupling . once chain extension and thioylation were complete , the crude mixture was then diluted with ch3cn , and poured directly into the osn apparatus ( see supporting information ) . however , along with the desired dmtr - dinucleotidyl homostar ( 7 ) , most of the building block related species , consisting of a mixture of amidates ( 8) and thioate salts ( 9 ) , were retained ( corresponding to n - op in figure 1 ) . osn of dinucleotidyl homostar monitored by p nmr spectroscopy : a ) tris(mumc - dmtr ) homostar 7 after permeating 12 diavolumes of ch3cn apart from the homostar , amidates 8 a and 8 b , and thioate salts 9 a and 9 b are present ; b ) tris(mumc - oh ) homostar 10 after permeating 5 diavolumes 1 % dca - ch3cn and 10 diavolumes ch3cn ; c ) expansion of b ) exhibiting the two diastereoisomers of the internucleotide linkage , plus a low level of possible n - deacetylation of cytosine ( see supporting information ) . to this were added pyrrole then dca , and after 30 min the detritylation was complete by hplc . suspecting that ion exchange could occur between the protonatable pbi membrane surface and thioate salts 12 , 1 vol % dca was added to the first five diavolumes . after the first chain extension cycle a moderate 75 % yield of tris(mumc - oh ) homostar 10 was isolated . thus , after chain extension , dmtr-3-mer homostar 13 was partially purified by osn ( 12 diavolumes ) then detritylated , after which all the nucleotidyl debris was separated by osn , and precipitation was again used to remove residual dmtr - pyrrole . it should be noted that chain extension cycle yields are calculated assuming 100 % purity of the product homostar . however , as low levels of side - reactions accumulate on the growing oligo , the purity can not be 100 % , so the molecular weight can not be precisely defined , and the yields are more correctly referred to as apparent yields . this protocol was used on 4-mer 16 and for all later chain extension cycles , following each reaction by hplc , and assaying the products by p nmr spectroscopy and maldi ms , both before and after detritylation ( see supporting information ) . although hplc usefully exhibited retention times lengthening in relation to the number of 5-dmtr ethers per homostar , both during chain extension and detritylation , by 5-mers 17 and 18 the peaks were too broad to be of further analytical use ( see supporting information ) , presumably due to the exponentially growing number of diastereoisomers at the p - centers of the oligo backbone . however , p nmr spectroscopy continued to demonstrate acceptably low levels of amidate contamination after each cycle . although these short - mers could be explained by lack of capping , we believe that they actually derive from chain extension of residual amidate building block 11 after osn . chain extension was continued from tris(pentanucleotidyl ) homostar 18 with the same protocol for another four cycles , and p nmr spectroscopy and ms now served as the principal methods of product characterization , to obtain 1.36 g of the desired tris(nonanucleotidyl ) homostar 27 . in this report we have demonstrated for the first time a new liquid - phase synthesis and separation paradigm for oligonucleotides : liquid - phase oligonucleotide synthesis / organic - solvent nanofiltration , lpos - osn . this promising technology has yet to equal the speed and purity of spos , requiring around two days per chain extension cycle with the limited area available from current laboratory - scale flat membrane cells . however , the fact that we were able to perform eight chain extension and detritylation cycles , with intermediate purifications , all in the liquid phase demonstrates that it has high potential . compared to competing precipitation strategies , lpos - osn is more amenable to industrial exploitation because liquid - phase handling is intrinsically scalable . lpos - osn also has the major advantage over spos that it is straightforward to sample and monitor every step of the process . apart from p nmr spectroscopy and hplc , the choice of a monodisperse support also allowed characterization of the growing oligonucleotidyl homostars by ms . from the above experience , several modifications can be suggested to improve future protocols : shortening the coupling time , and analysing only after thioylation , will reduce long - mer formation . the overall yield of fully protected 5-oh tris(oligonucleotidyl ) homostar 27 from loaded uridine homostar 4 is only about 39 % , mainly due to poor recovery from the early cycles of osn ; the first three couplings ( 44-mer 14 ) give a cumulative yield of only 52 % , but the next five couplings ( 4-mer 189-mer 27 ) have a combined yield of about 76 % . as with spos , we have recently demonstrated that an additional stage of diafiltration with a low - molecular - weight cut - off membrane can be used to recycle the permeate solvent , greatly reducing the potential cost on an industrial scale . as the scale of lpos - osn increases , an alternative analysis to direct hplc of the retentate will be required ; we believe that rapid ammonia - methylamine ( ama ) global deprotection , followed by hplc of the crude unblocked oligo will provide a suitable method to assay for complete chain extension . solid phase oligonucleotide synthesis ( spos ) was carried out on a ge akta oligopilot 10 , using preloaded 2ome u primer support 200 and manufacturer s standard protocols on a 30 mol scale . after stirring for 20 min , the activated succinate solution was transferred to the dropping funnel , rinsing out the flask with further ch2cl2 ( 5 ml ) , and this solution slowly added to homostar 1 over 45 min . to the residue was added 0.25 m ett in ch3cn ( 6.77 ml , 1.69 mmol , 9 equiv ) , and after 40 min , pads ( 1.71 g , 5.65 mmol , 30 equiv ) and pyridine ( 6.8 ml ) were added . tris(mupsmcpsmcpsmapsmupsmu - oh ) homostar ( 21)typical chain extension cycle : tris(ho-5-mer ) homostar 18 ( 1.286 g , 0.171 mmol ) was dissolved in dmf ( 4 ml ) to which was added ch3cn ( 20 ml ) and the solution evaporated in vacuo ; this was repeated with dmf ( 2 ml ) plus ch3cn ( 20 ml ) , and finally with neat ch3cn ( 20 ml ) . the retentate was evaporated to dryness , and the residual glass was re - dissolved in ch2cl2ch3oh ( 10 ml ) . the solution was added dropwise to briskly stirred diethyl ether ( 300 ml ) , and the precipitate collected to afford tris(ho-6-mer ) homostar 21 ( 1.394 g , 98 % ) as a brown powder . solid phase oligonucleotide synthesis ( spos ) was carried out on a ge akta oligopilot 10 , using preloaded 2ome u primer support 200 and manufacturer s standard protocols on a 30 mol scale . cleavage from solid support and deprotection of nucleobases was carried out in 0.88 aqueous ammonia at 55 c for 16 h. tris-1,3,5-{-[2-o - methyl-5-o-(4,4-dimethoxytriphenylmethyl)uridine-3-o - succinyloxy]octa(ethylene glycol)--oxymethyl}benzene ( 3 ) : compound 1 ( 1.125 g , 1.00 mmol ) was co - evaporated from ch3cn ( 315 ml ) , re - dissolved in ch2cl2 ( 7 ml ) and a dropping funnel was fitted to the flask . after stirring for 20 min , the activated succinate solution was transferred to the dropping funnel , rinsing out the flask with further ch2cl2 ( 5 ml ) , and this solution slowly added to homostar 1 over 45 min . to the residue was added 0.25 m ett in ch3cn ( 6.77 ml , 1.69 mmol , 9 equiv ) , and after 40 min , pads ( 1.71 g , 5.65 mmol , 30 equiv ) and pyridine ( 6.8 ml ) were added . tris(mupsmcpsmcpsmapsmupsmu - oh ) homostar ( 21)typical chain extension cycle : tris(ho-5-mer ) homostar 18 ( 1.286 g , 0.171 mmol ) was dissolved in dmf ( 4 ml ) to which was added ch3cn ( 20 ml ) and the solution evaporated in vacuo ; this was repeated with dmf ( 2 ml ) plus ch3cn ( 20 ml ) , and finally with neat ch3cn ( 20 ml ) . once 12 diavolumes had permeated , the retentate was evaporated to give crude 5-dmtr homostar 20 ( 1.779 g ) as a brown glass . the solution was added dropwise to briskly stirred diethyl ether ( 300 ml ) , and the precipitate collected to afford tris(ho-6-mer ) homostar 21 ( 1.394 g , 98 % ) as a brown powder .

as a genetic disease , cancer is primarily caused by mutations in oncogenes and tumor suppressors , which serve to control tissue homeostasis . altered function , in turn , leads to deregulated mitogenic survival and growth of tumors that frequently exhibit oncogene - activating genomic alterations such as gene amplification or gain - of - function point mutations . tumors may also exhibit tumor - suppressor inactivating mutations , including gene deletions , loss - of - function point mutations , or epigenetic silencing . taken together , these changes enable cells to acquire stereotypical capabilities termed by some scholars hallmarks of malignancy [ 1 , 2 ] . over the past 1020 years , increasing evidence has shown that the majority of oncogenes and tumor suppressors also play a role in the regulation of metabolism . mutations serve to orchestrate nutrient utilization in a manner that facilitates cell survival and growth a phenomenon exemplified in the work of warburg et al . the warburg effect named in his honor describes a process wherein cancer cells preferentially use fermentative glycolysis - based glucose metabolism instead of entering the tricarboxylic acid cycle ( tca ) and subsequent electron transport chain , even under aerobic conditions [ 36 ] . the causes and benefits of the effect have been the focus of study for many years [ 5 , 7 ] , yet despite advances in biochemical understanding , the physiological reason remains unclear . one hypothesis suggests that cancer cell proliferation is not limited by atp production but rather by the ability of cells to synthesize lipids , nucleic acids , and proteins needed to bolster an expanding biomass . preferential aerobic glycolysis would allow cancer cells to adapt metabolism to satisfy a resultantly increased biosynthetic need an idea supported by evidence which suggests that the enzyme pyruvate kinase , catalyzing the final step in glycolysis as shown in figure 1 , is inhibited in tumorigenic cells . the observed selection toward reduced pyruvate kinase activity may further enable upstream glycolytic intermediates ( e.g. , 3-phosphoglycerate ) to be diverted into other metabolic pathways used to produce the additional lipids , nucleic acids , and proteins that mullarky et al . blockage of the constituent metabolic activities in experimental systems has been found to suppress tumor cell growth in vitro and in vivo . metabolic reprograming in tumorigenic cells is not limited to deregulation by oncogenes and tumor suppressors but can also result from genomic modifications to the metabolic enzymes themselves , independently contributing to biomass accumulation and proliferative growth [ 914 ] . this later class of facilitative oncogenes has started to generate an increasing amount of interest in recent years as researchers return to classic studies to uncover the potential utility of seemingly overlooked historic results and apply them to human tumor growth . it is hoped that , by shedding light on the biological basis of malignancy , new applications of old discoveries may lead to the development of novel cancer therapeutics . to be an attractive candidate for cancer therapy , there must be a significant difference between the requirements for a particular enzyme 's activity in cancer and normally proliferating cells . it is here that putative metabolic oncogenes become both interesting and important given their propensity to display metabolic activities that differ significantly [ and ] , in some cases profoundly , from those of enzymes in normal cells . one example is the conversion of glycolytic intermediate 3-phosphoglycerate into phosphohydroxypyruvate by the enzyme phosphoglycerate dehydrogenase ( phgdh)a rate - limiting step in the conversion of 3-phosphoglycerate to serine ( figures 1 and 2 ) . in the paper that follows , i review the historic progression of our understanding of the role of phgdh in cancer from the classic work of snell et al . [ 1620 ] in the 1980s through its reemergence and rise to prominence in 2011 with near simultaneous publications by possemato et al . and locasale et al . , culminating in an assessment of subsequent work and what it means for the future of phgdh as a potential therapeutic target and putative metabolic oncogene . in a review of serine metabolism published in 1984 , snell provides what is widely regarded as one of the first in - depth discussions of the role of phgdh in tumorigenic cells . citing a number of prior laboratory results , he notes that the activities of enzymes involved in serine biosynthesis had been previously assessed in rat neoplastic tissue . specifically , enzymatic activity of phgdh assayed in four transplantable hepatomas ( morris series : 7794b , 7793 , 9121 , and 5123tc ) was elevated 1.710.6 times relative to control livers in rats of the same strain [ 16 , 27 ] . reductions in dietary protein induced adaptive increases in phgdh activity in control animals and healthy portions of the tumor - bearing host liver , presumably due to homeostatic efforts to support diminished nucleic and amino acid supplies , but failed to have any effect on the enzyme in hepatoma cells . snell hypothesized that the observation may suggest a loss of regulatory function or cancerous enzymatic adaptation . seemingly hesitant to make such a strong claim , he further noted that the observation may also reflect physiological independence between intramuscularly implanted hepatomas and the innate portal circulation of otherwise healthy rats . in the numerous assays performed by davis et al . in the morris lab , only two involving hepatomas 7793 and 5123tc were the subject of multiple analyses , both of which consistently found elevations of phgdh relative to healthy liver tissue . in 5123tc it was further shown that the overall rate of serine biosynthesis from carbon - labeled 3-phosphoglycerate closely correlated with the activity of phgdh , indicating that enhanced activity of phgdh in the four hepatomas was responsible for increased serine biosynthetic capacity a conclusion , which snell suggests , needs to be treated with caution in view of the above comment on protein content and , in any case , only relates to a very limited number and range of tumors . in the years that followed , snell 's tone began to change as his own research at the laboratory for experimental oncology at indiana university school of medicine led to the first real inclination of just how limited phgdh activity in cancer cells might be . two years later , snell and weber published a paper entitled enzymatic imbalance in serine metabolism in rat hepatomas which showed that the activity of phgdh was indeed increased in tissues with high cell - renewal capacity in addition to elevations in neonatal and regenerating liver cells . elevations in hepatomas were markedly higher still ( 8.811.9-fold in slow - growing hepatomas and up to 50.875.5-fold in fast - growing hepatomas ) , suggesting an apparent correlation of phgdh activity with the tumorigenic rate of growth and an important association with both neoplastic transformation ( slow - growth tumor ) and progression ( fast - growth tumor ) . relative to increased activity in neonatal ( 10-fold when adjusted for differences in cellularity between adult and neonatal livers ) and regenerating liver ( 2.6-fold ) cells , the enhanced activity observed , particularly in fast - growing tumor cells , was thought to point to a specificity in the changes of phgdh activity in cancer cells . examination of downstream enzyme activity revealed parallel increases in the activity of serine hydroxymethyltransferase and an absence of serine dehydratase and serine aminotransferase contributions ( figure 2 ) , resulting in preferential shunting of glucose via the serine biosynthetic pathway toward the formation of nucleic acid bases . such coordinated alterations coupled with the previous work of davis et al . and snell would seem to suggest that cancer cells ( at least in hepatomas ) have induced an enzymatic imbalance to meet tumorigenic needs that places phgdh in a regulatory position . capable of diverting glucose - derived carbon toward serine biosynthesis and , by way of hydroxymethyltransferase , toward nucleotide formation , such a change confers a selective growth advantage to neoplastic cells relative to their somatic counterparts . repeated transplantations of the hepatoma lines in vivo did not alter the effect , leading to what snell et al . would later describe as robust evidence for reprogramming of gene expression in rat hepatoma cells . some 19 months later in july 1987 , snell et al . published again this time looking at the activities of phgdh and hydroxymethyltransferase during the transition of hepatoma cells from a resting , nonproliferative state to induced proliferative growth . during the different phases of fast - growing hepatoma cells in culture , enzyme activities increased rapidly to reach peaks at 24 h during the early exponential or state - transitional phase and then declined as cells reached higher confluency and entered the plateau phase of growth . the burgeoning hypothesis of coordinated regulation was again confirmed and , in fact , further supported by a series of subsequent experiments in which matched changes in carbon - labeled serine incorporation into nucleic acids were observed . finally in january 1988 , snell et al . published their fourth and final paper on the role of phgdh in cancer cells , firmly calling into question snell 's 1984 supposition on the very limited number and range of tumors to which the observed reprogramming of gene expression applied [ 16 , 19 , 20 ] . their work showed that the patterns of serine metabolism in tumors established in the group 's previous studies [ 1618]enhanced phgdh and serine hydroxymethyltransferase , absent serine dehydratase and serine aminotransferase were consistent in both a transplantable rat sarcoma model and in human colon carcinoma . in tissues lacking serine dehydratase and serine aminotransferase , such as skeletal muscle where the rat sarcoma model was assayed , preferential reorientation for nucleotide formation can not be achieved by deletion of nonexistent competing enzymes and must instead rely only on substantially increased activity of serine hydroxymethyltransferase in tumors [ 17 , 20 ] . this final piece of work becomes significant moving forward , for as snell et al . importantly note , all of their previous studies relied on transplantable rat tumors in animal model systems . however , if the patterns are to have significance [ ] , particularly in relation to the development of strategies for enzyme - targeted anticancer drug therapy , then they must also be demonstrated in human cancers . much like the results from the rat sarcoma , the activity of phgdh was found to be elevated 10-fold , and that of serine hydroxymethyltransferase nearly 5-fold , in tumors relative to healthy colon mucosa . following snell et al . 's final publication in 1988 , the group 's work shifted to consider the therapeutic role of serine hydroxymethyltransferase , leaving the discussion of phgdh all but abandoned for more than a decade . . published in march 2000 that its potential utility as a metabolic oncogene begins to appreciably reemerge . . looked at nucleotide sequence and differential expression of the human phgdh gene . citing work by achouri et al . that reported successful cloning of the first mammalian phgdh gene from a rat hepatoma in 1997 , cho et al . used 300 cdna clones randomly sequenced from a zap human jurkat t - cell cdna library to look for similarity to the known rat phgdh gene . one 852 bp clone showed partial similarity to the 3-region and was subsequently used to rescreen the library as a novel probe . of five hybridization - positive clones , one contained an insert 2,478 bp long . comparison to other known phgdh sequences and 3d modeling allowed for the assignment of substrate - binding , nucleotide - binding , and regulatory domains . the overall sequence was found to share 94% homology with rat phgdh and 93% homology with murine phgdh . when the sequence was compared via individual functional domains , the nucleotide - binding domain , containing a consensus gly - xaa - gly - xaa - xaa - gly - xaa17-asp sequence known to be involved in binding the adenosine portion of nad , showed the highest degree of homology . the c - terminal regulatory domain was more variable , particularly in length , but did not contain alterations considered important for allosteric control [ 29 , 30 ] . tissue distribution of phgdh - specific mrna was then analyzed using a p - labeled version of the 2,478 bp phgdh cdna probe on a human multiple tissue northern blot assay . two transcripts , approximately 2,100 bp and 710 bp in size , were detected . the dominant 2.1 kb mrna transcript was found at high levels in the prostate , testis , ovary , brain , liver , kidney , and pancreas . lower levels were found in the mucosal lining of the colon and , weakly , in the thymus , small intestine , and heart . in contrast , achouri et al . found a single 2.1 kb mrna derived from the liver of a rat detectable only under conditions of a protein - free , carbohydrate - rich diet . pointing to this discrepancy , cho et al . note that dietary regulation akin to that mentioned by snell in 1984 may not be significant in human tissues given that the 2.1 kb mrna was found expressed at high levels in the human liver even under normal conditions . the secondary 710 bp mrna was detected as a minor transcript in most of the tissues where the dominant transcript was observed with notably higher levels relative to the dominant transcript in the heart . it was the only transcript found in human skeletal muscle . taken together , the normal physiological findings of cho et al . suggest that expression of human phgdh is not limited to tissues with a high proliferative capacity but rather expressed in a tissue - specific manner . the minor 710 bp mrna transcript is thought to potentially reflect differential splicing or use of a secondary internal ( 2.1 kb intron - contained ) transcriptional start . cho et al . then sought to determine what happens to the differential expression of phgdh - specific mrna in malignant human tumor cells and whether the anticipated elevation in phgdh protein expression is due to upregulation of phgdh mrna at the transcriptional level or is instead the result of modifications in enzymatic characteristics such as km , vmax , and/or stability . . used a northern blot assay with phgdh cdna to reveal that the dominant 2.1 kb mrna transcript was detectable in most continuously growing tumor cells . phgdh transcriptional expression was identified in several human leukemias ( jurkat , molt-3 , hl-60 , u937 , and thp-1 ) , t - cell lymphoblastic lymphoma ( sup - t1 ) , colon adenocarcinoma ( colo 320 dm ) , epithelioid carcinoma ( hela s3 ) , and murine lymphoma ( bw5147.g.1.4 ) . the physiological role for the minor transcript remains unclear , but , of note , its transcriptional expression in human tumors paralleled that of the dominant transcript . on the whole , the results of cho et al . establish that upregulation of phgdh at the transcriptional level may be responsible for the elevated enzymatic activity reported in neoplasia by snell et al . [ 1820 ] and suggest that their assertion of application to human expression can be generalized to include most leukemias and lymphomas of human and murine origin . studies of human phgdh began to appear in subsequent years , prior to prominently reemerging in 2011 with the work of possemato et al . and locasale et al . however , prior to their respective publications in nature and nature genetics , the work of pollari et al . set the stage . metastatic transcriptional expression revealed by cho et al . led to the identification of enhanced serine production in bone metastatic breast cancer cells and simulation of osteoclastogenesis . subsequent ( and prior ) contributions are summarized in table 1 . highlighting the incurable nature and significant morbidity of bone metastatic disease , pollari et al . used breast cancer cell line mda - md-231 ( derived from an estrogen receptor - negative strain ) to model breast cancer bone metastasis in vivo via intracardiac inoculation of immunodeficient mice . comparison of parental mda - md-231 and an enhanced daughter variant with heightened metastatic abilities , mda - mb-231(sa ) , revealed genetic aberrations that were highly conserved between the two lines . genome - wide expression profiles pointed to mere 315 genes ( 1.7% of genes ) that were more than 2.5-fold upregulated and 198 genes ( 1.1% of genes ) that were more than 2.5-fold downregulated in the highly metastatic variant relative to the parental strand . purported pathway associations for the upregulated genes pointed to changes in organic acid , amino acid , and amine metabolism in addition to alterations to nitrogen compound biosynthetic processes the most significantly enriched of which included the pathway for glycine , serine , and threonine metabolism . three genes and their corresponding enzymes are involved in the processes of all three : phgdh , phosphoserine aminotransferase ( psat1 ) , and phosphoserine phosphatase ( psph ) ( figure 2 ) . relative to the parental metastatic cell line , highly metastatic mda - mb-231(sa ) daughter cells exhibited upregulations of 5.1- , 5.8- , and 2.6-fold , respectively . a serine / alanine / cysteine/-threonine transporter known as slc1a4 was likewise elevated 3.4-fold with overexpression of each confirmed by quantitative rt - pcr ( mrna ) and western blot ( protein ) assays . subsequent statistical quantification using repeat probe sets further showed that while phgdh expression was significantly higher ( p < 0.001 ) in strongly versus weakly metastatic cells , psat1 expression was not significant and psph failed to correlate with metastatic ability at all . pollari et al . concluded their analysis with an assessment of phgdh , psat1 , and psph expression relative to time to relapse in 368 human clinical breast cancer sample cells as well as relative to overall survival time in 393 breast cancer sample cells . their secondary analysis presents the first well - documented assessment of clinical outcomes associated with serine biosynthetic elevation in cancer cells . what they found was a statistically significant association between high phgdh expression and shorter time to relapse ( p < 0.001 ) in addition to shorter overall survival time ( p = 0.002 ) . further assessment for clinically relevant features in a subset of 251 samples pointed to additional associations between enzymatic phgdh and psat1 expression and several recognized risk features , including estrogen and progesterone receptor negative status , mutated p53 , higher tumor grade , heightened expression of the cell proliferation markers pcna and ki-67 , and higher levels of erbb2 . in august 2011 , possemato et al . published a letter in nature . with the stated aim of identifying metabolic genes required for tumorigenesis , possemato et al . cross - referenced known maps of metabolic pathways with the kyoto encyclopedia of genes and genomic ( kegg ) database to compile what they considered a comprehensive list of 2,752 genes encoding all known human metabolic enzymes and transporters . high - priority set of 133 metabolic enzyme and transporter genes : ( 1 ) known to have high expression in tumors versus normal tissue , ( 2 ) known to have high expression in aggressive breast cancer , and ( 3 ) known to be associated with the stem - cell state . selected candidates had to fit two of the three categories or be at the top of a given category . among the chosen 133 genes , . constructed lentiviral short hairpin rna ( shrna ) vectors targeting the respective genes with a median of 5 shrna per gene and used them to generate 2 libraries1 targeting transporters and control genes ( 235 distinct shrna ) and 1 targeting metabolic enzymes and control genes ( 516 distinct shrna ) . when the libraries were screened for shrna that became depleted during breast cancer tumor formation in mice , 16 genes hit with at least 75% of the corresponding shrna targeting the respective genes . expected targets ( e.g. , the mitochondrial atp transporter vdac1 ) hit as did genes involved in control of oxidative stress , the pentose phosphate pathway , glycolysis , proline biosynthesis , and serine biosynthesis . for 5 of the 16 original hits ( including phgdh ) , 2 of the scoring shrna were tested for their effect on tumorigenesis . each suppressed target expression in tumor forming breast cancer cultured mcf10dcis.com cells and reduced the cell 's tumor - forming capacity . consultation of cancerous somatic genome - wide copy number alterations reported in the work of beroukhim et al . in an effort to prioritize genes revealed that phgdh exists in a region of chromosome 1p commonly amplified in several types of cancer , including cancers of the breast and skin ( melanoma ) . none of the other hit genes coincided with genomic regions of focal and recurrent copy number gain . moreover , three shrna that scored in the in vivo screen also decreased phgdh protein expression ; two of differing knockdown efficacies inhibited tumor growth . corroborate the findings of pollari et al . who , as previously described , reported elevated phgdh mrna levels in estrogen receptor - negative breast cancer cells . 's results in a distinct gene expression set before going on to find that phgdh was also elevated in estrogen receptor - negative breast cancer relative to normal breast tissue . in an effort to situate these observations within their larger body of work , possemato et al . note that , of all the genes identified as hits in their previous screen , phgdh had the most significantly elevated expression in estrogen receptor - negative breast cancer cells . among 82 human breast tumor samples assessed in an immunohistochemical assay ( not discussed in this review ) phgdh protein levels significantly correlated with estrogen receptor - negative status . in what became one of the paper 's most heavily cited results , possemato et al . conclusively state that , relative to estrogen receptor - positive breast tumors , estrogen receptor - negative tumors have approximately 68% and 70% elevations of phgdh , respectively , at the mrna and protein levels , accounting for an estimated 2025% of prevalent breast cancer cases and as much as 50% of breast cancer deaths within 5 years of diagnosis . turning to yet another line of evidence , the letter by possemato et al . continues to note that , across a set of 8 breast cancer cell lines , 4 with copy number amplifications of phgdh had 812-fold higher phgdh protein expression relative to nontransformed cell lines ( lacking gene - based amplification ) . where the subsidiary analysis becomes interesting is in its secondary observation that phgdh protein levels were elevated in two estrogen receptor - negative cell lines , lacking a phgdh copy number gain a finding which suggests that additional mechanisms beyond genomic amplification must exist to promote upregulation of phgdh expression at reported mrna and protein levels . to better understand the metabolic consequences associated with such increased phgdh expression , possemato et al . used metabolite profiling and an analysis of serine synthesis pathway flux to examine breast cancer cells with and without phgdh genomic amplification . cell lines with copy number gains ( bt-20 , mda - mb-468 , and hcc70 ) experienced increased flux through the serine biosynthetic pathway ( figure 2 ) relative to those without mda - mb-231 , mcf7 , and mcfc10a . moreover , cell lines with elevated phgdh and high pathway flux were capable of enhancing proliferation in medium lacking serine , while lines with lower levels of phgdh underwent significant blunting or cessation of proliferation . notably , rnai - mediated suppression of phgdh resulted in reduced serine pathway flux in both mda - mb-468 ( no amplification ) and bt-20 ( amplification ) cells . conversely , in mcf10a human mammary cells engineered to overexpress phgdh , serine pathway flux increased to levels similar to those seen in constitutively amplified cells . even in the absence of serine , the additional overexpression of phgdh in modified mcf10a cells was sufficient to drive glucose - originating flux through the biosynthetic serine pathway . in their penultimate experiment , possemato et al . sought to determine whether phgdh is required for cell survival and growth in cells exhibiting increased phgdh expression . they observed that , in cells with high phgdh expression ( whether or not they had copy number mutations ) , rnai - mediated suppression of phgdh caused an appreciable decrease in cell number and induced cell death in the absence of apoptotic markers . in cells with established tumorigenic growth , doxycycline treatment of inducible shrna reduced phgdh protein levels in murine mammary fat pad tumors established with transduced mda - md-468 cells at 25 days , indicating that phgdh suppression can adversely affect growth among established tumor cells . such seemingly contradictory results with respect to serine flux presented a conundrum for possemato et al . . on the one hand , serine , as shown in figure 2 , is a central metabolite in biosynthetic reactions for amino and nucleic acid production ; heightened expression of phgdh in cancer cells had been found to significantly enhance biosynthetic serine pathway flux . on the other hand , the work of possemato et al . thus far had shown that phgdh suppression also inhibited proliferation in cells growing in media with normal levels of extracellular serine ( an amino acid which can also be taken up ) . supplementation with additional serine or cell - permeable methyl - serine - ester did not blunt the suppressive effect . . confirmed that intra- and extracellular serine were in equilibrium and that import of extracellular serine was not defective in the cell lines considered . then , lacking an alternative explanation , they concluded that , perhaps , serine production is not the only important role for phgdh in cancer cells . thus , in their final experiment for the paper , the researchers sought to test the notion that phgdh , psat1 , and psph reactions may produce metabolites beyond serine critical for cell proliferation . they hypothesized that , in cells with high phgdh expression , the subsequent psat1 reaction might contribute a significant fraction of glutamate to alpha - ketoglutarate ( akg ) flux a notion supported by their finding that the serine pathway produces equimolar amounts of serine and akg . if true , as will be discussed in a subsequent section of the paper , serine biosynthesis would play an important role in anaplerosis of glutamine - derived carbon to the tca . consistent with this possibility , suppression of phgdh in mda - mb-468 cells caused a large reduction in levels of akg . moreover , of the major metabolites measured , akg had the most significant and largest change upon phgdh suppression , while serine levels were not significantly affected . confirmatory labeling studies using u - c - glutamine further revealed that absolute flux from glutamine to akg and to other tca intermediates was significantly reduced in cells with rnai - mediated suppression of phgdh . in cells with high phgdh expression , the serine biosynthetic pathway may be responsible for as much as 50% of the net conversion of glutamate to akg . suppression of phgdh would then result in a significant loss of tca - intermediate flux . parallel labeling studies in cell lines with phgdh amplification relative to those without found that flux through the serine biosynthetic pathway shunts 8 - 9% of glycolytic flux toward serine production as compared to 1 - 2% in cell lines with low phgdh expression . believe that increased flux through the serine biosynthetic pathway has a major impact on akg production but a smaller effect on glycolysis and serine bioavailability to tumorigenic cells ( at least in the breast ) than previously observed . in their nearly concurrent publication in nature genetics addressing phgdh expression in melanoma cells , locasale et al . challenge the conclusions of possemato et al . , suggesting that phgdh directly diverts glycolytic flux , thereby , contributing to oncogenesis in cancer cells . rather than a genomic database , locasale et al . began their investigation by monitoring the time course of conversion of u - c - glucose using targeted chromatography and mass spectrometry in hek293 t cells . labeled glucose was incorporated into 13 metabolites across multiple pathways over a 30 min span . importantly , flux to phosphoserine reached steady state on a time scale similar to that of phosphoenolpyruvate ( figures 1 and 2 ) , suggesting comparable relative flux . data further corroborated by nmr experiments which indicated that a substantial fraction of glucose is diverted from 3-phosphoglycerate toward the conversion of serine and glycine in these cells . to measure the total amount of glucose - derived serine being made , locasale et al . cultured cells in uniformly labeled c - glucose and measured metabolites in cell extracts using targeted chromatography and mass spectrometry . labeled serine accounted for about one - half with corresponding amounts of glucose incorporation detected in subsequent nucleotide and nucleoside intermediate formation . reported an approximately 30-fold increase in protein expression relative to nontumorigenic cells ( absent in mcf10a cells ) . indication of selective glucose diversion led to the notion that there may be a context in which pressure exists for tumors to increase phgdh activity ; much like possemato et al . , locasale et al . wondered whether the mechanism might involve genomic amplification ( a copy number gain ) at the locus containing the phgdh gene . to that end , they identified phgdh in the work of slamon et al . and noted that phgdh was found in a region of chromosome 1p ( 1p12 ) known to exhibit recurring copy number gains in 16% of all cancers . further inspection of the genomic region encoding phgdh revealed localized amplification within the coding region of the gene . in an effort to verify these findings , the researchers examined focal copy number gain using fluorescence in situ hybridization ( fish ) with an esophageal squamous cell carcinoma line ( t.t . stable phgdh knockdown using shrna reduced the proliferation rate . moving on to test whether the decreased proliferation was due to reduced ability to utilize the serine biosynthetic pathway , locasale et al . generated cell lines with reduced expression of psat1 and psph to find that shrna - mediated knockdown of these enzymes resulted in similarly diminished proliferation rates . echoing the need for human data first espoused by snell and cho et al , locasale et al . returned to their phgdh amplification results and noted that since amplification in a single tumor type was most commonly found in melanoma , it may be of use to consider phgdh expression and copy number gain in human melanoma tissue samples . to that end , they used immunohistochemistry to measure phgdh expression and found that high expression ( defined by an ihc score > 1 ) was observed in 21% of samples . corresponding copy number gains were detected with fish in a subsample of 21 out of 42 tumors assayed . to investigate whether melanoma cell lines containing phgdh amplification would be sensitive to decreased expression of phgdh , cell lines with and without copy number gains were assessed with a methodology similar to that of the studies conducted by possemato et al . . amplified cell lines revealed decreased proliferation in response to reduced phgdh , while nonamplified lines did not change on phgdh knockdown . verification of serine metabolic flux further showed that each amplified line did contain appreciable conversion of labeled glucose to serine . phgdh knockdown reduced phosphoserine levels in sk - me128 cells and globally altered metabolite levels , including those of glycolytic intermediates . more specifically , under phgdh knockdown conditions , heighted glycolytic metabolite levels were detected near the point of diversion onto the serine biosynthetic pathway the results which locasale et al . took to mean that the level of phgdh expressed in a cell alters glucose metabolism ( in sk - mel128 cells ) by modulating entry of glycolytic intermediates into serine metabolism . however , while copy number gain offers one mechanism to divert flux into serine biosynthesis , locasale et al . also note that other mechanisms to elevate phgdh expression likely exist and may be important in specific cancer contexts . for example , pointing to the work of possemato et al . which found that high phgdh mrna was associated with poor prognosis in breast cancer , locasale et al . undertook a bioinformatics analysis of multiple tumor microarray datasets in breast cancer and found strong associations ( p < 0.0001 ) with several clinical parameters . in an effort to validate and expand these results , locasale et al . assessed phgdh protein expression in 106 breast cancer tumor samples using immunohistochemistry correlated with mrna expression to find that high phgdh expression associated significantly with triple - negative ( p = 0.002 ) and basal subtypes ( p = 0.004 ) but did not associate with general parameters such as metastasis ( as was previously reported ) or with tumor size . consistent with a reliance of a subset of breast cancers on phgdh , protein expression was required for growth in a panel of three ( bt-20 , sk - br-3 , and mcf-7 ) breast cancer cell lines ( including the bt-20 cell line that carries amplification ) . reduced phgdh expression decreased phosphoserine levels in phgdh amplified bt-20 cells , while nontumorigenic breast cancer epithelial mcf-10a cells did not require phgdh for growth , exhibit alterations in glycolysis on shrna knockdown of phgdh , or show detectable labeling of phosphoserine from glucose . given the importance granted to these two papers and the dense nature of the results that they report , it seems prudent to take a moment to review . both studies published in mid-2011 report that the gene encoding phgdh is amplified in a significant subset of human tumors and underscore that diversion of glycolytic intermediates into the serine biosynthetic pathway may contribute to tumorigenesis in cancer cells [ 5 , 8 , 21 , 34 ] . convergence on phgdh happened through different means in different subsets of cancer - derived laboratory cells , leading to different understandings of the mechanisms involved . . 's loss - of - function rna - interference screen or locasale et al . 's glucose - derived carbon flux , both groups identified purported targets of interest and mined known databases of cancer copy number alterations to determine that phgdh on chromosome 1p12 is amplified in some 6% of breast cancers and 40% of melanomas . beyond genomic amplification , a larger fraction of tumors were found to have elevated phgdh protein levels , including 70% of estrogen receptor - negative breast cancers . high phgdh expression ( with or without genomic amplification ) was associated with dependence on the enzyme for growth either via serine utilization or a hypothesized benefit of akg to the tca a phenomenon that deberardinis and seton - rogers describe as tumorigenic addiction to flexible flux [ 5 , 8 , 21 , 34 ] . in a series of papers that followed liu et al . , jing et al . and noh et al . extend these ideas to consider phgdh 's purported role in astrocytoma , cervical cancer squamous cell carcinoma , and triple negative breast cancer cells . citing the work of possemato et al . and locasale et al . , liu et al . analysis of phgdh levels in specimens from glioma patients revealed that although phgdh is not normally expressed in healthy brain tissue , significant elevations were observed in astrocytic tumors in increasing correlation with progressively advanced tumor grade . mechanistic investigations revealed that inhibition of phgdh expression in glioma cells impaired proliferation , invasion , and tumorigenicity in vitro and in vivo . in nude mice injected with stable , phgdh shrna - silenced glioma cells , the oncogenic transcription factor foxm1 was also downregulated in phgdh shrna - silenced glioma cells . using liquid chromatography / liquid chromatography - mass spectrometry , liu et al . the interaction stabilized foxm1 protein levels and sequentially induced the expression of a series of oncogenes , including mmp-2 , vegf , chk2 , and cyclin d1a combined body of evidence which may suggest additional roles for phgdh in glioma tumorigenesis beyond serine biosynthetic flux akin to that described for akg in a subset of breast cancer cells by possemato et al . and , in this case , potentially beyond metabolic functions themselves . the work of jing et al . follows a similar trend highlighting the role for phgdh in cervical cancer . as with tumorigenic breast cancer , melanoma , and astrocytoma cells , phgdh was more strongly expressed in cervical cancer relative to normal cervical epithelium ( 72 versus 29% , p < 0.05 ) . expression levels positively correlated with serum squamous cervical cancer antigen in squamous cell carcinoma , a form of cervical cancer ( p < 0.05)both of which also associated with tumor progression , stage , and size ( p < 0.05 ) . finally , the work of noh et al . published in january 2014 echoes the work of pollari et al . with estrogen receptor - negative breast cancer cells ( later corroborated and expanded in the work of possemato et al . ) . using six different subtypes of triple negative breast cancer ( tnbc ) , noh et al . assembled microarrays of formalin - fixed and paraffin - embedded tissue from 129 tnbc patients . immunohistochemistry assays for enzymes of serine ( and glycine ) metabolism , including phgdh , psat1 , psph , and serine hydroxymethyltransferase , and surrogate markers for identification of molecular tumor type revealed that , among tnbc tumors , basal marker - positive patients exhibited increased expression of phgdh relative to basal marker - negative patients ( p = 0.029 ) . on the whole , protein expression of phgdh tended to be high in patients classified as mixed or basal - like subtype and low in patients classified as immune - related , molecular apocrine , or null subtype but was not statistically different when considered across all six tnbc types ( p = 0.070 ) . among mixed subtype cases , 89.3% showed partial expression of basal markers in their mix . historic progression aside , the question remains what do these findings actually mean for the utility of phgdh in cancer cells ? the answer is not clear , rendering phgdh a fascinating and , at first glance , unintuitive metabolic target in cancer . known to catalyze entry into a metabolic side street , diverting flux away from the superhighway of tumor cell glycolysis , it would appear to reduce energy formation from glucose in rapidly proliferating cells , yet as the works of warburg et al . [ 3 , 4 ] and others have shown , cancer metabolic pathophysiology is seldom so simple . phgdh copy number , transcriptional , and protein amplifications have been shown in multiple subsets of cancerous cells , clearly indicating that some selective advantage is being attained . while the precise nature of that advantage remains unclear and , in fact , may differ across types of cancerous cells , several hypotheses have been put forward . metabolic pathways downstream from serine metabolism contribute to growth , promoting biosynthesis and metabolic signaling involving the folate pool , amino acid / lipid intermediates , and redox regulation . the process of diverting glycolytic flux out from 3-phosphoglycerate serves to further alter cellular redox status via the oxidation of 3-phosphoglycerate and to aid in the generation of akg from glutamate all of which are reported to benefit cellular proliferation . considered in more detail , the first and perhaps most readily apparent benefit involves the products of serine biosynthesis itself de novo production of serine and , by way of serine hydroxymethyltransferase , glycine from which multiple amino and nucleic acids can be made . what is interesting about this aspect of the pathway is that cells grown in standard in vitro culture conditions consisting of abundant extracellular serine have been found to be sensitive to phgdh knockdown . perhaps , as mullarky et al . suggest , cells do not express the correct amino acid transporters to import enough serine or perhaps homeostatic mechanisms regulating metabolic flux are not coupled to extra- and intracellular serine pools . whatever the reason , de novo synthesis of an otherwise nonessential amino acid preferentially occurs such that in its absence cell proliferation suffers or does not occur . [ 5 , 34 ] leads to several of the other hypothesized reasons and , consequentially , purported benefits that heighted expression of phgdh is likely to confer . tying into the conversion of serine to glycine by the enzyme serine hydroxymethyltransferase , the second notion stems from recognition that this conversion provides a major source of methyl groups for the one carbon pools required for biosynthesis and dna methylation [ 5 , 6 ] . by contributing one - carbon units to the folate pool , serine biosynthesis regulated by phgdh effectively provides skeletons for the synthesis of purines and pyrimidines ( figure 2 ) , a consequence that led mullarky et al . to argue that the numerous resultant contributions of phgdh to biomass production may help to explain part of its protumorigenic effect . the work described by pollari et al . found that , in breast tumors , expression of multiple enzymes along the serine / glycine biosynthetic pathway was associated with metastasis in mice and poor clinical outcomes in humans . moreover , as noted by deberardinis and shown by nikiforov et al . two isoforms of serine hydroxymethyltransferase act as transcriptional targets for the oncogene c - myc with serine hydroxymethyltransferase overexpression leading to stimulated proliferation in c - myc - deficient cells . at the same time , both serine and glycine are abundant in the plasma , so as noted previously by mullarky et al . , it is not immediately apparent what can be gained by upregulating synthesis at the expense of glycolysis , despite the observation that that is precisely what some cancerous cells seem to do . perhaps the role of akg used by possemato et al . to explain their seemingly counterintuitive observation that silencing phgdh expression failed to deplete intracellular serine can offer insight to the benefit gained . as mentioned above , akg is a metabolite produced via the transamination reaction of phosphoserine aminotransferase during the conversion of phosphohydroxypyruvate to phosphoserine ( figure 2 ) . it is also the entry point through which glutamine supplies carbon to the tca cycle during cell growth , thereby enabling production of many essential biosynthetic precursors . as noted by possemato et al . , as much as half of all glutamine - derived akg in phgdh - overexpressing cells was generated via an offshoot of the phgdh - regulated serine biosynthetic pathway . , is that pghdh could act as a metabolic gatekeeper , aiding proliferative control of both macromolecular biosynthesis downstream of glutamine metabolism ( cell growth ) and serine - dependent dna synthesis ( cell proliferation ) . furthermore , as outlined by mullarky et al . , generation of akg through the serine biosynthetic pathway may help to alleviate ammonia toxicity stemming from amino acid catabolism . akg synthesis via the serine biosynthetic pathway bypasses a mechanism wherein glutamate derived from glutamine is catabolized by glutamate dehydrogenase to eventually derive akg , producing a unit of ammonia in the process . attest , it would be interesting to see whether cancer cells requiring phgdh to function have defects in autophagy after phgdh knockdown . the suggestion that akg synthesized from serine is critical for tumor growth is not easy to understand from a metabolic point of view given the numerous potential physiological sources of the compound . it is generated from transamination of a variety of amino acids and is synthesized directly in the tca cycle . mullen and deberardinis seemingly agree , stating that cancer cells ( at least in culture ) express a number of other highly active transaminases in addition to psat1 which often account for the majority of glutamine - derived akg . it will be interesting to see as future studies unfold why a subset of cancer cells appear to preferentially use psat1 as a source for akg . as for anaplerosis propagating the tca cycle , it is a role typically filled by conversion of pyruvate to oxaloacetate ( major reaction involving formation of hco3 ) and the reversible transamination of aspartate to form oxaloacetate ( including generation of glutamate and depletion of akg ) coupled with the oxidation of glutamate back to akg ( glutamate + nad + h2o nh4 + akg + nadh + h ) or the conversion of propionyl - coa to succinyl - coa ( propionyl - coa + atp + hco3 succinyl - coa + adp + pi ) in the -oxidation of fatty acids . nevertheless , in spite of the questions that remain and the need for detailed pathways to be satisfactorily drawn out , even kalhan and hanson acknowledge that it is clear serine is a major amino acid in the overall metabolism of a number of tumor - derived cell lines and is critical for cell growth and proliferation . allowing for a greater amount of speculation , mullarky et al . contend that an additional benefit may be derived via diversion of glycolytic flux into serine biosynthesis producing twice as much cytosolic nadh per glucose molecule as compared with the production of pyruvate alone . if true , their hypothesis contends that decreased nadh production in the face of phgdh knockdown would induce a form of redox stress . the problem is that , with less pyruvate produced as glucose is abstracted from glycolysis at the branch point of 3-phosphoglycerate , cells with elevated serine biosynthesis would need a means to regenerate nad . contend that perhaps the glycerol phosphate shuttle , which ultimately transfers electrons from cytosolic nadh to fad on mitochondrial electron transport chain complex ii , may provide a means . in such a situation , the purported pathway could provide an additional benefit to cancer cells by enabling mitochondrial atp synthesis to occur with less production of reactive oxygen species , passing the electrons from nadh to complex ii instead of conventional passage to complex i . finally , mullen and deberardinis succinctly note that beyond orchestrating a growth - promoting metabolic phenotype , evidence also suggests that phgdh , when expressed at high levels , may have properties that prime cells for transformation . citing the work of locasale et al . , they point out that overexpression of catalytically active phgdh but not a hypomorphic mutant in breast epithelial cells induced luminal filling , abnormal nuclear morphology , anchorage - independence , and disturbed cell polarity all changes associated with cellular transformation in cancerous cells . such observations , were they to be further assessed , may point to phgdh overexpression as a means of enhancing the acquisition of malignant properties . across all of these theories , the one thing that becomes collectively clear is that although glycolysis , the tca cycle , and glutamine metabolism are central to the functioning of normal , and at least to some extent cancerous , cells , they do not act alone . they are part of a much larger metabolic network through which enzymes such as phgdh act to alter the metabolism of the cell . bearing this consideration in mind , the oncogenic nature of phgdh amplification likely stems from a combinatorial effect of pathway flux toward biomass production , changes in redox status , energy metabolism , and possibly some signaling functions in a manner that likely varies based on environmental factors , tissue of origin , and cooperating oncogenic mutations . if the historic progression of phgdh understanding and subsequent hypotheses to explain its effect has taught us anything , it is that there is much that we still do not know . moving forward , the challenge will be to determine whether or not cancer cells expressing elevated levels of phgdh require additional serine for growth . evidence presented throughout this paper speaks to a complicated and uncertain result , for , as we have seen in the work of locasale et al . , higher levels of phgdh associated with enhanced serine production in melanoma cells an effect which could be reversed via rnai depletion of phgdh . at the same time , work by possemato et al . found that breast cancer cells with high levels of phgdh expression are resistant to serine withdrawal . rather , the authors noted a decrease in the level of akg produced , a finding that was not observed in melanoma cells . it is a situation that has led some scholars such as luo to suggest that purported addiction to phgdh observed in cancer cells might actually represent a set of distinct , albeit variable , metabolic addictions with some cells ( or cell lines ) requiring serine while others require akg . evidence from metabolic studies has shown levels of multiple metabolites affected by phgdh knockdown . where the discussion becomes interesting is in the correspondingly consistent finding across multiple types of cancer that phgdh overexpression may directly promote oncogenesis . using a mcf-10a breast epithelial cell morphogenesis 3d matrigel assay , locasale et al . revealed that overexpression of phgdh increased cellular proliferation rate and disrupted acinar structure such that a change in cellular serine metabolism by way of phgdh may directly lead to altered phenotypic behavior in a manner that favors transformation . if true , the requirement of additional serine for growth would hold weight , highlighting the need for future studies to determine how this happens and what it means in terms of the broader network of metabolic mechanisms in cancer and the potential for phgdh as a metabolic oncogene . ongoing work in the field has begun to consider intersecting pathways such as reported correlations between serine biosynthesis and p73 expression in human lung adenocarcinomas as well as possible regulation of phgdh by ebv - mir - bart1 in nasopharyngeal carcinomas involving concomitant overexpression of p130cas and erbb2 activation or by lack of repression from pkc . provides compelling evidence that loss of pkc function in mice results in increased tumorigenesis and heightened expression of phgdh as well as psat1 . mechanistically , crystal structures of phgdh dimers reveal phosphorylation sites at ser55 , thr57 , and thr78 thought to be highly conserved among humans , rats , monkeys , and mice . in prostate cancer , the enzyme has been found to regulate c - myc phosphorylation , and in human intestinal tumor cells , it correlates with caspase-3 . additional regulation of phgdh has further been reported in the work of zhang et al . and al - dhaheri et al . . in terms of using phgdh as a potential therapeutic target , one would need to establish a significant difference between the requirement of the enzyme 's activity in cancer and in normally proliferating cells . over the last 30 years , studies have satisfied this objective , strongly implicating phgdh as an attractive drug target in the subset of tumors that amplify and overexpress its gene . the important determination will lie in whether or not a sufficient therapeutic window exists , given that serine biosynthesis operates in all cells . mitigating this concern , luo maintains that phgdh inhibition remains a viable cancer therapeutic target for two reasons : ( 1 ) a phgdh inhibitor designed not to cross the blood - brain barrier would not interfere with serine homeostasis in the central nervous system , avoiding potential neurological effects reported for known phgdh mutations in humans , and ( 2 ) serine deficiency disorders can be treated by exogenous serine supplement , whereas tumors ' addiction to phgdh might not be associated with serine flux . thus , a phgdh inhibitor nonpermeable to the central nervous system coupled with serine supplement ( if it can be designed ) may provide the therapeutic index needed to selectively target tumorigenic cells . considerable work remains to be done before such a result could be achieved , particularly to elucidate the complex pathway and underlying control seemingly involved with phgdh . as exemplified in recent work by chen et al . , the results of such studies are far from clear and may ultimately depend on context- and stage - specific factors . in light of these considerations and the complexities revealed by previous attempts to understand metabolic phgdh function using cancer cell lines and tumor xenografts , it is thought that knock - in models in mice may provide an alternative choice as an animal model with a less confounded genetic background . regardless of the specific model and experimental methodology used , work remains to be done as researchers continue the ongoing pursuit of a potential therapeutic target and putative metabolic oncogene .

exemplified by cancer cells ' preference for glycolysis , for example , the warburg effect , altered metabolism in tumorigenesis has emerged as an important aspect of cancer in the past 1020 years . whether due to changes in regulatory tumor suppressors / oncogenes or by acting as metabolic oncogenes themselves , enzymes involved in the complex network of metabolic pathways are being studied to understand their role and assess their utility as therapeutic targets . conversion of glycolytic intermediate 3-phosphoglycerate into phosphohydroxypyruvate by the enzyme phosphoglycerate dehydrogenase ( phgdh)a rate - limiting step in the conversion of 3-phosphoglycerate to serine represents one such mechanism . forgotten since classic animal studies in the 1980s , the role of phgdh as a potential therapeutic target and putative metabolic oncogene has recently reemerged following publication of two prominent papers near - simultaneously in 2011 . since that time , numerous studies and a host of metabolic explanations have been put forward in an attempt to understand the results observed . in this paper , i review the historic progression of our understanding of the role of phgdh in cancer from the early work by snell through its reemergence and rise to prominence , culminating in an assessment of subsequent work and what it means for the future of phgdh .

1. Introduction 2. Classic Studies: Animal Models and Early Work by Snell 3. Expression in Humans 4. Reemergence of Phosphoglycerate Dehydrogenase: Breast Cancer 5. Extension to Other Cancers 6. Physiological Relevance of the Findings 7. Next Steps: Role as an Oncogene and Argument for Therapeutics

as a genetic disease , cancer is primarily caused by mutations in oncogenes and tumor suppressors , which serve to control tissue homeostasis . over the past 1020 years , increasing evidence has shown that the majority of oncogenes and tumor suppressors also play a role in the regulation of metabolism . the warburg effect named in his honor describes a process wherein cancer cells preferentially use fermentative glycolysis - based glucose metabolism instead of entering the tricarboxylic acid cycle ( tca ) and subsequent electron transport chain , even under aerobic conditions [ 36 ] . the causes and benefits of the effect have been the focus of study for many years [ 5 , 7 ] , yet despite advances in biochemical understanding , the physiological reason remains unclear . preferential aerobic glycolysis would allow cancer cells to adapt metabolism to satisfy a resultantly increased biosynthetic need an idea supported by evidence which suggests that the enzyme pyruvate kinase , catalyzing the final step in glycolysis as shown in figure 1 , is inhibited in tumorigenic cells . metabolic reprograming in tumorigenic cells is not limited to deregulation by oncogenes and tumor suppressors but can also result from genomic modifications to the metabolic enzymes themselves , independently contributing to biomass accumulation and proliferative growth [ 914 ] . one example is the conversion of glycolytic intermediate 3-phosphoglycerate into phosphohydroxypyruvate by the enzyme phosphoglycerate dehydrogenase ( phgdh)a rate - limiting step in the conversion of 3-phosphoglycerate to serine ( figures 1 and 2 ) . in the paper that follows , i review the historic progression of our understanding of the role of phgdh in cancer from the classic work of snell et al . [ 1620 ] in the 1980s through its reemergence and rise to prominence in 2011 with near simultaneous publications by possemato et al . , culminating in an assessment of subsequent work and what it means for the future of phgdh as a potential therapeutic target and putative metabolic oncogene . in a review of serine metabolism published in 1984 , snell provides what is widely regarded as one of the first in - depth discussions of the role of phgdh in tumorigenic cells . citing a number of prior laboratory results , he notes that the activities of enzymes involved in serine biosynthesis had been previously assessed in rat neoplastic tissue . specifically , enzymatic activity of phgdh assayed in four transplantable hepatomas ( morris series : 7794b , 7793 , 9121 , and 5123tc ) was elevated 1.710.6 times relative to control livers in rats of the same strain [ 16 , 27 ] . reductions in dietary protein induced adaptive increases in phgdh activity in control animals and healthy portions of the tumor - bearing host liver , presumably due to homeostatic efforts to support diminished nucleic and amino acid supplies , but failed to have any effect on the enzyme in hepatoma cells . in 5123tc it was further shown that the overall rate of serine biosynthesis from carbon - labeled 3-phosphoglycerate closely correlated with the activity of phgdh , indicating that enhanced activity of phgdh in the four hepatomas was responsible for increased serine biosynthetic capacity a conclusion , which snell suggests , needs to be treated with caution in view of the above comment on protein content and , in any case , only relates to a very limited number and range of tumors . in the years that followed , snell 's tone began to change as his own research at the laboratory for experimental oncology at indiana university school of medicine led to the first real inclination of just how limited phgdh activity in cancer cells might be . elevations in hepatomas were markedly higher still ( 8.811.9-fold in slow - growing hepatomas and up to 50.875.5-fold in fast - growing hepatomas ) , suggesting an apparent correlation of phgdh activity with the tumorigenic rate of growth and an important association with both neoplastic transformation ( slow - growth tumor ) and progression ( fast - growth tumor ) . relative to increased activity in neonatal ( 10-fold when adjusted for differences in cellularity between adult and neonatal livers ) and regenerating liver ( 2.6-fold ) cells , the enhanced activity observed , particularly in fast - growing tumor cells , was thought to point to a specificity in the changes of phgdh activity in cancer cells . and snell would seem to suggest that cancer cells ( at least in hepatomas ) have induced an enzymatic imbalance to meet tumorigenic needs that places phgdh in a regulatory position . repeated transplantations of the hepatoma lines in vivo did not alter the effect , leading to what snell et al . the burgeoning hypothesis of coordinated regulation was again confirmed and , in fact , further supported by a series of subsequent experiments in which matched changes in carbon - labeled serine incorporation into nucleic acids were observed . published their fourth and final paper on the role of phgdh in cancer cells , firmly calling into question snell 's 1984 supposition on the very limited number and range of tumors to which the observed reprogramming of gene expression applied [ 16 , 19 , 20 ] . their work showed that the patterns of serine metabolism in tumors established in the group 's previous studies [ 1618]enhanced phgdh and serine hydroxymethyltransferase , absent serine dehydratase and serine aminotransferase were consistent in both a transplantable rat sarcoma model and in human colon carcinoma . much like the results from the rat sarcoma , the activity of phgdh was found to be elevated 10-fold , and that of serine hydroxymethyltransferase nearly 5-fold , in tumors relative to healthy colon mucosa . 's final publication in 1988 , the group 's work shifted to consider the therapeutic role of serine hydroxymethyltransferase , leaving the discussion of phgdh all but abandoned for more than a decade . published in march 2000 that its potential utility as a metabolic oncogene begins to appreciably reemerge . when the sequence was compared via individual functional domains , the nucleotide - binding domain , containing a consensus gly - xaa - gly - xaa - xaa - gly - xaa17-asp sequence known to be involved in binding the adenosine portion of nad , showed the highest degree of homology . lower levels were found in the mucosal lining of the colon and , weakly , in the thymus , small intestine , and heart . note that dietary regulation akin to that mentioned by snell in 1984 may not be significant in human tissues given that the 2.1 kb mrna was found expressed at high levels in the human liver even under normal conditions . the secondary 710 bp mrna was detected as a minor transcript in most of the tissues where the dominant transcript was observed with notably higher levels relative to the dominant transcript in the heart . the physiological role for the minor transcript remains unclear , but , of note , its transcriptional expression in human tumors paralleled that of the dominant transcript . on the whole , the results of cho et al . establish that upregulation of phgdh at the transcriptional level may be responsible for the elevated enzymatic activity reported in neoplasia by snell et al . purported pathway associations for the upregulated genes pointed to changes in organic acid , amino acid , and amine metabolism in addition to alterations to nitrogen compound biosynthetic processes the most significantly enriched of which included the pathway for glycine , serine , and threonine metabolism . three genes and their corresponding enzymes are involved in the processes of all three : phgdh , phosphoserine aminotransferase ( psat1 ) , and phosphoserine phosphatase ( psph ) ( figure 2 ) . concluded their analysis with an assessment of phgdh , psat1 , and psph expression relative to time to relapse in 368 human clinical breast cancer sample cells as well as relative to overall survival time in 393 breast cancer sample cells . their secondary analysis presents the first well - documented assessment of clinical outcomes associated with serine biosynthetic elevation in cancer cells . , the mitochondrial atp transporter vdac1 ) hit as did genes involved in control of oxidative stress , the pentose phosphate pathway , glycolysis , proline biosynthesis , and serine biosynthesis . in an effort to prioritize genes revealed that phgdh exists in a region of chromosome 1p commonly amplified in several types of cancer , including cancers of the breast and skin ( melanoma ) . even in the absence of serine , the additional overexpression of phgdh in modified mcf10a cells was sufficient to drive glucose - originating flux through the biosynthetic serine pathway . on the one hand , serine , as shown in figure 2 , is a central metabolite in biosynthetic reactions for amino and nucleic acid production ; heightened expression of phgdh in cancer cells had been found to significantly enhance biosynthetic serine pathway flux . then , lacking an alternative explanation , they concluded that , perhaps , serine production is not the only important role for phgdh in cancer cells . thus , in their final experiment for the paper , the researchers sought to test the notion that phgdh , psat1 , and psph reactions may produce metabolites beyond serine critical for cell proliferation . if true , as will be discussed in a subsequent section of the paper , serine biosynthesis would play an important role in anaplerosis of glutamine - derived carbon to the tca . consistent with this possibility , suppression of phgdh in mda - mb-468 cells caused a large reduction in levels of akg . in cells with high phgdh expression , the serine biosynthetic pathway may be responsible for as much as 50% of the net conversion of glutamate to akg . parallel labeling studies in cell lines with phgdh amplification relative to those without found that flux through the serine biosynthetic pathway shunts 8 - 9% of glycolytic flux toward serine production as compared to 1 - 2% in cell lines with low phgdh expression . , suggesting that phgdh directly diverts glycolytic flux , thereby , contributing to oncogenesis in cancer cells . to that end , they identified phgdh in the work of slamon et al . in an effort to verify these findings , the researchers examined focal copy number gain using fluorescence in situ hybridization ( fish ) with an esophageal squamous cell carcinoma line ( t.t . to investigate whether melanoma cell lines containing phgdh amplification would be sensitive to decreased expression of phgdh , cell lines with and without copy number gains were assessed with a methodology similar to that of the studies conducted by possemato et al . took to mean that the level of phgdh expressed in a cell alters glucose metabolism ( in sk - mel128 cells ) by modulating entry of glycolytic intermediates into serine metabolism . for example , pointing to the work of possemato et al . given the importance granted to these two papers and the dense nature of the results that they report , it seems prudent to take a moment to review . both studies published in mid-2011 report that the gene encoding phgdh is amplified in a significant subset of human tumors and underscore that diversion of glycolytic intermediates into the serine biosynthetic pathway may contribute to tumorigenesis in cancer cells [ 5 , 8 , 21 , 34 ] . analysis of phgdh levels in specimens from glioma patients revealed that although phgdh is not normally expressed in healthy brain tissue , significant elevations were observed in astrocytic tumors in increasing correlation with progressively advanced tumor grade . the interaction stabilized foxm1 protein levels and sequentially induced the expression of a series of oncogenes , including mmp-2 , vegf , chk2 , and cyclin d1a combined body of evidence which may suggest additional roles for phgdh in glioma tumorigenesis beyond serine biosynthetic flux akin to that described for akg in a subset of breast cancer cells by possemato et al . follows a similar trend highlighting the role for phgdh in cervical cancer . with estrogen receptor - negative breast cancer cells ( later corroborated and expanded in the work of possemato et al . ) historic progression aside , the question remains what do these findings actually mean for the utility of phgdh in cancer cells ? known to catalyze entry into a metabolic side street , diverting flux away from the superhighway of tumor cell glycolysis , it would appear to reduce energy formation from glucose in rapidly proliferating cells , yet as the works of warburg et al . while the precise nature of that advantage remains unclear and , in fact , may differ across types of cancerous cells , several hypotheses have been put forward . the process of diverting glycolytic flux out from 3-phosphoglycerate serves to further alter cellular redox status via the oxidation of 3-phosphoglycerate and to aid in the generation of akg from glutamate all of which are reported to benefit cellular proliferation . what is interesting about this aspect of the pathway is that cells grown in standard in vitro culture conditions consisting of abundant extracellular serine have been found to be sensitive to phgdh knockdown . tying into the conversion of serine to glycine by the enzyme serine hydroxymethyltransferase , the second notion stems from recognition that this conversion provides a major source of methyl groups for the one carbon pools required for biosynthesis and dna methylation [ 5 , 6 ] . as mentioned above , akg is a metabolite produced via the transamination reaction of phosphoserine aminotransferase during the conversion of phosphohydroxypyruvate to phosphoserine ( figure 2 ) . the suggestion that akg synthesized from serine is critical for tumor growth is not easy to understand from a metabolic point of view given the numerous potential physiological sources of the compound . it will be interesting to see as future studies unfold why a subset of cancer cells appear to preferentially use psat1 as a source for akg . as for anaplerosis propagating the tca cycle , it is a role typically filled by conversion of pyruvate to oxaloacetate ( major reaction involving formation of hco3 ) and the reversible transamination of aspartate to form oxaloacetate ( including generation of glutamate and depletion of akg ) coupled with the oxidation of glutamate back to akg ( glutamate + nad + h2o nh4 + akg + nadh + h ) or the conversion of propionyl - coa to succinyl - coa ( propionyl - coa + atp + hco3 succinyl - coa + adp + pi ) in the -oxidation of fatty acids . nevertheless , in spite of the questions that remain and the need for detailed pathways to be satisfactorily drawn out , even kalhan and hanson acknowledge that it is clear serine is a major amino acid in the overall metabolism of a number of tumor - derived cell lines and is critical for cell growth and proliferation . across all of these theories , the one thing that becomes collectively clear is that although glycolysis , the tca cycle , and glutamine metabolism are central to the functioning of normal , and at least to some extent cancerous , cells , they do not act alone . bearing this consideration in mind , the oncogenic nature of phgdh amplification likely stems from a combinatorial effect of pathway flux toward biomass production , changes in redox status , energy metabolism , and possibly some signaling functions in a manner that likely varies based on environmental factors , tissue of origin , and cooperating oncogenic mutations . if the historic progression of phgdh understanding and subsequent hypotheses to explain its effect has taught us anything , it is that there is much that we still do not know . moving forward , the challenge will be to determine whether or not cancer cells expressing elevated levels of phgdh require additional serine for growth . evidence presented throughout this paper speaks to a complicated and uncertain result , for , as we have seen in the work of locasale et al . at the same time , work by possemato et al . found that breast cancer cells with high levels of phgdh expression are resistant to serine withdrawal . rather , the authors noted a decrease in the level of akg produced , a finding that was not observed in melanoma cells . it is a situation that has led some scholars such as luo to suggest that purported addiction to phgdh observed in cancer cells might actually represent a set of distinct , albeit variable , metabolic addictions with some cells ( or cell lines ) requiring serine while others require akg . where the discussion becomes interesting is in the correspondingly consistent finding across multiple types of cancer that phgdh overexpression may directly promote oncogenesis . if true , the requirement of additional serine for growth would hold weight , highlighting the need for future studies to determine how this happens and what it means in terms of the broader network of metabolic mechanisms in cancer and the potential for phgdh as a metabolic oncogene . ongoing work in the field has begun to consider intersecting pathways such as reported correlations between serine biosynthesis and p73 expression in human lung adenocarcinomas as well as possible regulation of phgdh by ebv - mir - bart1 in nasopharyngeal carcinomas involving concomitant overexpression of p130cas and erbb2 activation or by lack of repression from pkc . provides compelling evidence that loss of pkc function in mice results in increased tumorigenesis and heightened expression of phgdh as well as psat1 . additional regulation of phgdh has further been reported in the work of zhang et al . in terms of using phgdh as a potential therapeutic target , one would need to establish a significant difference between the requirement of the enzyme 's activity in cancer and in normally proliferating cells . over the last 30 years , studies have satisfied this objective , strongly implicating phgdh as an attractive drug target in the subset of tumors that amplify and overexpress its gene . mitigating this concern , luo maintains that phgdh inhibition remains a viable cancer therapeutic target for two reasons : ( 1 ) a phgdh inhibitor designed not to cross the blood - brain barrier would not interfere with serine homeostasis in the central nervous system , avoiding potential neurological effects reported for known phgdh mutations in humans , and ( 2 ) serine deficiency disorders can be treated by exogenous serine supplement , whereas tumors ' addiction to phgdh might not be associated with serine flux . in light of these considerations and the complexities revealed by previous attempts to understand metabolic phgdh function using cancer cell lines and tumor xenografts , it is thought that knock - in models in mice may provide an alternative choice as an animal model with a less confounded genetic background . regardless of the specific model and experimental methodology used , work remains to be done as researchers continue the ongoing pursuit of a potential therapeutic target and putative metabolic oncogene .

stem cells represent the building blocks of our bodies , functioning as the natural units of embryonic generation during development , and adult regeneration following tissue damage . they are defined by two distinct characteristics : the ability to maintain themselves through cell division , sometimes after long periods of inactivity ( self - renewal ) , and the ability to give rise to more specialized cell types ( differentiation ) . based on the stage in development they are derived from , stem cells are broadly classified as embryonic , umbilical cord , and adult stem cells . potency of stem cells decreases during development from totipotent stem cells at the morula stage , capable of differentiating into all embryonic and extraembryonic tissues , to pluripotent stem cells at the blastocyst stage , forming all embryonic tissues , and to multi- or uni - potent adult stem cells , forming tissues within their germ layer ( figure 1 ) . modern understanding of stem cell biology dates back to 1960s , when multipotent hematopoietic and stromal stem cells residing in the bone marrow were identified . from then on , a number of works have reported the isolation of stem cells from adult tissues . adult stem cells , also called somatic stem cells , prompt tissue homeostasis throughout life and ensure tissue regeneration following damage . they reside in specific anatomic locations ( stem cell niches ) and are regulated by a combination of cellular , molecular , and physical signals . numerous types of adult stem cells have been identified , including hematopoietic , mesenchymal , endothelial , intestinal , and neuronal stem cells , each displaying different properties and potency . mesenchymal stem cells , which have been isolated from a range of adult tissues including bone marrow , periosteum , fat , skeletal muscle , and synovial fluid , as well as from the cord blood , umbilical cord , and placenta , have been extensively explored as a source of cells for biomedical applications , owing to their broad regenerative potential , paracrine regulatory effects , and strong immunomodulatory activity . notably , cell populations isolated from different tissues exhibit phenotypic similarities , as well as specific differences in gene expression profiles and biosynthetic properties [ 14 , 15 ] . importantly , adult stem can be harvested and reimplanted in the same patient , therefore circumventing the immunological problems associated with allogeneic cell transplantation . however , adult stem cells exhibit limited proliferation potential and progressive loss of functionality upon in vitro expansion [ 1618 ] , and an age - associated decline in cellular fitness [ 19 , 20 ] , which limit their use for generating large amounts of functional cells for experimental and clinical applications . on the other hand , human embryonic stem cells ( hescs ) , which have been first derived from early embryos in 1998 , are characterized by virtually unlimited proliferation potential and ability to give rise to all tissues constituting the human body , thus holding a high potential for biomedical research and clinical applications as an allogeneic cell source . however , the broader differentiation potential also presents a challenge in directing / controlling the cell fate , and a higher risk of tumor formation after transplantation of hesc - derived tissue progenitors compared to adult stem cells . the ethical controversies involving isolation of hesc and the need for an autologous pluripotent stem cell source have fueled intense investigations into cellular reprogramming , leading to the generation of induced pluripotent stem cells ( ips ) in 2006 ( figure 1 ) . more recently , the new techniques of cellular reprogramming have also been adapted for transdifferentiation the ability of cells to switch from one specialized cell type to another directly , without reversal into a less specialized cell ( figure 1 ) . we are now at a point when our growing understanding of the stem cell regenerative potential , and the development of new technologies offer unprecedented possibilities to conduct biomedical research and to translate the findings into new treatments . in the current paper , we will present the state of the art in human pluripotent stem cells and transdifferentiation research , and discuss some of the biomedical applications that this emerging technology is expected to empower . thomson and colleagues were the first to derive hesc lines from the inner cell mass of human blastocysts generated during in vitro fertilization procedures . derivation from morula stage embryos , from developmentally arrested ( poor quality ) embryos , and from single blastomeres has also been reported , although with lower efficiencies [ 2326 ] . between 650% of normal embryos give rise to new hesc lines , whereas only up to 3% success has been reported with poor quality embryos . chen and colleagues systematically explored the timing of inner cell mass isolation and found that isolation on day 6 postfertilization results in the highest hesc derivation efficiency ( ~50% ) , about 10-fold increased compared to isolation on day 5 , and about 5-fold increased compared to hesc derivation from intact blastocysts . hesc typically grow in compact colonies on feeder layers of murine embryonic fibroblasts or human cells , which produce the extracellular matrix for cell attachment and condition the culture medium with paracrine factors [ 21 , 28 ] . protein substrates ( e.g. matrigel , laminin , vitronectin ) and synthetic matrices can also be used for cell derivation and/or culture , providing a more reproducible culture system [ 29 , 30 ] . hesc culture media were initially supplemented with fetal bovine serum of selected lots , or serum replacement , and with growth factors ( i.e. , basic fibroblast growth factor ) which activate the intracellular signaling networks maintaining pluripotency [ 21 , 31 ] . in 2006 , ludwig and colleagues developed a defined medium for feeder - free derivation and culture of hesc , accomplishing one of the critical steps to standardize cell production and banking . however , hesc cultivation and passaging are conducted manually , using enzymes ( collagenase iv , dispase , trypsin ) or mechanical dissociation of colonies . survival of hesc as single cells or small aggregates at passaging and after freezing - thawing cycles has been extremely low , until the discovery that application of a selective rho - associated kinase ( rock ) inhibitor , y-27632 markedly diminishes dissociation - induced apoptosis . further investigations are aimed at automating and scaling up hesc production , such as expansion and differentiation in three - dimensional carriers in suspension culture bioreactors . hescs are commonly characterized by probing the expression of pluripotency markers , including transcription factors ( oct4 , sox2 , nanog ) , surface antigens ( stage specific antigens ssea-4 , ssea-3 , proteoglycans tra-1 - 60 , tra-1 - 81 ) , and enzymes ( alkaline phosphatase and telomerase ) . pluripotency is confirmed by testing the formation of teratomas containing tissues of all three germ layers after injection into immunocompromised mice [ 21 , 35 ] . in addition , genetic status and microbiological status are screened to assess quality of the cultures . the described hesc culture environment has allowed extensive ( potentially unlimited ) expansion of undifferentiated cells , while maintaining a normal euploid karyotype . however , several studies showed that hesc cultivation can lead to accumulation of genomic abnormalities , including amplifications and deletions of whole chromosomes or large genomic regions , amplification of certain gene regions ( i.e. , myc oncogene ) , changes in single nucleotide polymorphism , mutations in mitochondrial genome , and aberrant methylation profiles [ 3638 ] . many of these alterations were observed in late passages , and are likely to provide a growth advantage to the cells . in the future , it will be important to evaluate how specific genomic changes might affect the differentiated progeny of hesc , and to regularly monitor the genomic status of hesc applying high - resolution techniques . first indications that nuclei of somatic cells can be reprogrammed to generate all germ layers of an adult animal date back to 1958 , when gurdon and colleagues cloned mature fertile xenopus laevis from cultured intestinal cells of tadpoles . it took until 1997 to clone the first mammalian , dolly the sheep , by transferring the nucleus of an epithelial cell into enucleated oocyte . reports of successful cloning of mice , cats , dogs , and other animals by somatic cell nuclear transfer followed [ 4143 ] . recently , it was shown that human somatic cell nuclei can be reprogrammed when intact oocytes are used as the recipient cells , resulting in the first triploid pluripotent cell lines . meanwhile , a groundbreaking discovery that has transformed the field came from an alternative approach in 2006/2007 , when takahashi and colleagues reprogrammed first mouse and then human skin fibroblasts to an induced pluripotent state by forced expression of four transcription factors : oct4 , sox2 , klf4 , c - myc , delivered by retroviruses [ 22 , 45 ] . understanding of esc biology was essential for the generation of induced pluripotent stem cells ( ips ) , guiding the selection of transcription factors and culture conditions . improvements of the reprogramming methods followed rapidly , including the use of alternative nonintegrating vectors [ 46 , 47 ] , reprogramming by recombinant proteins , using only two factors for reprogramming , and replacement of transcription factors with small molecules . in 2010 , warren and colleagues reported on cellular reprogramming using modified rna , avoiding genetic manipulation , and increasing the efficiency of the process . whereas optimization of techniques is still ongoing , generation of hips lines in a safe way opens the possibility for their use in replacement therapies . many of the culture protocols developed for hesc have been successfully adapted to hips , which resemble hesc in their morphology , feeder dependence , surface marker expression , and in vivo teratoma formation capacity . however , it remains an open question how similar are various hesc and hips lines . a study by the international stem cell initiative characterizing 59 hesc lines from 17 laboratories showed similarities in the expression patterns of most hesc markers , as well as differences in expression patterns of some lineage markers and imprinted genes . variable efficiencies to form specific lineages , such as cardiac , neuronal , and pancreatic in vitro , were also observed [ 52 , 53 ] . in hips , the problem of variability resulting from different genetic backgrounds and culture conditions is compounded by the differences in reprogramming protocols , which involve major transcriptional and epigenetic shifts . perhaps not surprisingly , studies showed larger heterogeneity of hips compared to hesc on a single - cell level , distinct gene expression and methylation signatures , epigenetic memory of somatic cell origin , and differences in the yield and properties of differentiated progeny , such as early senescence of ips - derived cells . there have also been reports of similarities between cohorts of hesc and hips , such at the study of boulting and colleagues , who have found a similar range of efficiencies for motor neuron differentiation between hesc and hips lines . bock and colleagues have approached the problem by establishing genome - wide reference maps for dna methylation and gene expression of 20 hesc and 12 hips lines , and combined these with assaying in vitro differentiation propensity and evaluating 500 lineage - specific transcripts . the assays were combined in a scorecard for a quick and comprehensive characterization of pluripotent cell lines . their results suggest that hesc and hips should be regarded as two partially overlapping groups , with inherent variability among both hesc and hips lines . some studies have indicated that hips retain certain epigenetic memory from the parent cells , and it is largely unclear to what extent the origin of the reprogrammed cells could affect their safety and function . ideally , the source cells would be easily accessible with minimal risk procedures for the patients ( i.e. , blood sample ) , available in large quantities , and allow relatively high reprogramming efficiency and fast hips derivation process . the elucidation of the molecular and cellular events occurring during embryogenesis is of paramount importance to understand the key pathways regulating cell differentiation and tissue formation under normal and pathological conditions . historically , early development was studied in experimental animals , with mouse being the most popular due to its defined genetics and reproductive ability . strong conservation of genes and signaling pathways regulating development between the species has justified the use of animal models as research platform . mouse studies led to identification of several genes and signaling pathways , including wnt , nodal and bmp pathways , which display specific spatiotemporal expression profiles during embryogenesis and play an important role in germ layer specification and tissue formation ( reviewed by tam and loebel ) . subsequent studies in mesc cultures revealed that the same pathways are involved in the regulation of germ layer development in vitro . despite the similarities observed , there are also differences between mouse and human development , including the timing and profile of expression of control genes , as well as the onset and patterning of tissue formation [ 62 , 63 ] . in addition , discrepancies exist between mesc and hesc , which differ in morphology , cell surface markers , responsiveness to specific factors ( including the leukemia inhibitory factor signaling pathway ) , and pluripotent state . noteworthy , recent studies demonstrated that hesc display a closer similarity with mouse epiblast stem cells ( episc ) , which are derived from postimplantation embryos , suggesting that mouse episc might be a closer equivalent for comparative developmental studies between the two species . today , the ability to culture human pluripotent stem cells and to apply developmentally relevant inductive signals offers an unprecedented possibility to establish human models of embryonic development in vitro . early lineage specification is recapitulated when esc are induced to form three - dimensional cell aggregates embryoid bodies ( ebs ) . in order to direct differentiation toward specific cell lineages , ebs are stimulated with specific growth factors and signaling molecules in tightly controlled concentrations and temporal sequences . typical factors currently used to promote human ebs differentiation include egf , fgf , ra , bmp-4 for ectodermal - mesodermal induction , tgf- and activin - a for mesodermal induction , and a set of small molecules for endoderm specification [ 61 , 67 ] . examples of specific lineages derived from hesc using directed differentiation approach include functional motor neurons [ 68 , 69 ] , human cardiovascular progenitors , pancreatic endoderm , insulin - secreting -cells , hepatocytes , and others . importantly , cells exhibited functionality following in vivo implantation , such as improvement in cardiac function in rodent models of myocardial infarction and development of glucose - responsive endocrine cells from implanted pancreatic endoderm . similarly , stepwise induction protocols have been successfully used for differentiation of ips lines toward different specialized cell types , although the authors often report differences in differentiation efficiency and properties of differentiated progeny [ 48 , 74 , 75 ] . factors such as size of ebs and variability of initial hesc colonies have also been shown to affect differentiation outcomes [ 76 , 77 ] . for some lineages , differentiation has been achieved in monolayer culture , thereby simplifying the protocols and avoiding the variability associated with eb cultures . frequently , a combination of ebs and monolayer culture is used , utilizing the effects of three - dimensional environment on cell differentiation , and accessibility of monolayer culture for subsequent cell characterization . hesc and hips technologies allow the generation of novel in vitro models to study the underlying mechanisms of disease development , and establish platforms for the screening of new drugs to prevent or reverse disease progression . in one of the first examples , digiorgio and colleagues have shown that motor neurons derived from hesc are sensitive to toxic effects of glial cells that carry a mutation in sod1 gene , causing amyotrophic lateral sclerosis ( als ) , a fatal neurodegenerative disease . to study the familial forms of the disease , the group has established hips lines from patients with als and shown development of models to study inherited and degenerative disorders , including alzheimer 's disease , parkinson 's disease , schizophrenia , diabetes , gaucher 's disease , and muscular dystrophy is ongoing and expected to significantly affect our understanding of a variety of medical conditions . creation of disease - specific hips is especially valuable when the underlying mechanisms of the disease are not well understood , and the affected cells are not available from the patients , for example in neurodegenerative diseases or cardiac conditions . generation of hesc / hips libraries from specific patient populations and healthy individuals also allows the development of screening platforms to evaluate toxicity and safety of existing and new drugs . during recent years , a considerable number of drugs were withdrawn from the market due to unforeseen cardiotoxic side effects , and cardiotoxicity is one of the major reasons for late - stage attrition of drug candidates . another serious complication is drug - induced liver injury , which is among the most frequent reasons for withdrawal of approved drugs from the market . the early detection of fatal side effects of new drugs could prevent the continuation of a useless and cost - intensive developing process . also , exclusion of compounds proving false negative in suboptimal test systems could be prevented by improving detection of potential toxicity . currently , negative effects are not detected during in vitro and preclinical studies , due to the limited functional capacity and genetic diversity of cellular models used , and due to the interspecies differences between experimental animals and humans in pharmacotoxicological effects . the potential usefulness of hesc - derived cardiac cells for safe pharmacology was shown recently [ 86 , 87 ] , where cells were reactive to several drugs in a manner that resembles mature cardiomyocytes . in addition , there has been progress in the establishment of protocols for the generation of hepatocyte - like cells from hesc and hips . with respect to evaluating hepatotoxicity using these cells , it will be important to establish the degree of their similarity to primary cells , as well as to identify specific assays to evaluate the functionality of the cells and to detect the drug - induced liver injury in vitro . a large number of medical conditions are associated with loss or malfunction of tissues and organs , and result in patient distress , disability , and death . treatments for these patients currently rely on the transplantation from living and deceased donors , or implantation of medical devices that are limited in their functionality and availability . the ability of stem cells to self - renew and differentiate into specialized cells offers great possibilities in the field of regenerative medicine , both for the ex vivo construction of tissue substitutes ( tissue engineering ) and for transplantation of healthy , potentially genetically - corrected cells ( cell therapy ) . in tissue engineering , stem cells are combined with biomaterial scaffolds , which act as a functional template for regeneration and tissue maturation under appropriate culture conditions . to date , adult stem cells have largely been used for experimental and clinical tissue engineering applications . however , adult stem cells display limited proliferation and differentiation potential , progressive loss of functionality upon in vitro expansion , and age - associated decline in cellular fitness [ 16 , 1820 , 90 ] . in contrast , pluripotent stem cells allow the generation of an unlimited supply of reparative cells ( which are patient - matched in the case of hips ) . to avoid the risk of teratoma formation following in vivo implantation , hesc and hips are induced into lineage - specific progenitors , which display differentiation potential restricted within the germ layer [ 91 , 92 ] . in one example , our group and others have derived mesodermal progenitors with similarity to adult mesenchymal stem cells in the expression of surface markers , global gene expression profile , and potential to differentiate into osteogenic , chondrogenic , and adipogenic lineage [ 91 , 9395 ] . hesc - mesenchymal progenitors have displayed excellent potential for formation of bone - like tissue when seeded in three - dimensional scaffolds and cultured in dynamic culture systems , suggesting they could be a promising cell source for regeneration of the skeletal system . in some studies , in vivo transplantation of hesc - mesenchymal progenitors showed restricted developmental potential of the cells , as teratoma formation was not observed [ 94 , 95 ] . in addition , hesc - mesenchymal progenitors injection improved the outcome of ischemic hindlimb injuries . in light of the variability detected between hesc and hips lines , the current challenges lay in the development of robust protocols that will allow effective and reproducible differentiation of various hips lines . in addition , therapeutic applications of hesc and hips derivatives will critically depend on the development of stringent protocols for selection and/or purification of well - defined , stable tissue progenitors , that will demonstrate limited developmental potential within the selected lineage . advances in the hesc field have already resulted in first clinical trials using hesc - derived cells . in 2010 , geron corporation started a phase i clinical trial to evaluate the safety of hesc - derived neural progenitor cells administration in patients with neurologically complete subacute spinal cord injuries ( http://clinicaltrials.gov/ , number nct01217008 ) . unfortunately the trial has been stopped in 2011 due to financial difficulties of the company . currently , advanced cell technology has two ongoing prospective clinical trials ( phases i / ii ) to evaluate the effects of subretinal injection of hesc - derived retinal pigment epithelium cells in patients with dry age - related macular degeneration and patients with stargardt 's macular dystrophy ( http://clinicaltrials.gov/ , no . preliminary studies indicated measurable improvements in the vision of two nearly blind patients that lasted four months following the treatment . a similar clinical trial for treatment of age - related macular degeneration with hesc - derived retinal pigment epithelium cells is expected to start in the uk in 2012 by the london project to cure blindness ( http://www.thelondonproject.org/ ) . in addition to tissue replacement , stem cells represent a valuable tool in cell therapy for the correction of genetic diseases . in one of the recent studies , deyle et al . reported the disruption of the allele carrying the dominant mutation in the type i collagen genes causing osteogenesis imperfecta , using a combination of gene - targeting and ips technology . mesenchymal stem cells derived from the bone of patients affected by the disease were gene targeted to disrupt the mutant alleles causing the disease , converted to hips , expanded in vitro , and then induced to differentiate toward the osteogenic lineage . following in vivo implantation on ceramic scaffolds , gene - targeted osteogenic cells were able to form bone tissue in experimental animals . correction of genetic mutations was reported for other diseases , including 1-antitrypsin deficiency and x - linked chronic granulomatous disease , using the zinc finger nuclease technology , which holds great potential for efficient and precise gene manipulation at the desired genomic locations [ 99 , 100 ] . in summary , stem cell technology , along with the accurate genetic manipulation of hips , opens new possibilities for the generation of clinically relevant cells for autologous cell - based therapies . our growing understanding of the stem cell biology and the development of specific cell lineages has allowed the generation of multiple specialized cell types from stem cells , and the identification of distinct developmental stages that are governed by genetic and epigenetic regulatory networks . in parallel , an alternative approach is being investigated , in which one somatic cell type could be directly converted transdifferentiated into another somatic cell type . this way , abundant adult cells , such as dermal fibroblasts or adipocytes , could be used to produce other therapeutically important cells , such as neurons , cardiomyocytes or pancreatic beta cells . natural transdifferentiation involves a stepwise dedifferentiation of the primary cell to an intermediate cell type , that can then differentiate into the new lineage . in contrast , experimentally induced transdifferentiation involves a direct conversion through a simultaneous downregulation of one genetic programme and a concomitant upregulation of the new genetic programme . in one of the first experiments , muscle - specific genes were induced in pigmented epithelium , nerve , fat , liver , and fibroblast cells by forced expression of myod , a master regulator of muscle differentiation . however , it was not clear until 2008 how the procedure could be applied more generally to other cell types . following the ips reprogramming experiments , zhou and colleagues found that reexpressing a combination of 3 transcription factors , tested from a list of 20 key developmental regulators , can turn pancreatic exocrine cells into pancreatic beta - like cells in adult mice . using a similar approach , vierbuchen and colleagues showed that a combination of 3 factors with important roles in neural development converts mouse embryonic and postnatal fibroblasts into functional neurons in vitro . in a later study , it was shown that the same factors work in converting terminally differentiated hepatocytes into neurons , thereby demonstrating the possibility to reprogram cells between different germ layers . interestingly , the resulting neurons silenced the donor cell transcriptional program , suggesting a binary lineage switch rather than an induction of hybrid cell phenotypes . however , a small but detectable epigenetic memory of the donor cells remained in the induced neurons . current examples of experimentally induced transdifferentiation rely on the ectopic expression of master regulator genes , which are essential for lineage specification during development and can direct the establishment of a new epigenetic state when expressed in the host cell . to achieve a complete phenotypic switch between the two mature cells and generate functional cells for potential clinical applications , the activation of a new epigenetic state in the host cells must be associated with full deactivation of the original epigenetic state . this might be more cumbersome to achieve between more distantly related cells owing to their larger epigenetic differences , resulting in relatively inaccessible chromosomal regions . possible strategies to convert more distant cell types could involve the use of chemicals that loosen the chromatin structure and favor epigenetic rearrangements . in addition , reduction of factors that support the original epigenetic state of the mature host cells , and the use of factors that promote the reentry into cell cycle could facilitate the transdifferentiation . taken together , transdifferentiation studies underline the importance of specific factors in lineage specification , and overturn the long - believed notion of stable and irreversible lineage commitment events . with further development and optimization , transdifferentiation protocols could offer a shortcut to production of differentiated cells for biomedical research and clinical applications , potentially avoiding the extensive cell proliferation required for hips derivation and differentiation . in addition , transdifferentiation opens the possibility to convert different cell types directly in vivo to drive tissue repair and regeneration in situ , an approach that would be impossible using ips technology due to the teratoma - forming ability of pluripotent stem cells . on the other hand , direct conversion between terminally differentiated cells , which are characterized by limited proliferative potential , may not be suitable for experimental and clinical applications where large amounts of cells are needed , and in situations where disease conditions result from genetic mutations . hitherto , only a few instructive factor combinations have been reported to directly convert mature cell types , and it is likely that these will depend on the selected target and final cell types . as with the ips , a number of questions regarding the transdifferentiated cell stability , functionality , and safety need to be addressed , and a deeper understanding of the reprogramming process is needed before transdifferentiation can be translated into a therapeutic setting . advances in pluripotent stem cell biology and transdifferentiation have generated great enthusiasm in the scientific community over the last years , and are expected to affect numerous biomedical applications . hesc and hips allow the study and modeling of human development and tissue formation under diverse experimental conditions , and therefore represent an excellent platform for understanding human diseases and developing innovative therapeutic solutions . in parallel , development of transdifferentiation technology offers new insights into adult cell plasticity and could lead to expedited protocols for differentiated cell production . in the context of regenerative medicine , hesc and hips offer the possibility to generate an unlimited number of functional cells for reconstructive and reparative therapies . creation of hips libraries is underway , and will expedite both safety pharmacology studies , as well as development of novel therapies . approval of the first hesc - based clinical trials in the usa is encouraging for the field , and signifies the potential of pluripotent stem cells to cure the most devastating diseases of our time . however , there is a lot of work to be done to fully determine the clinical potential , efficacy , and safety of hesc and hips - based treatments . as with other medical breakthroughs , it is only via scrupulous scientific investigation that the true potential of human pluripotent stem cells may be realized .

stem cells divide by asymmetric division and display different degrees of potency , or ability to differentiate into various specialized cell types . owing to their unique regenerative capacity , stem cells have generated great enthusiasm worldwide and represent an invaluable tool with unprecedented potential for biomedical research and therapeutic applications . stem cells play a central role in the understanding of molecular mechanisms regulating tissue development and regeneration in normal and pathological conditions and open large possibilities for the discovery of innovative pharmaceuticals to treat the most devastating diseases of our time . not least , their intrinsic characteristics allow the engineering of functional tissues for replacement therapies that promise to revolutionize the medical practice in the near future . in this paper , the authors present the characteristics of pluripotent stem cells and new developments of transdifferentiation technologies and explore some of the biomedical applications that this emerging technology is expected to empower .

1. Introduction 2. Derivation, Culture, and Characteristics of 3. Applications of Pluripotent Stem Cells 4. Transdifferentiation 5. Conclusions

stem cells represent the building blocks of our bodies , functioning as the natural units of embryonic generation during development , and adult regeneration following tissue damage . they are defined by two distinct characteristics : the ability to maintain themselves through cell division , sometimes after long periods of inactivity ( self - renewal ) , and the ability to give rise to more specialized cell types ( differentiation ) . based on the stage in development they are derived from , stem cells are broadly classified as embryonic , umbilical cord , and adult stem cells . potency of stem cells decreases during development from totipotent stem cells at the morula stage , capable of differentiating into all embryonic and extraembryonic tissues , to pluripotent stem cells at the blastocyst stage , forming all embryonic tissues , and to multi- or uni - potent adult stem cells , forming tissues within their germ layer ( figure 1 ) . modern understanding of stem cell biology dates back to 1960s , when multipotent hematopoietic and stromal stem cells residing in the bone marrow were identified . from then on , a number of works have reported the isolation of stem cells from adult tissues . adult stem cells , also called somatic stem cells , prompt tissue homeostasis throughout life and ensure tissue regeneration following damage . numerous types of adult stem cells have been identified , including hematopoietic , mesenchymal , endothelial , intestinal , and neuronal stem cells , each displaying different properties and potency . mesenchymal stem cells , which have been isolated from a range of adult tissues including bone marrow , periosteum , fat , skeletal muscle , and synovial fluid , as well as from the cord blood , umbilical cord , and placenta , have been extensively explored as a source of cells for biomedical applications , owing to their broad regenerative potential , paracrine regulatory effects , and strong immunomodulatory activity . importantly , adult stem can be harvested and reimplanted in the same patient , therefore circumventing the immunological problems associated with allogeneic cell transplantation . however , adult stem cells exhibit limited proliferation potential and progressive loss of functionality upon in vitro expansion [ 1618 ] , and an age - associated decline in cellular fitness [ 19 , 20 ] , which limit their use for generating large amounts of functional cells for experimental and clinical applications . on the other hand , human embryonic stem cells ( hescs ) , which have been first derived from early embryos in 1998 , are characterized by virtually unlimited proliferation potential and ability to give rise to all tissues constituting the human body , thus holding a high potential for biomedical research and clinical applications as an allogeneic cell source . however , the broader differentiation potential also presents a challenge in directing / controlling the cell fate , and a higher risk of tumor formation after transplantation of hesc - derived tissue progenitors compared to adult stem cells . the ethical controversies involving isolation of hesc and the need for an autologous pluripotent stem cell source have fueled intense investigations into cellular reprogramming , leading to the generation of induced pluripotent stem cells ( ips ) in 2006 ( figure 1 ) . more recently , the new techniques of cellular reprogramming have also been adapted for transdifferentiation the ability of cells to switch from one specialized cell type to another directly , without reversal into a less specialized cell ( figure 1 ) . we are now at a point when our growing understanding of the stem cell regenerative potential , and the development of new technologies offer unprecedented possibilities to conduct biomedical research and to translate the findings into new treatments . in the current paper , we will present the state of the art in human pluripotent stem cells and transdifferentiation research , and discuss some of the biomedical applications that this emerging technology is expected to empower . derivation from morula stage embryos , from developmentally arrested ( poor quality ) embryos , and from single blastomeres has also been reported , although with lower efficiencies [ 2326 ] . chen and colleagues systematically explored the timing of inner cell mass isolation and found that isolation on day 6 postfertilization results in the highest hesc derivation efficiency ( ~50% ) , about 10-fold increased compared to isolation on day 5 , and about 5-fold increased compared to hesc derivation from intact blastocysts . hesc culture media were initially supplemented with fetal bovine serum of selected lots , or serum replacement , and with growth factors ( i.e. in 2006 , ludwig and colleagues developed a defined medium for feeder - free derivation and culture of hesc , accomplishing one of the critical steps to standardize cell production and banking . survival of hesc as single cells or small aggregates at passaging and after freezing - thawing cycles has been extremely low , until the discovery that application of a selective rho - associated kinase ( rock ) inhibitor , y-27632 markedly diminishes dissociation - induced apoptosis . further investigations are aimed at automating and scaling up hesc production , such as expansion and differentiation in three - dimensional carriers in suspension culture bioreactors . in addition , genetic status and microbiological status are screened to assess quality of the cultures . , myc oncogene ) , changes in single nucleotide polymorphism , mutations in mitochondrial genome , and aberrant methylation profiles [ 3638 ] . in the future , it will be important to evaluate how specific genomic changes might affect the differentiated progeny of hesc , and to regularly monitor the genomic status of hesc applying high - resolution techniques . recently , it was shown that human somatic cell nuclei can be reprogrammed when intact oocytes are used as the recipient cells , resulting in the first triploid pluripotent cell lines . understanding of esc biology was essential for the generation of induced pluripotent stem cells ( ips ) , guiding the selection of transcription factors and culture conditions . improvements of the reprogramming methods followed rapidly , including the use of alternative nonintegrating vectors [ 46 , 47 ] , reprogramming by recombinant proteins , using only two factors for reprogramming , and replacement of transcription factors with small molecules . in 2010 , warren and colleagues reported on cellular reprogramming using modified rna , avoiding genetic manipulation , and increasing the efficiency of the process . whereas optimization of techniques is still ongoing , generation of hips lines in a safe way opens the possibility for their use in replacement therapies . many of the culture protocols developed for hesc have been successfully adapted to hips , which resemble hesc in their morphology , feeder dependence , surface marker expression , and in vivo teratoma formation capacity . a study by the international stem cell initiative characterizing 59 hesc lines from 17 laboratories showed similarities in the expression patterns of most hesc markers , as well as differences in expression patterns of some lineage markers and imprinted genes . in hips , the problem of variability resulting from different genetic backgrounds and culture conditions is compounded by the differences in reprogramming protocols , which involve major transcriptional and epigenetic shifts . perhaps not surprisingly , studies showed larger heterogeneity of hips compared to hesc on a single - cell level , distinct gene expression and methylation signatures , epigenetic memory of somatic cell origin , and differences in the yield and properties of differentiated progeny , such as early senescence of ips - derived cells . there have also been reports of similarities between cohorts of hesc and hips , such at the study of boulting and colleagues , who have found a similar range of efficiencies for motor neuron differentiation between hesc and hips lines . the assays were combined in a scorecard for a quick and comprehensive characterization of pluripotent cell lines . some studies have indicated that hips retain certain epigenetic memory from the parent cells , and it is largely unclear to what extent the origin of the reprogrammed cells could affect their safety and function . ideally , the source cells would be easily accessible with minimal risk procedures for the patients ( i.e. the elucidation of the molecular and cellular events occurring during embryogenesis is of paramount importance to understand the key pathways regulating cell differentiation and tissue formation under normal and pathological conditions . historically , early development was studied in experimental animals , with mouse being the most popular due to its defined genetics and reproductive ability . mouse studies led to identification of several genes and signaling pathways , including wnt , nodal and bmp pathways , which display specific spatiotemporal expression profiles during embryogenesis and play an important role in germ layer specification and tissue formation ( reviewed by tam and loebel ) . subsequent studies in mesc cultures revealed that the same pathways are involved in the regulation of germ layer development in vitro . in addition , discrepancies exist between mesc and hesc , which differ in morphology , cell surface markers , responsiveness to specific factors ( including the leukemia inhibitory factor signaling pathway ) , and pluripotent state . noteworthy , recent studies demonstrated that hesc display a closer similarity with mouse epiblast stem cells ( episc ) , which are derived from postimplantation embryos , suggesting that mouse episc might be a closer equivalent for comparative developmental studies between the two species . today , the ability to culture human pluripotent stem cells and to apply developmentally relevant inductive signals offers an unprecedented possibility to establish human models of embryonic development in vitro . examples of specific lineages derived from hesc using directed differentiation approach include functional motor neurons [ 68 , 69 ] , human cardiovascular progenitors , pancreatic endoderm , insulin - secreting -cells , hepatocytes , and others . similarly , stepwise induction protocols have been successfully used for differentiation of ips lines toward different specialized cell types , although the authors often report differences in differentiation efficiency and properties of differentiated progeny [ 48 , 74 , 75 ] . hesc and hips technologies allow the generation of novel in vitro models to study the underlying mechanisms of disease development , and establish platforms for the screening of new drugs to prevent or reverse disease progression . in one of the first examples , digiorgio and colleagues have shown that motor neurons derived from hesc are sensitive to toxic effects of glial cells that carry a mutation in sod1 gene , causing amyotrophic lateral sclerosis ( als ) , a fatal neurodegenerative disease . to study the familial forms of the disease , the group has established hips lines from patients with als and shown development of models to study inherited and degenerative disorders , including alzheimer 's disease , parkinson 's disease , schizophrenia , diabetes , gaucher 's disease , and muscular dystrophy is ongoing and expected to significantly affect our understanding of a variety of medical conditions . creation of disease - specific hips is especially valuable when the underlying mechanisms of the disease are not well understood , and the affected cells are not available from the patients , for example in neurodegenerative diseases or cardiac conditions . generation of hesc / hips libraries from specific patient populations and healthy individuals also allows the development of screening platforms to evaluate toxicity and safety of existing and new drugs . during recent years , a considerable number of drugs were withdrawn from the market due to unforeseen cardiotoxic side effects , and cardiotoxicity is one of the major reasons for late - stage attrition of drug candidates . another serious complication is drug - induced liver injury , which is among the most frequent reasons for withdrawal of approved drugs from the market . the early detection of fatal side effects of new drugs could prevent the continuation of a useless and cost - intensive developing process . also , exclusion of compounds proving false negative in suboptimal test systems could be prevented by improving detection of potential toxicity . in addition , there has been progress in the establishment of protocols for the generation of hepatocyte - like cells from hesc and hips . with respect to evaluating hepatotoxicity using these cells , it will be important to establish the degree of their similarity to primary cells , as well as to identify specific assays to evaluate the functionality of the cells and to detect the drug - induced liver injury in vitro . treatments for these patients currently rely on the transplantation from living and deceased donors , or implantation of medical devices that are limited in their functionality and availability . the ability of stem cells to self - renew and differentiate into specialized cells offers great possibilities in the field of regenerative medicine , both for the ex vivo construction of tissue substitutes ( tissue engineering ) and for transplantation of healthy , potentially genetically - corrected cells ( cell therapy ) . in tissue engineering , stem cells are combined with biomaterial scaffolds , which act as a functional template for regeneration and tissue maturation under appropriate culture conditions . to date , adult stem cells have largely been used for experimental and clinical tissue engineering applications . however , adult stem cells display limited proliferation and differentiation potential , progressive loss of functionality upon in vitro expansion , and age - associated decline in cellular fitness [ 16 , 1820 , 90 ] . in contrast , pluripotent stem cells allow the generation of an unlimited supply of reparative cells ( which are patient - matched in the case of hips ) . in one example , our group and others have derived mesodermal progenitors with similarity to adult mesenchymal stem cells in the expression of surface markers , global gene expression profile , and potential to differentiate into osteogenic , chondrogenic , and adipogenic lineage [ 91 , 9395 ] . hesc - mesenchymal progenitors have displayed excellent potential for formation of bone - like tissue when seeded in three - dimensional scaffolds and cultured in dynamic culture systems , suggesting they could be a promising cell source for regeneration of the skeletal system . in some studies , in vivo transplantation of hesc - mesenchymal progenitors showed restricted developmental potential of the cells , as teratoma formation was not observed [ 94 , 95 ] . in light of the variability detected between hesc and hips lines , the current challenges lay in the development of robust protocols that will allow effective and reproducible differentiation of various hips lines . in addition , therapeutic applications of hesc and hips derivatives will critically depend on the development of stringent protocols for selection and/or purification of well - defined , stable tissue progenitors , that will demonstrate limited developmental potential within the selected lineage . advances in the hesc field have already resulted in first clinical trials using hesc - derived cells . unfortunately the trial has been stopped in 2011 due to financial difficulties of the company . preliminary studies indicated measurable improvements in the vision of two nearly blind patients that lasted four months following the treatment . a similar clinical trial for treatment of age - related macular degeneration with hesc - derived retinal pigment epithelium cells is expected to start in the uk in 2012 by the london project to cure blindness ( http://www.thelondonproject.org/ ) . in addition to tissue replacement , stem cells represent a valuable tool in cell therapy for the correction of genetic diseases . in one of the recent studies , deyle et al . reported the disruption of the allele carrying the dominant mutation in the type i collagen genes causing osteogenesis imperfecta , using a combination of gene - targeting and ips technology . mesenchymal stem cells derived from the bone of patients affected by the disease were gene targeted to disrupt the mutant alleles causing the disease , converted to hips , expanded in vitro , and then induced to differentiate toward the osteogenic lineage . following in vivo implantation on ceramic scaffolds , gene - targeted osteogenic cells were able to form bone tissue in experimental animals . correction of genetic mutations was reported for other diseases , including 1-antitrypsin deficiency and x - linked chronic granulomatous disease , using the zinc finger nuclease technology , which holds great potential for efficient and precise gene manipulation at the desired genomic locations [ 99 , 100 ] . in summary , stem cell technology , along with the accurate genetic manipulation of hips , opens new possibilities for the generation of clinically relevant cells for autologous cell - based therapies . our growing understanding of the stem cell biology and the development of specific cell lineages has allowed the generation of multiple specialized cell types from stem cells , and the identification of distinct developmental stages that are governed by genetic and epigenetic regulatory networks . this way , abundant adult cells , such as dermal fibroblasts or adipocytes , could be used to produce other therapeutically important cells , such as neurons , cardiomyocytes or pancreatic beta cells . natural transdifferentiation involves a stepwise dedifferentiation of the primary cell to an intermediate cell type , that can then differentiate into the new lineage . in contrast , experimentally induced transdifferentiation involves a direct conversion through a simultaneous downregulation of one genetic programme and a concomitant upregulation of the new genetic programme . in one of the first experiments , muscle - specific genes were induced in pigmented epithelium , nerve , fat , liver , and fibroblast cells by forced expression of myod , a master regulator of muscle differentiation . however , it was not clear until 2008 how the procedure could be applied more generally to other cell types . interestingly , the resulting neurons silenced the donor cell transcriptional program , suggesting a binary lineage switch rather than an induction of hybrid cell phenotypes . however , a small but detectable epigenetic memory of the donor cells remained in the induced neurons . current examples of experimentally induced transdifferentiation rely on the ectopic expression of master regulator genes , which are essential for lineage specification during development and can direct the establishment of a new epigenetic state when expressed in the host cell . to achieve a complete phenotypic switch between the two mature cells and generate functional cells for potential clinical applications , the activation of a new epigenetic state in the host cells must be associated with full deactivation of the original epigenetic state . this might be more cumbersome to achieve between more distantly related cells owing to their larger epigenetic differences , resulting in relatively inaccessible chromosomal regions . possible strategies to convert more distant cell types could involve the use of chemicals that loosen the chromatin structure and favor epigenetic rearrangements . in addition , reduction of factors that support the original epigenetic state of the mature host cells , and the use of factors that promote the reentry into cell cycle could facilitate the transdifferentiation . with further development and optimization , transdifferentiation protocols could offer a shortcut to production of differentiated cells for biomedical research and clinical applications , potentially avoiding the extensive cell proliferation required for hips derivation and differentiation . in addition , transdifferentiation opens the possibility to convert different cell types directly in vivo to drive tissue repair and regeneration in situ , an approach that would be impossible using ips technology due to the teratoma - forming ability of pluripotent stem cells . hitherto , only a few instructive factor combinations have been reported to directly convert mature cell types , and it is likely that these will depend on the selected target and final cell types . as with the ips , a number of questions regarding the transdifferentiated cell stability , functionality , and safety need to be addressed , and a deeper understanding of the reprogramming process is needed before transdifferentiation can be translated into a therapeutic setting . advances in pluripotent stem cell biology and transdifferentiation have generated great enthusiasm in the scientific community over the last years , and are expected to affect numerous biomedical applications . hesc and hips allow the study and modeling of human development and tissue formation under diverse experimental conditions , and therefore represent an excellent platform for understanding human diseases and developing innovative therapeutic solutions . in parallel , development of transdifferentiation technology offers new insights into adult cell plasticity and could lead to expedited protocols for differentiated cell production . in the context of regenerative medicine , hesc and hips offer the possibility to generate an unlimited number of functional cells for reconstructive and reparative therapies . approval of the first hesc - based clinical trials in the usa is encouraging for the field , and signifies the potential of pluripotent stem cells to cure the most devastating diseases of our time . as with other medical breakthroughs , it is only via scrupulous scientific investigation that the true potential of human pluripotent stem cells may be realized .

solid - state fermentation which involves growth of microorganisms on moist solid substrates in the absence of free flowing water , recently , has received a considerable attention of researchers due to its several advantages over the submerged fermentation . solid substrates and wastes from different origins could be treated by solid - state fermentation and useful products could be produced . solid - state fermentation had a long history of production of traditional foods by using different organisms and is also found to have an increasing application in the production of enzymes , antibiotics , surfactants , biocides , and so forth . it had been reported that in solid - state fermentation , especially in fungal one , the productions of metabolites , such as enzymes , antibiotics , organic acids , and aroma compounds , are higher than that in submerged fermentation . bacillus subtilis strains produced a broad spectrum of bioactive compounds with great potential for biotechnological and biopharmaceutical applications including surfactin , fengycin , iturin , mycosubtilins , and bacillomycins , which are amphiphilic membrane - active biosurfactants with potent antimicrobial activities . these biosurfactants possessed , also , antiviral , antitumor , hemolytic , blood anticoagulant , and fibrinolytic activities . moreover , biosurfactants of bacillus subtilis have numerous environmental and biotechnological applications and have shown particular utility in oil recovery , remediation of soil contaminated by heavy metals , and biocontrol against phytopathogens and insects . the high production cost of biosurfactants has been a major concern towards restricted use in commercial applications [ 13 , 14 ] . the present study aimed at the optimization of fermentation conditions in the solid substrate fermentation . we have screened twelve solid substrates for the production of b. subtilis ( spb1 ) biosurfactant and its antimicrobial activity was determined against multidrug - resistant bacteria . a plackett - burman design was used to identify the most significant variables influencing biosurfactant production . a response surface methodology combined with a central composite design was then used to further optimize those variables , including temperature , inoculum age , and moisture content . b. subtilis spb1 ( hq392822 ) , used through this work , was a wild type strain isolated from tunisian soil . surfactant compounds were tested against a group of multiresistant bacteria isolated from clinical specimens : pseudomonas aeruginosa , pseudomonas stutzeri , escherichia coli , staphylococcus aureus , enteroccoccus faecalis , salmonella typhimirium , klebsiella pneumoniae , enterobacter cloacae , enterobacter faecium , and brevibacterium flavum , a group of fungi belonging to the culture collection of our laboratory : penicillium notatum , penicillium italicum , alternaria alternaria , puccinia allii , peronospora destructor , aspergillus oryzae , aspergillus niger , rhizopus oryzae , and two species of yeast : candida albicans and saccharomyces cerevisiae . the screening of biosurfactants producers was performed by blood haemolysis technique as described by master . it was identified by physiological - biochemical characterization and by 16s rna gene ribotyping . for the determination of 16s rdna sequences , this dna was used as template for the pcr using the universal primers rd1 ( 5aaggaggtgatccagcc3 ) and fd1 ( 5agtttgatcctggctcag3 ) and a gene amp pcr system 2700 ( applied biosystems ) . the 25 l of pcr mixture contained : 0.2 mm of deoxynucleoside triphosphate , 1.32 m concentration each primer , 0.5 u of dna polymerase , 5 l of 5x buffer and 1 l ( 5 ng ) of total dna template . the pcr programme was 94c for 3 min followed by 35 cycles consisting of denaturation at 94c for 45 s , annealing at 59c for 1 min and extension at 72c for 2 min . following amplification , the pcr product was purified ( promega gel extraction kit , biogne , tunisia ) and sequenced with abi prismtm 3100 genetic analyzer ( applied biosystems , usa ) . the clustalw program was used to make multiple alignments between all sequences using default parameters . b. subtilis spb1 strain was streaked on a nutrient agar slant and incubated at 37c . after 24 h , one loop of cells was dispensed in 3 ml of lb medium and incubated overnight at 37c . aliquots ( 0.2 ml ) were used to inoculate 250 ml erlenmeyer flasks containing 50 ml lb medium and incubated in a rotatory shaker at 200 rpm and 37c ( 0.5 ) during 4 , 11 , 14 , 23 , 24 , 31 h in accordance with the purpose . solid - state fermentation was conducted as follows : fifteen grams of substrates ( banana peels , potato peels , chick - pea flour , corn bran , corn starch , millet , soya meal , barley flour , barley bran , rice flour , cornstarch , and orange peels ) were placed into a 100 ml erlenmeyer flask , the mouth of which was stoppered with a cotton plug . then those substrates were autoclaved twice at 120c ( 1 bar ) for 30 min at an interval of 812 h to kill the spore - forming microorganisms inhabiting the material . after autoclaving , 75 l of kh2po4 ( 1 m ) , 225 l of mgso4 ( 1 m ) , and 367 l of deionised distilled water were aseptically added for the fortification of nutrients [ 20 , 21 ] , and three ml of a preculture of b. subtilis spb1 strain grown in the lb medium was inoculated . cultivation temperature varied from 18 , 25 , 28 , 30 , 35 , 37 , 42 , 45 , to 52c depending on the purpose . samples were collected at time - defined intervals for the determination of biosurfactant production yield . the crude biosurfactant was isolated from the cell - free broth of 24 h grown culture . the bacterial cells were removed from surfactant containing culture broth by centrifugation at 1062 g at 4c for 20 min . the supernatant was precipitated overnight at 4c by adding concentrated hcl to achieve a final ph of 2.0 in order to precipitate lipids and proteins . grey white pellets formed by precipitation were collected by centrifugation at 1062 g at 4c for 20 min . the crude surfactant was lyophilized and weighted for quantification . for the extraction of biosurfactant compounds , 50 ml of chloroform - methanol ( 2 : 1 v / v ) was added to 500 mg of the dry product and incubated in a rotatory shaker at 250 rpm , 30c ( 0.5 ) for 15 min . culture without inoculation was used as a negative control to take account possible contribution of lipids and proteins from substrates . crude biosurfactant weight was calculated as the result of subtracting the weight obtained with the negative control from that measured with the culture containing the biosurfactant producing strain . the values presented are the three determinations ' average results of the two separate experiments for each cultural condition . the antimicrobial activity of the biosurfactant was tested against several gram - positive and gram - negative bacteria using the agar spot assay method described by paik and et al . . each strain , used as an indicator , had been previously subcultured in its appropriate medium and temperature . a quantity of 5 ml of soft agar ( 0.7% agar ) containing about 10 cells of indicator strain was overlaid on 2.5% lb agar plates . the appearance of the inhibition zone was determined after 24 h of incubation using the appropriate medium and temperature . the values presented are the average of the results of three determinations of two separate experiments . samples ( 0.5 ml ) of cell - free supernatant were added to a screw - capped tube containing 7.5 ml of tris - mg ( 20 mm tris hcl ( ph 7.0 ) and 10 mm mgso4 ) and 0.1 ml of kerosene . after a vigorous vortex , the tubes were allowed to sit for 1 hour . the optimum temperature of the emulsification activity of the crude biosurfactant was determined by incubating 0.5 ml of supernatant of the culture with 7.5 ml of tris - mg ( 20 mm tris hcl ( ph 7.0 ) and 10 mm mgso4 ) and 0.1 ml of kerosene for 1 hour at different temperatures ranging from 25 to 90c . thermostability of spb1 biosurfactant was studied by the determination of the residual activity after a heat treatment during 60 min of crude biosurfactant at a range of temperatures ( 0 , 20 , 30 , 40 , 50 , 60 , 70 , and 80c ) . the optimum ph for biosurfactant activity was determined by incubating the supernatant in different buffers : 50 mmol l citrate buffer ( ph 3.06.0 ) , 50 mmol l phosphate buffer ( ph 6.08.0 ) , and 50 mmol l glycine - naoh buffer ( ph 9.0 - 10.0 ) . the stability at 4c in various ph conditions was assessed by incubating the biosurfactant at different ph and by measuring the residual activity after 24 h using the standard protocol . various industries byproducts like banana peels , potato peels , chick - pea flour , corn bran , corn flour , orange peels flour , soya meal , barley flour , barley its , rice flour , cornstarch , and millet were screened for b. subtilis spb1 biosurfactant production . the substrate yielding the maximum biosurfactant production was identified and selected for further studies using response surface methodology . moisture content of the substrate was estimated by drying 15 g of substrate to constant weight at 105c and the dry weight was recorded . to fix the initial moisture content of the solid medium the sample was then dried as described above , and moisture content ( % ) was calculated as follows : moisture content ( initial ) of solid medium ( 1)(%)=[(wt.of substratedry wt.)dry wt.]100 . the optimization of b. subtilis spb1 biosurfactant production was carried out using a statistical experimental design that consisted of two major steps . the first concerned the screening of the significant variables that affected the biosurfactant production and the second involved the further optimization of the relatively important variables . plackett - burman design , an efficient technique for medium component optimization , was used to screen the plackett - burman design was then used to evaluate the relative importance of five parameters for b. subtilis spb1 biosurfactant production in solid - state fermentation . in fact , this design does not consider the interaction effects among the variables and was used only to screen the important variables affecting biosurfactant production yield . the variables that were chosen for the present study were ( a ) temperature , ( b ) moisture , ( c ) initial ph , ( d ) inoculum age , and ( e ) inoculum size . the experimental design , with the names and actual levels of the variables , is presented in table 1 . the plackett - burman experimental design was based on the following first order model equation : ( 2)y^=b0+bixi , where y^ is the response ( biosurfactant production yield mg / g ) , b0 is the model intercepts , bi is the linear coefficient , and xi is the level of the independent variable . the effects of each variable on biosurfactant production were estimated as the difference between both averages of measurements made at the higher level and at the lower level . the next step in the optimization of solid - state fermentation conditions was to determine the optimum levels of the significant variables for b. subtilis spb1 biosurfactant production . for this purpose , a response surface methodology was adopted to maximize biosurfactant production using a central composite design . the significant variables that were selected were ( x1 ) temperature , ( x2 ) moisture , and ( x3 ) inoculum age . each variable was assessed at five coded levels ( 1.682 , 1 , 0 , + 1 , and + 1.682 ) . a total of 24 experiments were conducted including 15 experiments for the central composite design , three supplementary experiments in the domain centre to estimate the pure error and four other experiments to check the validity of the model . the response values ( y^ ) used in each trial were the average of three determinations of two separate experiments ( table 2 ) . the data obtained from the response surface methodology with regards to b. subtilis spb1 biosurfactant production were subjected to analysis of variance ( anova ) to check the errors and the significance of each parameter . the data were then subjected to a multiple regression analysis to obtain an empirical model that could relate the response measured to the independent variables . the behaviour of the system was explained by the following quadratic equation : ( 3)y^=b0+b1x1+b2x2+b3x3+b11x12+b22x22 + b33x32+b12x1x2+b13x1x3+b23x2x3 , where y^ refers to the predicted response , x1 , x2 , x3 to the independent coded variables , b0 to the offset term , b1 , b2 , b3 to the linear effects , b11 , b22 , b33 to the squared effects and b12 , b23 , b13 to the interaction terms . the statistical software package , ( nemrod - w by lprai marseilles , france ) was used to conduct a regression analysis on the experimental data and to plot the response surface graphs . the statistical significance of the model was determined by the application of fisher 's f test . the two - dimensional graphical representation of the system behaviour , called the isoresponse contour plot , was used to describe the individual and cumulative effects of the variables as well as the possible correlations existed between them . comparison was performed using a factorial anova with subsequent post hoc test ( ryan , einot , gabriel , & welsch ) . two hundred strains isolated from different locations of the south tunisia 's soil were screened for their ability to produce biosurfactants by using blood hemolysis assay [ 23 , 28 ] . several strains were selected on the basis of their high -hemolysis activities , since the size of the clear zone developed is in proportion to the amount of the produced biosurfactant . the 16s rdna sequence of strain spb1 was determined and deposited in the genebank database under accession number hq392822 . zones of inhibition diameter produced by different concentrations of biosurfactant were measured using agar spot assay method . as shown in table 3 , the compound showed higher activity against gram - positive cocci than that of gram - negative bacilli ( p < 0.05 ) . its activity was very effective against enterococcus faecalis , the halos presented a mean value of 18 mm and 20 mm when the compound was used at a concentration of 1 mg / ml and 2 mg / ml , respectively . it is important to note that this strain is resistant to at least two -lactams . moreover , the mean halo diameter obtained with enterobacter faecium and brevibacterium flavum was of 13 mm , while those obtained with escherichia coli , pseudomonas aeruginosa , pseudomonas stutzeri , klebsiella pneumoniae , enterobacter cloacae , and salmonella typhimirium varied from 9 to 15 mm with 1 mg / ml of the compound . indeed , it was clear that the activity against gram - negative bacteria was lower than that obtained with gram - positive bacteria ( p < 0.0001 ) . furthermore , as shown in table 3 , the biosurfactant produced by b. subtilis spb1 was also characterized by an important antifungal activity , especially , against penicillium notatum , penicillium italicum , and aspergillus niger and lower activity against rhizopus oryzae and aspergillus oryzae . moreover , it was clear that this compound presented an important antiyeast activity , mainly , against candida albicans . biosurfactant production by b. subtilis spb1 was studied with different solid substrates supplemented with nutrient medium . the studies on the production of spb1 biosurfactant using various solid substrates were carried for 72 h. in the case of potato peels , chick - pea flour , corn bran , corn flour , millet , soya bean meal , barley flour , and orange peels , the maximum biosurfactant production was observed on the first 24 h ( figure 1 ) , whereas in case of banana peels , barley its , rice flour and corn starch it was noted on the third day . among all the solid substrates used for our study , the highest biosurfactant production was found using millet . it was evaluated to 14 mg of biosurfactant per g of substrate after 24 h of culture . when varying millet concentrations , it was clear that the optimal production ( 14.5 mg of biosurfactant per g of substrate ) was obtained when using only 10 g of dehydrated substrate ( data not shown ) . the purpose of the first optimization step is to identify which factors , representing culture conditions , have significant effect on b. subtilis spb1 biosurfactant production . the adequacy of the model was calculated , and the variables evidencing statistically significant effects were screened via student 's t - test for anova ( table 4 ) . factors evidencing p values of less than 0.05 were considered to have significant effects on the response and were , therefore , selected for further optimization studies . among the variables screened , temperature , moisture , and inoculum age were identified as the most significant variables influencing biosurfactant production by b. subtilis spb1 . the values of the other variables were statistically insignificant and were , therefore , not considered in the subsequent analysis . the optimum levels of the three significant variables selected were further determined by performing a central composite design . the central composite experimental design was applied to find out the optimum conditions for b. subtilis spb1 biosurfactant production through the determination of the optimum levels of the significant factors temperature ( x1 ) , moisture ( x2 ) , and inoculums ( x3 ) . the results obtained were subjected to an anova to determine the significant differences . as shown in table 2 , there was a considerable variation in spb1 biosurfactant production yield which was heavily depended on the levels of the three independent variables in the medium . in fact , biosurfactant production yield varied from 12.47 mg / g ( run 2 ) to 19.50 mg / g ( run 12 ) . by applying a least - squares method to the experimental data , the following second - order polynomial equation was found to adequately explain the biosurfactant production yield by considering only the significant terms ( table 5 ) : ( 4)y=14,256 + 0,313x1 + 1,070x2 + 0,607x3 + 0,746x12 + 1,119x220,379x32 + 1,064x1x2 + 0,396x1x3 + 0,138x2x3 . the fit of the model was evaluated by the coefficient of determination r , which was 0.983 , indicating that 98.3% of the variability in the response could be explained by the model . the predicted r of 0.983 was in reasonable agreement with the adjusted r of 0.941 indicating that the regression model could be used to analyze trends of responses . the closer the value of r ( multiple correlation coefficient ) to 1 , the better the correlation between the observed and the predicted values is . the coefficient of variation cv indicates the degree of accuracy with which the treatments are compared . usually , the higher the value of cv , the lower the reliability of the experiment is . in this experiment the results from this test as well as those from anova yielded a very low p value , thus indicating that the model was highly significant and reliable ( table 6 ) . when the magnitude of the t test value is large and the p value is small , this indicates that the corresponding coefficient is highly significant . as far as the current study is concerned , the estimated parameters and the corresponding p values suggest that , among all the independent variables , x1 ( temperature ) , x2 ( moisture ) , and x3 ( inoculum age ) had a significant effect on spb1 biosurfactant production . the effect of the interaction of various physicochemical parameters on biosurfactant production by b. subtilis was investigated by plotting the response surface curves against any two independent variables while keeping the third independent variable at the 0 level . the interactive roles of temperature , moisture , and inoculum on biosurfactant production by b. subtilis spb1 are illustrated in the three - dimensional curves of the calculated response surface shown in figure 2 . these plots are helpful in studying the effects of the factors ' variation in the studied field and consequently , in determining the optimal experimental conditions . the 3d response surface curves and their respective contour plots provided information about the interaction between two parameters and allowing an easy prediction and interpretation of the optimum experimental conditions . figure 2(a ) indicated an interaction between moisture and temperature level . however a decrease was observed in spb1 biosurfactant production at low moisture level percentage . it showed that moisture content ( 93% ) was required with 35c temperature for high yield of the response due to the maximum microbial activity which might be related to a balance between temperature activity for oxygen penetration and water availability . figure 2(b ) , showing the response surface plot , indicated no significant interaction between moisture and inoculum age . it depicted that a linear increase in spb1 biosurfactant production was observed when moisture was increased up to 93% . figure 2(c ) showed an interaction between temperature and inoculum age indicating that a maximum biosurfactant production was observed when temperature was at 35c and inoculum age was at 25 h. the model was verified for the three variables within the design space . four combinations of production conditions were selected by the software and were then tested for b. subtilis spb1 biosurfactant production ( table 2 ) . the experimental values of biosurfactant production that were determined were found to be in a good agreement with those that were statistically predicted by the model ( r = 0.983 ) , thus confirming the authenticity and reliability of the model . in addition , the average error ( difference between observed and predicted value ) was close to zero , indicating the absence of bias in the predictions made by the model . the value of x1 ( temperature ) , x2 ( moisture ) , and x3 ( inoculum age ) were found to be 37c , 88% , and 14 h , respectively . the biosurfactant production yield was 20.8 mg / g while the predicted value was 19.15 mg / g 1.36 . it is the highest reported to date for b. subtilis species in solid - state fermentation . the time course of the biosurfactant production by b. subtilis spb1 for both the optimized and the unoptimized media are shown in figure 3 . in the optimum conditions , the biosynthesis of the biosurfactant started since the first hour of culture and reached a maximum after 24 hours . the maximum of the biosurfactant production obtained under unoptimized conditions was only 12.5 after 24 h. by optimising the medium composition and the culture conditions , the production of spb1 biosurfactant was enhanced from 12.5 mg / g to 20.8 mg / g . the thermoactivity and thermostability of the crude biosurfactant from b. subtilis are shown in figure 4(a ) . this bioemulsifier retained more than 45% and 25% of its activity when incubated at 60 and 80c , respectively . moreover , up to 60c , it was completely stable and retained more than 80% and 50% of its activity after incubation during 60 min at 70 and 80c , respectively . the optimum of biosurfactant activity was shown for ph ranging from 5 to 9 and b. subtilis spb1 biosurfactant was very stable over a considerable ph range from 2 to 9 ( figure 4(b ) ) . the production of surface active compounds , or biosurfactants , by microorganisms has been a subject of increasing interest in recent years , especially due to their potential applications in biotechnological and biopharmaceutical domains . the hydrophilic part of biosurfactant ( the cationic part ) is proposed to initiate electrostatic interaction with the negatively charged component of the membrane . thus , hemolytic activity appears to be a good screening criterion for surfactant producing strains . therefore , using such strategy , b. subtilis spb1 strain was selected as the highest biosurfactant producer strain based on the size of the created hemolysis zone . several biosurfactants which exhibit antimicrobial activity against various microorganisms they include surfactin and iturin produced by b. subtilis strains , rhamnolipids from pseudomonas species , mannosylerythritol lipids from candida antarctica , and biosurfactants produced by some fungi . moreover , there are few reports about the antimicrobial activity of biosurfactants isolated from lactobacilli ; only biosurfactants obtained from s. thermophilus a and l. lactis 53 showed significant antimicrobial activity against several bacterial and yeast strains isolated from explanted voice prostheses . the biosurfactant isolated in the present study exhibited a broad spectrum of action , including antimicrobial activity against microorganisms with multidrug - resistant profiles . the compound showed higher activity against gram - positive cocci than against gram - negative bacilli . these results are of great interest , since these microorganisms have natural resistance to aztreonam , co - trimoxazole , cephalosporins , chloramphenicol , and clindamycin and low sensibility to aminoglycosides and penicillin g as discussed by furtado et al . . the antimicrobial activity of the compounds was also considerable against staphylococcus aureus which is known to be resistant to at least two -lactams . moreover , it was clear that the activity against gram - negative bacteria was lower when compared to gram - positive bacteria . singh and cameotra have also observed that the lipopeptide n1 , produced by b. subtilis c1 , was active against several microorganisms , especially s. aureus and mycobacterium sp . recently , interest in the potential industrial application of b. subtilis biosurfactants in medicine has increased . this is partially because their antiadhesive activity against a variety of pathogens was indicative of their potential use in the protection of biomaterials . furthermore , the biosurfactant produced by b. subtilis spb1 was also characterized by an important antifungal activity , especially , against penicillium notatum , penicillium italicum , and aspergillus niger and lower activity against rhizopus oryzae and aspergillus oryzae . this activity was clearly negative against alternaria alternata , puccinia allii , and peronospora destructor . moreover , it was clear that this compound presented also an important antiyeast activity , mainly , against candida albicans . despite possessing many commercially attractive properties and clear advantages compared with their synthetic counterparts , the production of microbial surfactants on a commercial scale has not been realized because of their low yields and high production costs . in light of the economic constraints associated with biosurfactant production , essentially two basic strategies have been explored to a greater extent and have been reported to be effective in substantially increasing the production of biosurfactants . they corresponded , respectively , to the use of cheaper and waste substrates to lower the initial raw material costs involved in the process and the development of efficient bioprocesses , including optimization of the culture conditions for maximum biosurfactant production . maximizing productivity or minimizing production costs the classical method of medium optimization is laborious , time consuming , and does not guarantee the determination of the optimal conditions for metabolite production . to tackle this problem and make the optimization process easier , a statistical optimization strategy based on response surface methodology has been used by various investigators . this method was successfully used to determine the optimum media , inoculum and environmental conditions for the enhanced biosurfactant production by b. subtilis . these optimization methods would help the industry to design the best media containing cheaper substrates and to use the most favourable environmental conditions for improved biosurfactant production . there were many reports on the production of lipopeptide biosurfactants by b. subtilis species in solid - state fermentation [ 43 , 44 ] . biosurfactant production was 9.26 mg / g when b. subtilis s3 was cultivated for five days in solid - state fermentation containing high gluten flour and rice bran . b. subtilis rb14-cs produced 14 mg / g dry solid material after four days of fermentation using soybean crude residue ( okara ) . after that , response surface methodology was applied to predict the optimum amounts of the carbon and nitrogen source for biosurfactant production by b. subtilis in solid - state fermentation , in that the maximum production yield was 15.591 mg / g under optimized condition . in the present study , b. subtilis spb1 biosurfactant production yield was increased significantly through application of the solid - state fermentation and the response surface methodology . the central composite design exploited in the present study enabled us to investigate the culture conditions that support biosurfactant overproduction . a high degree of similarity was observed between the predicted and experimental values that reflected the accuracy and applicability of the response surface methodology to optimise the process for biosurfactant production . the maximum production was achieved at temperature of 37c , moisture of 88% , and inoculum age of 14 h. excess or diminution in one of these values may cause a disruption in the corresponding production yield . the yield of the biosurfactant ( 20.8 mg / g ) produced by b. subtilis spb1 reported in this study is the highest reported to date for b. subtilis species in solid - state fermentation . in addition , the use of millet as a substrate in solid - state fermentation for biosurfactants production by b. subtilis is unique . by increasing the biosurfactant yield via this experimental design approach , the production cost would markedly be reduced , enhancing feasibility of commercial application of this powerful biosurfactant . showing that biosurfactant from bacillus licheniformis 86 was stable in the temperatures range of 25120c . therefore , the stability of this biosurfactant over a wide range of ph and after heat treatment indicates that it preserves its activity at extreme conditions , which is an extremely interesting feature in view of its potential use in detergent industries and bioremediation of hydrocarbons .

during the last years , several applications of biosurfactants with medical purposes have been reported . biosurfactants are considered relevant molecules for applications in combating many diseases . however , their use is currently extremely limited due to their high cost in relation to that of chemical surfactants . use of inexpensive substrates can drastically decrease its production cost . here , twelve solid substrates were screened for the production of bacillus subtilis spb1 biosurfactant and the maximum yield was found with millet . a plackett - burman design was then used to evaluate the effects of five variables ( temperature , moisture , initial ph , inoculum age , and inoculum size ) . statistical analyses showed that temperature , inoculum age , and moisture content had significantly positive effect on spb1 biosurfactant production . their values were further optimized using a central composite design and a response surface methodology . the optimal conditions of temperature , inoculum age , and moisture content obtained under the conditions of study were 37c , 14 h , and 88% , respectively . the evaluation of the antimicrobial activity of this compound was carried out against 11 bacteria and 8 fungi . the results demonstrated that this biosurfactant exhibited an important antimicrobial activity against microorganisms with multidrug - resistant profiles . its activity was very effective against staphylococcus aureus , staphylococcus xylosus , enterococcus faecalis , klebsiella pneumonia , and so forth .

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

solid - state fermentation which involves growth of microorganisms on moist solid substrates in the absence of free flowing water , recently , has received a considerable attention of researchers due to its several advantages over the submerged fermentation . solid - state fermentation had a long history of production of traditional foods by using different organisms and is also found to have an increasing application in the production of enzymes , antibiotics , surfactants , biocides , and so forth . bacillus subtilis strains produced a broad spectrum of bioactive compounds with great potential for biotechnological and biopharmaceutical applications including surfactin , fengycin , iturin , mycosubtilins , and bacillomycins , which are amphiphilic membrane - active biosurfactants with potent antimicrobial activities . moreover , biosurfactants of bacillus subtilis have numerous environmental and biotechnological applications and have shown particular utility in oil recovery , remediation of soil contaminated by heavy metals , and biocontrol against phytopathogens and insects . the high production cost of biosurfactants has been a major concern towards restricted use in commercial applications [ 13 , 14 ] . we have screened twelve solid substrates for the production of b. subtilis ( spb1 ) biosurfactant and its antimicrobial activity was determined against multidrug - resistant bacteria . a plackett - burman design was used to identify the most significant variables influencing biosurfactant production . a response surface methodology combined with a central composite design was then used to further optimize those variables , including temperature , inoculum age , and moisture content . surfactant compounds were tested against a group of multiresistant bacteria isolated from clinical specimens : pseudomonas aeruginosa , pseudomonas stutzeri , escherichia coli , staphylococcus aureus , enteroccoccus faecalis , salmonella typhimirium , klebsiella pneumoniae , enterobacter cloacae , enterobacter faecium , and brevibacterium flavum , a group of fungi belonging to the culture collection of our laboratory : penicillium notatum , penicillium italicum , alternaria alternaria , puccinia allii , peronospora destructor , aspergillus oryzae , aspergillus niger , rhizopus oryzae , and two species of yeast : candida albicans and saccharomyces cerevisiae . for the determination of 16s rdna sequences , this dna was used as template for the pcr using the universal primers rd1 ( 5aaggaggtgatccagcc3 ) and fd1 ( 5agtttgatcctggctcag3 ) and a gene amp pcr system 2700 ( applied biosystems ) . aliquots ( 0.2 ml ) were used to inoculate 250 ml erlenmeyer flasks containing 50 ml lb medium and incubated in a rotatory shaker at 200 rpm and 37c ( 0.5 ) during 4 , 11 , 14 , 23 , 24 , 31 h in accordance with the purpose . after autoclaving , 75 l of kh2po4 ( 1 m ) , 225 l of mgso4 ( 1 m ) , and 367 l of deionised distilled water were aseptically added for the fortification of nutrients [ 20 , 21 ] , and three ml of a preculture of b. subtilis spb1 strain grown in the lb medium was inoculated . the antimicrobial activity of the biosurfactant was tested against several gram - positive and gram - negative bacteria using the agar spot assay method described by paik and et al . the values presented are the average of the results of three determinations of two separate experiments . thermostability of spb1 biosurfactant was studied by the determination of the residual activity after a heat treatment during 60 min of crude biosurfactant at a range of temperatures ( 0 , 20 , 30 , 40 , 50 , 60 , 70 , and 80c ) . the optimum ph for biosurfactant activity was determined by incubating the supernatant in different buffers : 50 mmol l citrate buffer ( ph 3.06.0 ) , 50 mmol l phosphate buffer ( ph 6.08.0 ) , and 50 mmol l glycine - naoh buffer ( ph 9.0 - 10.0 ) . various industries byproducts like banana peels , potato peels , chick - pea flour , corn bran , corn flour , orange peels flour , soya meal , barley flour , barley its , rice flour , cornstarch , and millet were screened for b. subtilis spb1 biosurfactant production . the substrate yielding the maximum biosurfactant production was identified and selected for further studies using response surface methodology . moisture content of the substrate was estimated by drying 15 g of substrate to constant weight at 105c and the dry weight was recorded . to fix the initial moisture content of the solid medium the sample was then dried as described above , and moisture content ( % ) was calculated as follows : moisture content ( initial ) of solid medium ( 1)(%)=[(wt.of substratedry wt. the optimization of b. subtilis spb1 biosurfactant production was carried out using a statistical experimental design that consisted of two major steps . the first concerned the screening of the significant variables that affected the biosurfactant production and the second involved the further optimization of the relatively important variables . plackett - burman design , an efficient technique for medium component optimization , was used to screen the plackett - burman design was then used to evaluate the relative importance of five parameters for b. subtilis spb1 biosurfactant production in solid - state fermentation . the variables that were chosen for the present study were ( a ) temperature , ( b ) moisture , ( c ) initial ph , ( d ) inoculum age , and ( e ) inoculum size . the plackett - burman experimental design was based on the following first order model equation : ( 2)y^=b0+bixi , where y^ is the response ( biosurfactant production yield mg / g ) , b0 is the model intercepts , bi is the linear coefficient , and xi is the level of the independent variable . the effects of each variable on biosurfactant production were estimated as the difference between both averages of measurements made at the higher level and at the lower level . the next step in the optimization of solid - state fermentation conditions was to determine the optimum levels of the significant variables for b. subtilis spb1 biosurfactant production . for this purpose , a response surface methodology was adopted to maximize biosurfactant production using a central composite design . the significant variables that were selected were ( x1 ) temperature , ( x2 ) moisture , and ( x3 ) inoculum age . a total of 24 experiments were conducted including 15 experiments for the central composite design , three supplementary experiments in the domain centre to estimate the pure error and four other experiments to check the validity of the model . the data obtained from the response surface methodology with regards to b. subtilis spb1 biosurfactant production were subjected to analysis of variance ( anova ) to check the errors and the significance of each parameter . the statistical software package , ( nemrod - w by lprai marseilles , france ) was used to conduct a regression analysis on the experimental data and to plot the response surface graphs . the two - dimensional graphical representation of the system behaviour , called the isoresponse contour plot , was used to describe the individual and cumulative effects of the variables as well as the possible correlations existed between them . two hundred strains isolated from different locations of the south tunisia 's soil were screened for their ability to produce biosurfactants by using blood hemolysis assay [ 23 , 28 ] . several strains were selected on the basis of their high -hemolysis activities , since the size of the clear zone developed is in proportion to the amount of the produced biosurfactant . as shown in table 3 , the compound showed higher activity against gram - positive cocci than that of gram - negative bacilli ( p < 0.05 ) . its activity was very effective against enterococcus faecalis , the halos presented a mean value of 18 mm and 20 mm when the compound was used at a concentration of 1 mg / ml and 2 mg / ml , respectively . moreover , the mean halo diameter obtained with enterobacter faecium and brevibacterium flavum was of 13 mm , while those obtained with escherichia coli , pseudomonas aeruginosa , pseudomonas stutzeri , klebsiella pneumoniae , enterobacter cloacae , and salmonella typhimirium varied from 9 to 15 mm with 1 mg / ml of the compound . furthermore , as shown in table 3 , the biosurfactant produced by b. subtilis spb1 was also characterized by an important antifungal activity , especially , against penicillium notatum , penicillium italicum , and aspergillus niger and lower activity against rhizopus oryzae and aspergillus oryzae . moreover , it was clear that this compound presented an important antiyeast activity , mainly , against candida albicans . biosurfactant production by b. subtilis spb1 was studied with different solid substrates supplemented with nutrient medium . the studies on the production of spb1 biosurfactant using various solid substrates were carried for 72 h. in the case of potato peels , chick - pea flour , corn bran , corn flour , millet , soya bean meal , barley flour , and orange peels , the maximum biosurfactant production was observed on the first 24 h ( figure 1 ) , whereas in case of banana peels , barley its , rice flour and corn starch it was noted on the third day . among all the solid substrates used for our study , the highest biosurfactant production was found using millet . the purpose of the first optimization step is to identify which factors , representing culture conditions , have significant effect on b. subtilis spb1 biosurfactant production . the adequacy of the model was calculated , and the variables evidencing statistically significant effects were screened via student 's t - test for anova ( table 4 ) . among the variables screened , temperature , moisture , and inoculum age were identified as the most significant variables influencing biosurfactant production by b. subtilis spb1 . the optimum levels of the three significant variables selected were further determined by performing a central composite design . the central composite experimental design was applied to find out the optimum conditions for b. subtilis spb1 biosurfactant production through the determination of the optimum levels of the significant factors temperature ( x1 ) , moisture ( x2 ) , and inoculums ( x3 ) . as shown in table 2 , there was a considerable variation in spb1 biosurfactant production yield which was heavily depended on the levels of the three independent variables in the medium . by applying a least - squares method to the experimental data , the following second - order polynomial equation was found to adequately explain the biosurfactant production yield by considering only the significant terms ( table 5 ) : ( 4)y=14,256 + 0,313x1 + 1,070x2 + 0,607x3 + 0,746x12 + 1,119x220,379x32 + 1,064x1x2 + 0,396x1x3 + 0,138x2x3 . when the magnitude of the t test value is large and the p value is small , this indicates that the corresponding coefficient is highly significant . as far as the current study is concerned , the estimated parameters and the corresponding p values suggest that , among all the independent variables , x1 ( temperature ) , x2 ( moisture ) , and x3 ( inoculum age ) had a significant effect on spb1 biosurfactant production . the effect of the interaction of various physicochemical parameters on biosurfactant production by b. subtilis was investigated by plotting the response surface curves against any two independent variables while keeping the third independent variable at the 0 level . the interactive roles of temperature , moisture , and inoculum on biosurfactant production by b. subtilis spb1 are illustrated in the three - dimensional curves of the calculated response surface shown in figure 2 . these plots are helpful in studying the effects of the factors ' variation in the studied field and consequently , in determining the optimal experimental conditions . however a decrease was observed in spb1 biosurfactant production at low moisture level percentage . it showed that moisture content ( 93% ) was required with 35c temperature for high yield of the response due to the maximum microbial activity which might be related to a balance between temperature activity for oxygen penetration and water availability . figure 2(b ) , showing the response surface plot , indicated no significant interaction between moisture and inoculum age . it depicted that a linear increase in spb1 biosurfactant production was observed when moisture was increased up to 93% . figure 2(c ) showed an interaction between temperature and inoculum age indicating that a maximum biosurfactant production was observed when temperature was at 35c and inoculum age was at 25 h. the model was verified for the three variables within the design space . four combinations of production conditions were selected by the software and were then tested for b. subtilis spb1 biosurfactant production ( table 2 ) . the experimental values of biosurfactant production that were determined were found to be in a good agreement with those that were statistically predicted by the model ( r = 0.983 ) , thus confirming the authenticity and reliability of the model . the value of x1 ( temperature ) , x2 ( moisture ) , and x3 ( inoculum age ) were found to be 37c , 88% , and 14 h , respectively . the time course of the biosurfactant production by b. subtilis spb1 for both the optimized and the unoptimized media are shown in figure 3 . the maximum of the biosurfactant production obtained under unoptimized conditions was only 12.5 after 24 h. by optimising the medium composition and the culture conditions , the production of spb1 biosurfactant was enhanced from 12.5 mg / g to 20.8 mg / g . this bioemulsifier retained more than 45% and 25% of its activity when incubated at 60 and 80c , respectively . the optimum of biosurfactant activity was shown for ph ranging from 5 to 9 and b. subtilis spb1 biosurfactant was very stable over a considerable ph range from 2 to 9 ( figure 4(b ) ) . the production of surface active compounds , or biosurfactants , by microorganisms has been a subject of increasing interest in recent years , especially due to their potential applications in biotechnological and biopharmaceutical domains . therefore , using such strategy , b. subtilis spb1 strain was selected as the highest biosurfactant producer strain based on the size of the created hemolysis zone . several biosurfactants which exhibit antimicrobial activity against various microorganisms they include surfactin and iturin produced by b. subtilis strains , rhamnolipids from pseudomonas species , mannosylerythritol lipids from candida antarctica , and biosurfactants produced by some fungi . moreover , there are few reports about the antimicrobial activity of biosurfactants isolated from lactobacilli ; only biosurfactants obtained from s. thermophilus a and l. lactis 53 showed significant antimicrobial activity against several bacterial and yeast strains isolated from explanted voice prostheses . the biosurfactant isolated in the present study exhibited a broad spectrum of action , including antimicrobial activity against microorganisms with multidrug - resistant profiles . the antimicrobial activity of the compounds was also considerable against staphylococcus aureus which is known to be resistant to at least two -lactams . furthermore , the biosurfactant produced by b. subtilis spb1 was also characterized by an important antifungal activity , especially , against penicillium notatum , penicillium italicum , and aspergillus niger and lower activity against rhizopus oryzae and aspergillus oryzae . moreover , it was clear that this compound presented also an important antiyeast activity , mainly , against candida albicans . in light of the economic constraints associated with biosurfactant production , essentially two basic strategies have been explored to a greater extent and have been reported to be effective in substantially increasing the production of biosurfactants . they corresponded , respectively , to the use of cheaper and waste substrates to lower the initial raw material costs involved in the process and the development of efficient bioprocesses , including optimization of the culture conditions for maximum biosurfactant production . maximizing productivity or minimizing production costs the classical method of medium optimization is laborious , time consuming , and does not guarantee the determination of the optimal conditions for metabolite production . to tackle this problem and make the optimization process easier , a statistical optimization strategy based on response surface methodology has been used by various investigators . this method was successfully used to determine the optimum media , inoculum and environmental conditions for the enhanced biosurfactant production by b. subtilis . there were many reports on the production of lipopeptide biosurfactants by b. subtilis species in solid - state fermentation [ 43 , 44 ] . after that , response surface methodology was applied to predict the optimum amounts of the carbon and nitrogen source for biosurfactant production by b. subtilis in solid - state fermentation , in that the maximum production yield was 15.591 mg / g under optimized condition . in the present study , b. subtilis spb1 biosurfactant production yield was increased significantly through application of the solid - state fermentation and the response surface methodology . the central composite design exploited in the present study enabled us to investigate the culture conditions that support biosurfactant overproduction . a high degree of similarity was observed between the predicted and experimental values that reflected the accuracy and applicability of the response surface methodology to optimise the process for biosurfactant production . the maximum production was achieved at temperature of 37c , moisture of 88% , and inoculum age of 14 h. excess or diminution in one of these values may cause a disruption in the corresponding production yield . by increasing the biosurfactant yield via this experimental design approach , the production cost would markedly be reduced , enhancing feasibility of commercial application of this powerful biosurfactant . therefore , the stability of this biosurfactant over a wide range of ph and after heat treatment indicates that it preserves its activity at extreme conditions , which is an extremely interesting feature in view of its potential use in detergent industries and bioremediation of hydrocarbons .

there is a growing armamentarium of effective medical therapies to treat neurological diseases.1,2 national guidelines have discussed ten us food and drug administration ( fda)-approved disease - modifying therapies for multiple sclerosis , > 20 fda - approved antiepileptic drugs for seizure prophylaxis , and 23 drugs involved in stroke prevention , including novel oral anticoagulants.35 most of these therapies are expensive and adherence is critical to their effectiveness.68 medication adherence depends on many factors , including affordable access to drugs , which varies by drug type and insurance - benefit design . for instance , medicare beneficiaries obtain outpatient drug coverage through the part d program , which is administered by private insurance plans , i.e. , part d drug plans . the formulary protections of these plans apply to formulary inclusion , but not tier placement . the resulting differences in out - of - pocket payments can be challenging for beneficiaries with limited incomes or impaired cognition.9 in addition , since adherence depends mainly on patient reporting and perception , it has been demonstrated that many patients who claim to have been adherent to their medications are in fact not.10,11 according to a recent study on therapeutic drug monitoring in consecutive emergency hospital admissions for seizures , > 40% of nonadherent patients claimed to have been adherent when further asked about medication failure.12 medication nonadherence has been shown to seriously hurt health outcomes in neurology , and hence efforts to understand the causes and solutions to this problem have become increasingly important . some studies have shown a moderate association between drug choice , adherence , and satisfaction with treatment.7,13 previous studies of medication nonadherence in neurology have been limited to population - wide databases and reliable claims - based algorithms.1424 these large - scale approaches to the evaluation of medication adherence using retrospective electronic health records ( ehrs ) abstraction or claims data have missed critical information and fallen short in representing the real - world complexity.25 in an effort to capture more accurate , specific , and informative data on medication nonadherence , systematic collection of patient - reported outcomes offers great promise.26,27 the term patient - reported outcomes refers to data directly collected from patients such as self - administered surveys and other attempts to quantify patients behavior and their subjective experience of health . patient - reported data have been shown to correlate with and augment more typical clinical measurements , proving useful in the assessment of treatment outcomes and determinants of health.2831 this study examined the frequency and determinants of financial barriers to medication adherence among individuals treated for neurological disorders using patient - reported data . this study was a retrospective review of data collected as part of a quality improvement project implemented in neurology ambulatory clinical practices that began in july 2015 and continues to date . since july 2015 , all ambulatory neurology patients in the outpatient waiting room aged 18 + years have been offered an ipad survey upon arrival at the office , except for unaccompanied non - english speakers , whose eligibility was determined by front desk staff based on the patient s observed inability to understand very simple survey directions . the front desk staff had received standard institutional training to determine patients ability to read and sign check - in - related documents ( eg , insurance - driven pre - office forms ) . all patients were given the opportunity to verbally opt out of the survey and were reminded that the quality of their care would not be adversely affected by survey completion . patients who arrived late for appointments or had other essential tasks to complete prior to the physician visit were at times unable to complete the survey prior to the end of the office visit . although longitudinal data were captured for patients with several visits during this period , only the first visit was included in the analysis.7,32,33 this study combines information from the survey merged by deterministic linkage using the medical record number to administrative and clinical data gathered using a research patient data registry ( rpdr ) query tool . in ambulatory neurology clinics , patients were checked in by front desk staff before being seen by the provider . after determining patient appropriateness , the front desk staff handed patients an ipad tablet pre - loaded with the survey questions . patients completed the survey in the waiting room . before beginning , they were prompted to read and agree to an informed consent disclaimer , which they signed on the apple ipad2 ( apple , ca , usa ) screen . a report summarizing the patients survey responses became available in the ehr immediately upon completion , and providers were encouraged to review this information with the patient . the survey had four parts an introduction , demographic questions , and the medication adherence question , how often in the past 12 months did you decide not to fill or refill your prescription because the medicines cost too much ? with the answers often , sometimes , never , and prefer not to answer . after this question , the survey included an additional questionnaire to measure global health function , which is not the focus of this article and is detailed in the supplementary materials . to determine the primary predictors of participation and examine the potential for selection bias , we gathered additional demographic information using the partners healthcare clinical data registry for all arriving patients ( both participating and nonparticipating in the survey ) seen in the ambulatory neurology clinics during the study time frame . the rpdr is a clinical data registry that aggregates data from sources throughout the partners healthcare system ( phs ) , including the medical record , reports , claims , and administrative systems . queried variables included age , gender , preferred language , insurance status , ethnicity , marital status , and clinic type ( eg , general neurology vs subspecialty area ) . results of the rpdr query identified patients scheduled for an ambulatory neurology clinical visit from july 2015 to september 2015 . a member of the study staff reviewed the rpdr query output to exclude ineligible patients or encounters . eligible encounters were the patients > 18 years of age seen at the neurology outpatient clinic ( main campus only ) , who had a first visit or were established patients who came for a follow - up visit within the study time frame ( subsequent visits were excluded ) . we defined cost - related medication nonadherence as an answer of often or sometimes to the question how often in the past 12 months did you decide not to fill or refill a prescription because the medicines cost too much ? . we performed univariate comparisons of the baseline characteristics of the adherence groups using two - group two - sided t - tests for continuous variables and chi - square tests for categorical variables . in multivariable analysis , we utilized logistic regression models to assess the associations between sociodemographic and clinical characteristics associated with the report of cost - related medication nonadherence . the independent variables were selected based on prior knowledge and examination of the distribution of the univariate analysis . conservatively , no step - wise approach was used . to address the potential for selection bias in our respondents , we performed univariate analysis of covariates associated with survey participation ( participants vs nonparticipants , including age , gender , ethnicity , preferred language , insurance type , marital status , and clinic type ) . this study was conducted under a protocol approved by the partners healthcare institutional review board . this study was a retrospective review of data collected as part of a quality improvement project implemented in neurology ambulatory clinical practices that began in july 2015 and continues to date . since july 2015 , all ambulatory neurology patients in the outpatient waiting room aged 18 + years have been offered an ipad survey upon arrival at the office , except for unaccompanied non - english speakers , whose eligibility was determined by front desk staff based on the patient s observed inability to understand very simple survey directions . the front desk staff had received standard institutional training to determine patients ability to read and sign check - in - related documents ( eg , insurance - driven pre - office forms ) . all patients were given the opportunity to verbally opt out of the survey and were reminded that the quality of their care would not be adversely affected by survey completion . patients who arrived late for appointments or had other essential tasks to complete prior to the physician visit were at times unable to complete the survey prior to the end of the office visit . although longitudinal data were captured for patients with several visits during this period , only the first visit was included in the analysis.7,32,33 this study combines information from the survey merged by deterministic linkage using the medical record number to administrative and clinical data gathered using a research patient data registry ( rpdr ) query tool . in ambulatory neurology clinics , patients were checked in by front desk staff before being seen by the provider . after determining patient appropriateness , the front desk staff handed patients an ipad tablet pre - loaded with the survey questions . patients completed the survey in the waiting room . before beginning , they were prompted to read and agree to an informed consent disclaimer , which they signed on the apple ipad2 ( apple , ca , usa ) screen . a report summarizing the patients survey responses became available in the ehr immediately upon completion , and providers were encouraged to review this information with the patient . the survey had four parts an introduction , demographic questions , and the medication adherence question , how often in the past 12 months did you decide not to fill or refill your prescription because the medicines cost too much ? with the answers often , sometimes , never , and prefer not to answer . after this question , the survey included an additional questionnaire to measure global health function , which is not the focus of this article and is detailed in the supplementary materials . we anticipated that not all patients would be able to participate in the survey . to determine the primary predictors of participation and examine the potential for selection bias , we gathered additional demographic information using the partners healthcare clinical data registry for all arriving patients ( both participating and nonparticipating in the survey ) seen in the ambulatory neurology clinics during the study time frame . the rpdr is a clinical data registry that aggregates data from sources throughout the partners healthcare system ( phs ) , including the medical record , reports , claims , and administrative systems . queried variables included age , gender , preferred language , insurance status , ethnicity , marital status , and clinic type ( eg , general neurology vs subspecialty area ) . results of the rpdr query identified patients scheduled for an ambulatory neurology clinical visit from july 2015 to september 2015 . a member of the study staff reviewed the rpdr query output to exclude ineligible patients or encounters . eligible encounters were the patients > 18 years of age seen at the neurology outpatient clinic ( main campus only ) , who had a first visit or were established patients who came for a follow - up visit within the study time frame ( subsequent visits were excluded ) . in ambulatory neurology clinics , patients were checked in by front desk staff before being seen by the provider . after determining patient appropriateness , the front desk staff handed patients an ipad tablet pre - loaded with the survey questions . patients completed the survey in the waiting room . before beginning , they were prompted to read and agree to an informed consent disclaimer , which they signed on the apple ipad2 ( apple , ca , usa ) screen . a report summarizing the patients survey responses became available in the ehr immediately upon completion , and providers were encouraged to review this information with the patient . the survey had four parts an introduction , demographic questions , and the medication adherence question , how often in the past 12 months did you decide not to fill or refill your prescription because the medicines cost too much ? with the answers often , sometimes , never , and prefer not to answer . after this question , the survey included an additional questionnaire to measure global health function , which is not the focus of this article and is detailed in the supplementary materials . we anticipated that not all patients would be able to participate in the survey . to determine the primary predictors of participation and examine the potential for selection bias , we gathered additional demographic information using the partners healthcare clinical data registry for all arriving patients ( both participating and nonparticipating in the survey ) seen in the ambulatory neurology clinics during the study time frame . the rpdr is a clinical data registry that aggregates data from sources throughout the partners healthcare system ( phs ) , including the medical record , reports , claims , and administrative systems . queried variables included age , gender , preferred language , insurance status , ethnicity , marital status , and clinic type ( eg , general neurology vs subspecialty area ) . results of the rpdr query identified patients scheduled for an ambulatory neurology clinical visit from july 2015 to september 2015 . a member of the study staff reviewed the rpdr query output to exclude ineligible patients or encounters . eligible encounters were the patients > 18 years of age seen at the neurology outpatient clinic ( main campus only ) , who had a first visit or were established patients who came for a follow - up visit within the study time frame ( subsequent visits were excluded ) . we defined cost - related medication nonadherence as an answer of often or sometimes to the question how often in the past 12 months did you decide not to fill or refill a prescription because the medicines cost too much ? . we performed univariate comparisons of the baseline characteristics of the adherence groups using two - group two - sided t - tests for continuous variables and chi - square tests for categorical variables . in multivariable analysis , we utilized logistic regression models to assess the associations between sociodemographic and clinical characteristics associated with the report of cost - related medication nonadherence . the independent variables were selected based on prior knowledge and examination of the distribution of the univariate analysis . conservatively , no step - wise approach was used . to address the potential for selection bias in our respondents , we performed univariate analysis of covariates associated with survey participation ( participants vs nonparticipants , including age , gender , ethnicity , preferred language , insurance type , marital status , and clinic type ) . this study was conducted under a protocol approved by the partners healthcare institutional review board . a total of 2992 out of 6075 patients participated in the survey and 2716 fully completed the survey questions , yielding a participation rate of 49% and a completion rate of 45% ( figure s1 ) . the mean age of participants was 56 ( sd : 18 ) years and 1613 ( 54% ) were females . survey responders more often identified english as their preferred language ( 90.6% , p=0.02 ; table s1 ) . among the participants , 9.8% ( n=265 ) of patients reported cost - related medication nonadherence . more specifically , nonadherent patients often ( 40 or 15.1% ) or sometimes ( 225 or 84.9% ) failed to fill or refill a prescription ( figure 1 ) . patients who reported cost - related nonadherence were younger ( mean age : 5316 years vs 5618 years , p<0.01 ) , more often hispanic ( 16.7% vs 9.8% , p<0.01 ) , had a native language that was not english ( 22% vs 9.4% , p=0.02 ) , and were not currently partnered ( single , separated , divorced , or widowed vs married ; 11.9% vs 8.5% , p<0.01 ) . in multivariable analysis , care in subspecialty clinics , compared to general neurology clinic as the reference , was associated with higher rates of patient - reported nonadherence . stroke clinical patients had twice the odds ratio ( or ) of nonadherence ( or = 2.09 , 95% ci : 1.054.16 ) , and memory disorder clinical patients had more than threefold increase ( or = 3.73 , 95% ci : 1.3110.59 ; figure s2 ) . a sensitivity analysis using both marital status and cohabitation in a multivariable logistic regression did not significantly impact the reported results . we have presented a cross - sectional study of cost - related medication nonadherence to use in a patient - reported survey conducted in a large cohort of neurology patients . our results suggest that the prevalence of financial barriers to medication adherence is modest ( < 10% ) . these findings were consistent with those presented in the study of campbell et al,34 who found that trouble affording medications was the fourth leading reason for medication nonadherence among elderly patients . however , the comparison of our cost - related non - adherence rates with the current literature is limited by the general lack of congruence in the way adherence is measured and reported . most other medication adherence studies have either evaluated patients in controlled settings with small sample sizes or used claims data to measure patient prescription refills . in comparison , our survey describes the patient report of the burden of financial barriers to medication adherence.35,36 in addition , to reduce potential for both recall and interviewer bias , we collected data from patients immediately before their clinical visits . we believe that collecting data from patients immediately before their clinical visits yielded more accurate results because the patients were already prompted to think about their health problems and medications in use . also , having to complete the survey in the waiting room allowed the patients to have ready access to staff members and clarify the meaning of any question they found hard to interpret . in comparison , this would not be possible in a paper - based form sent out after clinic.3741 of note , our participation and completion rates ( 49% and 45% , respectively ) were similar to the rates obtained in previous similar studies.4246 a second interesting finding was that patients seen at the memory disorders clinic , which includes primarily patients with alzheimer s disease and related dementias who were almost always accompanied by a care giver , were especially vulnerable to financial barriers to medication adherence , even after adjusting for pertinent socioeconomic factors . this finding is particularly concerning because these patients tend to be older , and medication adherence by older patients is lower compared to younger patients due to increased side effects as well.4749 these older- and cognitively impaired adults are clinically vulnerable due to concurrent medical illnesses and drug interactions.4749 studies have shown that the number and types of co - morbidities , such as depression , are predictors of cost - related nonadherence.36,37 this is especially striking since most of these patients are medicare eligible and so would not be expected to have large out - of - pocket expenses for many tests , procedures , or hospitalizations . observational retrospective studies using claims data ( prescription fills ) have suggested that drug - benefit design and tier strategies ( i.e. , prescription drug coverage ) may also affect adherence to treatment.50 adding complexity to this problem , mcwilliams et al9 showed that medicare beneficiaries with low cognitive function were less responsive to the generosity of medicare advantage benefits in their decisions ( i.e. , multiple and complex insurance plan benefit choices negatively impacted seniors with impaired decision making ) . our study findings build upon the existing literature and highlight the complex interaction of multiple socioeconomic and clinical factors in cost - related nonadherence.35 in this study , patients from socially disadvantaged backgrounds , such as hispanics and non - english speakers , also appear to be more likely to report having financial barriers to medication adherence.51 our study supports other groups that have presented data on how people of lower socioeconomic status and ethnic minorities report higher rates of cost - related medication nonadherence.37,47,5257 while many studies focused on examining the adherence to a specific medication , we asked a broad question about any medication , which allowed for a cross - clinic comparison.58 in the case of patients with alzheimer s disease in our memory disorders clinic , our study findings remain consistent with studies about the recent rising costs of neurology prescription drugs , which ultimately indicates more out - of - pocket spending for this population.5 for example , an analysis of medicare part d data revealed that neurologists comprised only 1.2% of prescribing providers but expenditures with neurology - prescribed medications were the third highest of all specialties . medications for multiple sclerosis , epilepsy , and alzheimer s disease and related dementias were the major drivers of the exceptional costs ( $ 5 billion in 2013).5 finally , this study is aligned with the existing literature that suggests that patient reporting and the linking of multiple sources of data have been shown to improve the accuracy of research findings.59,60 this study had many important limitations . most notably , the trade - off in asking a broad medication adherence question and absence of data from chart abstraction is that we may not have captured the specific medication that is the principal reason for the financial barrier in each clinical scenario . this medication is relatively of low cost and is classified as tier 1 , designating medications that are largely covered , according to the most common insurance policies . for patients with stroke , we believe that the culprit might be the related medications often prescribed in patients with stroke , such as anticoagulants and medications for hypertension , hyperlipidemia , and pain management.58,61 in addition , other broad wordings that serve as a limitation of our study were such as the categorical scale with frequency options ( eg , often , sometimes , and never ) . perhaps , an open - ended question could yield a more specific measurement ( eg , how many days in a month would you miss a medication ? ) . however , the likelihood that a patient would be able to provide an accurate numerical value with respect to adherence over the past 12 months would be low . therefore , we considered categorical scales easier to respond and opted to use simple frequency words . a second major limitation was that we were unable to distinguish between patients who declined to participate and patients who were not approached , due to the aforementioned exclusion criteria and administrative errors . the absence of these data prevents further characterization of potentially confounding participant vs nonparticipant differences . in addition , we excluded unaccompanied non - english speakers and unaccompanied patients with severe cognitive impairment . both the criteria were determined after a patient - handed survey based on self - reported or observed inability to follow basic survey directions . also , 275 patients ( 9% ) started the survey but did not complete the medication adherence question . the reasons for leaving the survey incomplete include the early call from the treating physician ( eg , when there is < 10 minutes of waiting time from check - in to the encounter with the physician ) , fatigue ( eg , patients get tired and give up ) , and distractions ( eg , patients answer phone calls and leave the survey aside ) . another limitation that exists with the study population is that the research team did not consider other impairments , such as visual impairment , as one of the exclusion criteria due to the fact that patients were likely to visit with a designated proxy . because the patients were self - answering the survey in the context of a busy neurology clinic this study has limited generalizability to nonacademic medical centers as is common to many similar studies.11,22 however , it is hoped that the findings are more generalizable due to the high participation and completion rates . we did not identify patients who were nonadherent because of nonfinancial reasons , and therefore the comparison arm of our analysis includes some patients who were nonadherent due to other reasons . next , this study did not prospectively capture adherence following the prescription of a specific medication , making causal association in our study impossible . the clinical value of our study was demonstrated in the secondary analysis . as outlined earlier , this study made note of a potential association between patients being seen in certain clinics that may prescribe more expensive medications , such as those for patients with alzheimer s disease and related dementias , suggesting that they may have a higher cost - sharing burden . finally , residual measured and unmeasured confounding may exist , such as the cost per drug tier in each patient s insurance coverage formulary . we would expect that the patients with cost - related nonadherence would have been most often prescribed medications in the highest cost tier , as described in previous studies.62 however , we were not able to examine if nonadherence was related to drug tier and co - pay as we did not have reliable access to each patient s pharmacy benefit to determine these interactions . despite these limitations , this study offers opportunities to improve outcomes in neurology patients through medication adherence and drive health policy in neurology . 1 ) patients could be encouraged to reduce their medication costs by requesting generic medications or using mail order pharmacies . 2 ) more assistance for patients in navigating health insurance and therapeutic choices might reduce nonadherence to medication regimens among patients with neurological conditions . 3 ) value - based drug pricing ( varying payments for medications based on outcomes or magnitude of clinical benefits ) may be another means of engaging patients , physicians , and payers toward improving adherence and outcomes . our study also sought to improve the discussions between patients and treating physicians about prices of medications . we present the medication adherence question to the patients before their visit in order to prompt the patients to discuss any prescription filling problems with the treating physician during the encounter . however , patients still have difficulty starting this conversation and may only realize the amount of co - pay when they go to pick up their refill after the clinical visit . we encourage quality improvement strategies , such as using electronic notifications of answers to the cost - related nonadherence question , in order to encourage the physicians to incite these discussions about financial issues . future studies should also integrate patient - reported information into insurance - benefit designs in order to measure the actual ratio of adherence to out - of - pocket spending , per patient and by treatment period . this study suggests that while the overall level of poor adherence due to costs is limited , cost - related barriers to medication adherence appear to be concentrated among the most cognitively- and socioeconomically vulnerable patients . multiple strategies are needed to prospectively identify these vulnerable patients and to alter prescribing practices to enhance affordability and thereby long - term efficacy . the survey was administered on an apple ipad2 ( apple , cupertino , ca , usa ) , with a plastic cover to allow for easy grip and identification in the busy outpatient setting of a major academic neurology clinic . the survey questions were built using the tonic for health platform ( tonic solutions inc . , ca , usa ) , a survey software tool that allowed intuitive survey construction , and integration with the apple ipad ios ( version 8 and beyond ) and the hospital s electronic health record ( ehr ) . in addition to the measures described in the main manuscript , we also collected the nih promis-10 . the nih promis-10 is a short form that measures a patient s perceived physical and mental health . the promis-10 has been validated in populations with different neurological diseases ( eg , stroke , epilepsy , and parkinson s disease ) as well as without neurological diseases and attempt to measure patient - reported physical and mental function.1 note : it demonstrates the attrition from 6075 to 2716 patients . notes : it graphically represents the logistic regression using demographic and clinical characteristics in the model to predict the odds of cost - related medication nonadherence . the patients whose preferred language was english ( or : 0.46 [ 0.250.87 ] ) , the patients seen at the memory disorders clinic ( or : 3.72 [ 1.310.6 ] ) , and the patients seen at the stroke clinic ( or : 2.1 [ 1.04.2 ] ) had higher odds for reporting cost - related medication nonadherence . characteristics of the eligible population by survey participation notes : two - sided t - tests were used to calculate p - values to compare the mean values of patient age between the groups . the chi - squared test of independence was used to calculate the p - values to test whether the categorical variables were associated with increased rates of adherence . the survey was administered on an apple ipad2 ( apple , cupertino , ca , usa ) , with a plastic cover to allow for easy grip and identification in the busy outpatient setting of a major academic neurology clinic . the survey questions were built using the tonic for health platform ( tonic solutions inc . , ca , usa ) , a survey software tool that allowed intuitive survey construction , and integration with the apple ipad ios ( version 8 and beyond ) and the hospital s electronic health record ( ehr ) . in addition to the measures described in the main manuscript , we also collected the nih promis-10 . the nih promis-10 is a short form that measures a patient s perceived physical and mental health . the promis-10 has been validated in populations with different neurological diseases ( eg , stroke , epilepsy , and parkinson s disease ) as well as without neurological diseases and attempt to measure patient - reported physical and mental function.1 note : it demonstrates the attrition from 6075 to 2716 patients . notes : it graphically represents the logistic regression using demographic and clinical characteristics in the model to predict the odds of cost - related medication nonadherence . the patients whose preferred language was english ( or : 0.46 [ 0.250.87 ] ) , the patients seen at the memory disorders clinic ( or : 3.72 [ 1.310.6 ] ) , and the patients seen at the stroke clinic ( or : 2.1 [ 1.04.2 ] ) had higher odds for reporting cost - related medication nonadherence . characteristics of the eligible population by survey participation notes : two - sided t - tests were used to calculate p - values to compare the mean values of patient age between the groups . the chi - squared test of independence was used to calculate the p - values to test whether the categorical variables were associated with increased rates of adherence .

objectivemany effective medical therapies are available for treating neurological diseases , but these therapies tend to be expensive and adherence is critical to their effectiveness . we used patient - reported data to examine the frequency and determinants of financial barriers to medication adherence among individuals treated for neurological disorders.patients and methodspatients completed cross - sectional surveys on ipads as part of routine outpatient care in a neurology clinic . survey responses from a 3-month period were collected and merged with administrative sources of demographic and clinical information ( eg , insurance type ) . we explored the association between patient characteristics and patient - reported failure to refill prescription medication due to cost in the previous 12 months , termed here as nonadherence.resultsthe population studied comprised 6075 adults who were presented between july and september 2015 for outpatient neurology appointments . the mean age of participants was 56 ( standard deviation : 18 ) years , and 1613 ( 54% ) were females . the patients who participated in the surveys ( 2992 , 49% ) were comparable to nonparticipants with respect to gender and ethnicity but more often identified english as their preferred language ( 94% vs 6% , p<0.01 ) . among respondents , 9.8% ( n=265 ) reported nonadherence that varied by condition . these patients were more frequently hispanic ( 16.7% vs 9.8% white , p=0.01 ) , living alone ( 13.9% vs 8.9% cohabitating , p<0.01 ) , and preferred a language other than english ( 15.3% vs 9.4% , p=0.02).conclusionoverall , the magnitude of financial barriers to medication adherence appears to vary across neurological conditions and demographic characteristics .

Introduction Patients and methods Participants Procedures and measurements Survey RPDR query tool Statistical analysis Results Discussion Conclusion Supplementary materials Additional survey details

there is a growing armamentarium of effective medical therapies to treat neurological diseases.1,2 national guidelines have discussed ten us food and drug administration ( fda)-approved disease - modifying therapies for multiple sclerosis , > 20 fda - approved antiepileptic drugs for seizure prophylaxis , and 23 drugs involved in stroke prevention , including novel oral anticoagulants.35 most of these therapies are expensive and adherence is critical to their effectiveness.68 medication adherence depends on many factors , including affordable access to drugs , which varies by drug type and insurance - benefit design . the resulting differences in out - of - pocket payments can be challenging for beneficiaries with limited incomes or impaired cognition.9 in addition , since adherence depends mainly on patient reporting and perception , it has been demonstrated that many patients who claim to have been adherent to their medications are in fact not.10,11 according to a recent study on therapeutic drug monitoring in consecutive emergency hospital admissions for seizures , > 40% of nonadherent patients claimed to have been adherent when further asked about medication failure.12 medication nonadherence has been shown to seriously hurt health outcomes in neurology , and hence efforts to understand the causes and solutions to this problem have become increasingly important . some studies have shown a moderate association between drug choice , adherence , and satisfaction with treatment.7,13 previous studies of medication nonadherence in neurology have been limited to population - wide databases and reliable claims - based algorithms.1424 these large - scale approaches to the evaluation of medication adherence using retrospective electronic health records ( ehrs ) abstraction or claims data have missed critical information and fallen short in representing the real - world complexity.25 in an effort to capture more accurate , specific , and informative data on medication nonadherence , systematic collection of patient - reported outcomes offers great promise.26,27 the term patient - reported outcomes refers to data directly collected from patients such as self - administered surveys and other attempts to quantify patients behavior and their subjective experience of health . patient - reported data have been shown to correlate with and augment more typical clinical measurements , proving useful in the assessment of treatment outcomes and determinants of health.2831 this study examined the frequency and determinants of financial barriers to medication adherence among individuals treated for neurological disorders using patient - reported data . this study was a retrospective review of data collected as part of a quality improvement project implemented in neurology ambulatory clinical practices that began in july 2015 and continues to date . the front desk staff had received standard institutional training to determine patients ability to read and sign check - in - related documents ( eg , insurance - driven pre - office forms ) . although longitudinal data were captured for patients with several visits during this period , only the first visit was included in the analysis.7,32,33 this study combines information from the survey merged by deterministic linkage using the medical record number to administrative and clinical data gathered using a research patient data registry ( rpdr ) query tool . a report summarizing the patients survey responses became available in the ehr immediately upon completion , and providers were encouraged to review this information with the patient . the survey had four parts an introduction , demographic questions , and the medication adherence question , how often in the past 12 months did you decide not to fill or refill your prescription because the medicines cost too much ? after this question , the survey included an additional questionnaire to measure global health function , which is not the focus of this article and is detailed in the supplementary materials . to determine the primary predictors of participation and examine the potential for selection bias , we gathered additional demographic information using the partners healthcare clinical data registry for all arriving patients ( both participating and nonparticipating in the survey ) seen in the ambulatory neurology clinics during the study time frame . the rpdr is a clinical data registry that aggregates data from sources throughout the partners healthcare system ( phs ) , including the medical record , reports , claims , and administrative systems . queried variables included age , gender , preferred language , insurance status , ethnicity , marital status , and clinic type ( eg , general neurology vs subspecialty area ) . eligible encounters were the patients > 18 years of age seen at the neurology outpatient clinic ( main campus only ) , who had a first visit or were established patients who came for a follow - up visit within the study time frame ( subsequent visits were excluded ) . we defined cost - related medication nonadherence as an answer of often or sometimes to the question how often in the past 12 months did you decide not to fill or refill a prescription because the medicines cost too much ? to address the potential for selection bias in our respondents , we performed univariate analysis of covariates associated with survey participation ( participants vs nonparticipants , including age , gender , ethnicity , preferred language , insurance type , marital status , and clinic type ) . this study was a retrospective review of data collected as part of a quality improvement project implemented in neurology ambulatory clinical practices that began in july 2015 and continues to date . the front desk staff had received standard institutional training to determine patients ability to read and sign check - in - related documents ( eg , insurance - driven pre - office forms ) . although longitudinal data were captured for patients with several visits during this period , only the first visit was included in the analysis.7,32,33 this study combines information from the survey merged by deterministic linkage using the medical record number to administrative and clinical data gathered using a research patient data registry ( rpdr ) query tool . a report summarizing the patients survey responses became available in the ehr immediately upon completion , and providers were encouraged to review this information with the patient . the survey had four parts an introduction , demographic questions , and the medication adherence question , how often in the past 12 months did you decide not to fill or refill your prescription because the medicines cost too much ? after this question , the survey included an additional questionnaire to measure global health function , which is not the focus of this article and is detailed in the supplementary materials . to determine the primary predictors of participation and examine the potential for selection bias , we gathered additional demographic information using the partners healthcare clinical data registry for all arriving patients ( both participating and nonparticipating in the survey ) seen in the ambulatory neurology clinics during the study time frame . queried variables included age , gender , preferred language , insurance status , ethnicity , marital status , and clinic type ( eg , general neurology vs subspecialty area ) . eligible encounters were the patients > 18 years of age seen at the neurology outpatient clinic ( main campus only ) , who had a first visit or were established patients who came for a follow - up visit within the study time frame ( subsequent visits were excluded ) . a report summarizing the patients survey responses became available in the ehr immediately upon completion , and providers were encouraged to review this information with the patient . the survey had four parts an introduction , demographic questions , and the medication adherence question , how often in the past 12 months did you decide not to fill or refill your prescription because the medicines cost too much ? after this question , the survey included an additional questionnaire to measure global health function , which is not the focus of this article and is detailed in the supplementary materials . to determine the primary predictors of participation and examine the potential for selection bias , we gathered additional demographic information using the partners healthcare clinical data registry for all arriving patients ( both participating and nonparticipating in the survey ) seen in the ambulatory neurology clinics during the study time frame . the rpdr is a clinical data registry that aggregates data from sources throughout the partners healthcare system ( phs ) , including the medical record , reports , claims , and administrative systems . queried variables included age , gender , preferred language , insurance status , ethnicity , marital status , and clinic type ( eg , general neurology vs subspecialty area ) . eligible encounters were the patients > 18 years of age seen at the neurology outpatient clinic ( main campus only ) , who had a first visit or were established patients who came for a follow - up visit within the study time frame ( subsequent visits were excluded ) . we defined cost - related medication nonadherence as an answer of often or sometimes to the question how often in the past 12 months did you decide not to fill or refill a prescription because the medicines cost too much ? to address the potential for selection bias in our respondents , we performed univariate analysis of covariates associated with survey participation ( participants vs nonparticipants , including age , gender , ethnicity , preferred language , insurance type , marital status , and clinic type ) . a total of 2992 out of 6075 patients participated in the survey and 2716 fully completed the survey questions , yielding a participation rate of 49% and a completion rate of 45% ( figure s1 ) . the mean age of participants was 56 ( sd : 18 ) years and 1613 ( 54% ) were females . survey responders more often identified english as their preferred language ( 90.6% , p=0.02 ; table s1 ) . among the participants , 9.8% ( n=265 ) of patients reported cost - related medication nonadherence . patients who reported cost - related nonadherence were younger ( mean age : 5316 years vs 5618 years , p<0.01 ) , more often hispanic ( 16.7% vs 9.8% , p<0.01 ) , had a native language that was not english ( 22% vs 9.4% , p=0.02 ) , and were not currently partnered ( single , separated , divorced , or widowed vs married ; 11.9% vs 8.5% , p<0.01 ) . in multivariable analysis , care in subspecialty clinics , compared to general neurology clinic as the reference , was associated with higher rates of patient - reported nonadherence . stroke clinical patients had twice the odds ratio ( or ) of nonadherence ( or = 2.09 , 95% ci : 1.054.16 ) , and memory disorder clinical patients had more than threefold increase ( or = 3.73 , 95% ci : 1.3110.59 ; figure s2 ) . we have presented a cross - sectional study of cost - related medication nonadherence to use in a patient - reported survey conducted in a large cohort of neurology patients . our results suggest that the prevalence of financial barriers to medication adherence is modest ( < 10% ) . however , the comparison of our cost - related non - adherence rates with the current literature is limited by the general lack of congruence in the way adherence is measured and reported . most other medication adherence studies have either evaluated patients in controlled settings with small sample sizes or used claims data to measure patient prescription refills . in comparison , our survey describes the patient report of the burden of financial barriers to medication adherence.35,36 in addition , to reduce potential for both recall and interviewer bias , we collected data from patients immediately before their clinical visits . we believe that collecting data from patients immediately before their clinical visits yielded more accurate results because the patients were already prompted to think about their health problems and medications in use . also , having to complete the survey in the waiting room allowed the patients to have ready access to staff members and clarify the meaning of any question they found hard to interpret . in comparison , this would not be possible in a paper - based form sent out after clinic.3741 of note , our participation and completion rates ( 49% and 45% , respectively ) were similar to the rates obtained in previous similar studies.4246 a second interesting finding was that patients seen at the memory disorders clinic , which includes primarily patients with alzheimer s disease and related dementias who were almost always accompanied by a care giver , were especially vulnerable to financial barriers to medication adherence , even after adjusting for pertinent socioeconomic factors . this finding is particularly concerning because these patients tend to be older , and medication adherence by older patients is lower compared to younger patients due to increased side effects as well.4749 these older- and cognitively impaired adults are clinically vulnerable due to concurrent medical illnesses and drug interactions.4749 studies have shown that the number and types of co - morbidities , such as depression , are predictors of cost - related nonadherence.36,37 this is especially striking since most of these patients are medicare eligible and so would not be expected to have large out - of - pocket expenses for many tests , procedures , or hospitalizations . our study findings build upon the existing literature and highlight the complex interaction of multiple socioeconomic and clinical factors in cost - related nonadherence.35 in this study , patients from socially disadvantaged backgrounds , such as hispanics and non - english speakers , also appear to be more likely to report having financial barriers to medication adherence.51 our study supports other groups that have presented data on how people of lower socioeconomic status and ethnic minorities report higher rates of cost - related medication nonadherence.37,47,5257 while many studies focused on examining the adherence to a specific medication , we asked a broad question about any medication , which allowed for a cross - clinic comparison.58 in the case of patients with alzheimer s disease in our memory disorders clinic , our study findings remain consistent with studies about the recent rising costs of neurology prescription drugs , which ultimately indicates more out - of - pocket spending for this population.5 for example , an analysis of medicare part d data revealed that neurologists comprised only 1.2% of prescribing providers but expenditures with neurology - prescribed medications were the third highest of all specialties . medications for multiple sclerosis , epilepsy , and alzheimer s disease and related dementias were the major drivers of the exceptional costs ( $ 5 billion in 2013).5 finally , this study is aligned with the existing literature that suggests that patient reporting and the linking of multiple sources of data have been shown to improve the accuracy of research findings.59,60 this study had many important limitations . most notably , the trade - off in asking a broad medication adherence question and absence of data from chart abstraction is that we may not have captured the specific medication that is the principal reason for the financial barrier in each clinical scenario . for patients with stroke , we believe that the culprit might be the related medications often prescribed in patients with stroke , such as anticoagulants and medications for hypertension , hyperlipidemia , and pain management.58,61 in addition , other broad wordings that serve as a limitation of our study were such as the categorical scale with frequency options ( eg , often , sometimes , and never ) . perhaps , an open - ended question could yield a more specific measurement ( eg , how many days in a month would you miss a medication ? ) however , the likelihood that a patient would be able to provide an accurate numerical value with respect to adherence over the past 12 months would be low . a second major limitation was that we were unable to distinguish between patients who declined to participate and patients who were not approached , due to the aforementioned exclusion criteria and administrative errors . both the criteria were determined after a patient - handed survey based on self - reported or observed inability to follow basic survey directions . the reasons for leaving the survey incomplete include the early call from the treating physician ( eg , when there is < 10 minutes of waiting time from check - in to the encounter with the physician ) , fatigue ( eg , patients get tired and give up ) , and distractions ( eg , patients answer phone calls and leave the survey aside ) . another limitation that exists with the study population is that the research team did not consider other impairments , such as visual impairment , as one of the exclusion criteria due to the fact that patients were likely to visit with a designated proxy . because the patients were self - answering the survey in the context of a busy neurology clinic this study has limited generalizability to nonacademic medical centers as is common to many similar studies.11,22 however , it is hoped that the findings are more generalizable due to the high participation and completion rates . we did not identify patients who were nonadherent because of nonfinancial reasons , and therefore the comparison arm of our analysis includes some patients who were nonadherent due to other reasons . we would expect that the patients with cost - related nonadherence would have been most often prescribed medications in the highest cost tier , as described in previous studies.62 however , we were not able to examine if nonadherence was related to drug tier and co - pay as we did not have reliable access to each patient s pharmacy benefit to determine these interactions . 2 ) more assistance for patients in navigating health insurance and therapeutic choices might reduce nonadherence to medication regimens among patients with neurological conditions . 3 ) value - based drug pricing ( varying payments for medications based on outcomes or magnitude of clinical benefits ) may be another means of engaging patients , physicians , and payers toward improving adherence and outcomes . we present the medication adherence question to the patients before their visit in order to prompt the patients to discuss any prescription filling problems with the treating physician during the encounter . future studies should also integrate patient - reported information into insurance - benefit designs in order to measure the actual ratio of adherence to out - of - pocket spending , per patient and by treatment period . this study suggests that while the overall level of poor adherence due to costs is limited , cost - related barriers to medication adherence appear to be concentrated among the most cognitively- and socioeconomically vulnerable patients . the survey was administered on an apple ipad2 ( apple , cupertino , ca , usa ) , with a plastic cover to allow for easy grip and identification in the busy outpatient setting of a major academic neurology clinic . , ca , usa ) , a survey software tool that allowed intuitive survey construction , and integration with the apple ipad ios ( version 8 and beyond ) and the hospital s electronic health record ( ehr ) . the promis-10 has been validated in populations with different neurological diseases ( eg , stroke , epilepsy , and parkinson s disease ) as well as without neurological diseases and attempt to measure patient - reported physical and mental function.1 note : it demonstrates the attrition from 6075 to 2716 patients . notes : it graphically represents the logistic regression using demographic and clinical characteristics in the model to predict the odds of cost - related medication nonadherence . the patients whose preferred language was english ( or : 0.46 [ 0.250.87 ] ) , the patients seen at the memory disorders clinic ( or : 3.72 [ 1.310.6 ] ) , and the patients seen at the stroke clinic ( or : 2.1 [ 1.04.2 ] ) had higher odds for reporting cost - related medication nonadherence . the survey was administered on an apple ipad2 ( apple , cupertino , ca , usa ) , with a plastic cover to allow for easy grip and identification in the busy outpatient setting of a major academic neurology clinic . , ca , usa ) , a survey software tool that allowed intuitive survey construction , and integration with the apple ipad ios ( version 8 and beyond ) and the hospital s electronic health record ( ehr ) . the promis-10 has been validated in populations with different neurological diseases ( eg , stroke , epilepsy , and parkinson s disease ) as well as without neurological diseases and attempt to measure patient - reported physical and mental function.1 note : it demonstrates the attrition from 6075 to 2716 patients . notes : it graphically represents the logistic regression using demographic and clinical characteristics in the model to predict the odds of cost - related medication nonadherence . the patients whose preferred language was english ( or : 0.46 [ 0.250.87 ] ) , the patients seen at the memory disorders clinic ( or : 3.72 [ 1.310.6 ] ) , and the patients seen at the stroke clinic ( or : 2.1 [ 1.04.2 ] ) had higher odds for reporting cost - related medication nonadherence .

the paradox of meritocracy is alive and well under conditions where careers are primarily evaluated in terms of upward mobility ( vinkenburg & weber , 2012 ) . a prime example of such conditions is an up - or - out career system , common to academia , the military , and professional service firms ( psfs ) . careers in such settings are subjected to a linear promotion process shaped by regular and elaborate performance measurements based on predetermined criteria and fixed time frames . in order to sustain the pyramidal shape of the hierarchy , those not advancing to the next career level according to the given parameters are counseled to leave generally , an up - or - out system strongly suggests meritocracy through its reliance on formal procedures and transparent criteria . indeed , in many high - prestige settings such as academia and law , performance evaluations are based on relatively measurable criteria or indicators such as billable hours or research productivity ( joshi et al . , 2015 ) . however , as in other types of organizations , the selection and promotion decision - making process remains rather subjective ( perry , davis - blake , & kulik , 1994 ) . scores on seemingly objective criteria ( e.g. , billable hours , impact factor ) , are not decisive in the process , but only serve as a threshold for being considered , are systematically overestimated for dominant group members , or even dropped from the deliberations altogether ( van den brink , holgersson , linghag , & de , 2016 ; vinkenburg , jansen , dries , & pepermans , 2014 ) . decision making on promotion to the highest levels of organizational hierarchies , due to structural conditions and situational components , can be characterized as especially imperfect ( vinkenburg et al . room for bias appears when managers have ( or use ) discretion in translating performance scores to pay and promotion decisions in psfs ( ossenkop et al . , 2015b ) . in trying to explain discrepancies between outcomes of performance evaluations versus reward allocations , joshi et al . ( 2015 ) label decisions on pay and promotion in prestigious settings as highly subjective , opaque , and adversarial up - or - out promotion norms exacerbate the situation by raising the stakes as not getting promoted on time means leaving . under such conditions , and regardless of actual merit , the likelihood of upward mobility is higher for dominant group members than for others . the consequence is the typical image of almost exclusively white men at the top of the hierarchy . in a career system that thus creates and maintains outcome disparities between members of different social groups , and where the paradox of meritocracy feeds into a limited awareness of biased decision making ( block , 2016 ) , breaking the cycle is complicated . while many organizations that embrace an up - or - out career system recruit and select a heterogeneous or diverse pool of entry - level candidates , after 12 to 15 years typically a disproportionate number of dominant group members will have made it to the highest level . nondominant group members will be absent at this level , as the career system does not allow up - or - stay . taking a systems perspective , martell , emrich , and robison - cox ( 2012 ) conceptualize the process by which micro - level forces such as bias in performance assessment and macro - level forces such as demographic composition operate together to ( re)produce gender segregation at the top of the organizational hierarchy . in upward mobility systems , promotion signals such as early success and velocity , as well as promotion models shaped as tournaments and gatekeeping exacerbate segregation ( martell et al . , 2012 ) . erfurt sandhu ( 2013 ) explains the persistent homogeneity at the highest levels in psfs as a consequence of self - reinforcing mechanisms . organizational members increasingly adapt to and adopt behavioral and interactional rules for reasons of efficiency and uncertainty reduction , and over time the unwritten rules of the game ( scott - morgan , 1994 ; sydow , schreygg , & koch , 2009 ) become so normalized that members are locked - in and no longer see alternative , broader scopes of action . as an example , the rule to spend friday in the office instead of at the client site ( invented for efficiency reasons ) becomes a way to increase visibility with gatekeepers , and emerges as a criterion for promotion , effectively excluding those who do not spend friday in the office for religious or care - related reasons . obrien , scheffer , van nes , and van der lee ( 2015 ) effectively illustrate that because of system dynamics and self - reinforcing mechanisms , achieving more diversity at all hierarchical levels will take much longer than most decision makers tend to think . as a system tends to reproduce and reinforce itself , targeted interventions common diversity interventions in organizations such as training , mentoring , and networking are criticized because they fail to transform organizational structures and cultures , because they aim to fix other employees rather than fix the system , and because they do not target dominant group members ( benschop , holgersson , van den brink , & wahl , 2016 ; bird , 2011 ; ely & meyerson , 2000 ) . designing systemic diversity interventions with transformative power , such as the examples described later on in this article , requires basic knowledge of system dynamics , an approach that is useful in understanding the behavior of complex systems over time . experts in system dynamics draw causal loop diagrams that show the structure of the system , including its components or variables and various relationships between components . an example of such a model or causal loop diagram is found in figure 1 . when drawing a causal loop diagram representing diversity in a career system , stocks represent the relative representation of members of various social groups at different levels in the hierarchy , flows represent the relative advancement rates from one level in the hierarchy to the next , and feedback loops represent self - reinforcing positive or negative feedback processes in the diagram ( bleijenbergh & van engen , 2015 ) . 2015 ) use system dynamics , including stocks and flows , to predict the rate of change in workforce composition at different levels of the hierarchy depending on the rate at which different categories of employees enter , are promoted , and leave the organization . taking a systems perspective model developed by technical university , reprinted with permission emerald group publishing , from bleijenbergh and van engen ( 2015 ) . the main challenge in designing an effective diversity intervention lies in the identification of the main lever for change . this identification is tricky because the relationships between system components are characterized by dynamic complexity , as in constantly changing , tightly coupled , nonlinear , and feedback - driven ( sterman , 2001 ) . while there are many possible levers , most of these are and should be context specific . this requires mapping the system under consideration in detail , to make sure we are not pushing the wrong buttons . for the purpose of this article , however , i would like to choose a lever that is universal and has been identified as one of the most tenacious causes of homogeneity and inequality bias . bias in the strictest definition of the term is a cognitive distortion and is evidenced in decision making . bias is especially prominent in the construction , operationalization , and application of criteria for selection and promotion . i will show how bias plays a role in reproducing the overrepresentation of a homogeneous group at the top of organizations by looking at cognitive distortions in general and gender bias in particular . while racioethnic bias is another common and tenacious kind of bias , i do not discuss it here , because underlying stereotypes of various racioethnic categories are first more particular to the societal and organizational context than gender stereotypes ( roberson & block , 2001 ) , second are more conflated with stereotypes about immigration and religion than gender stereotypes ( vinkenburg , 2014 ) , third are more ambivalent in terms of content ( fiske , 2012 ) than gender stereotypes , and fourth are less well described in the literature in terms of career consequences ( ossenkop , vinkenburg , jansen , & ghorashi , 2015a ) than gender stereotypes . bias reflects limits to our cognitive ability to make objective informed decisions about people s careers , on account of bounded rationality ( kahneman , 2003 ) , intransitivity ( suppes & zinnes , 1963 ) , high cognitive loads and time pressure ( chugh , 2004 ) , and the influence of emotion and gut feelings ( rivera , 2015 ) . career - related decision making ( e.g. , selection , performance evaluation , promotion ) is rarely objective and decidedly suboptimal ( vinkenburg et al . , 2014 ) , especially so at higher hierarchical levels . regardless of group membership , this type of bias limits the effectiveness and predictive validity of our decisions about people . gender bias is defined as our prejudice in favor of one gender over the other , generally used as bias against women . gender bias follows from gender stereotypes , which show a greater degree of stereotype fit or larger role congruency between men and work and between women and care than the other way around ( eagly & heilman , 2016 ; heilman , 2012 ) . this lack of stereotype fit or incongruence results in women being presumed incompetent in professional roles ( heilman , block , & lucas , 1992 ; heilman , manzi , & braun , 2015 ) , and in shifting standards in performance evaluation ( biernat & fuegen , 2001 ) . women who display stereotypically male behavior such as assertiveness and self - promotion in a work environment may experience penalties and backlash ( rudman & phelan , 2008 ) . gender bias affects careers in limiting chances of being promoted ( vinkenburg , van engen , eagly , & johannesen - schmidt , 2011 ) . as gender bias is often implicit and subtle , it is more difficult to recognize and thus harder to counter than blatant and explicit discrimination ( biernat , tocci , & williams , 2011 ) . in up - or - out career systems , where white men are relatively overrepresented at the top of the hierarchy , the biased assessment of merit is in fact more a matter of favoritism ( bias for white men ) than of discrimination ( bias against all others ) . evidence of bias is not readily accepted by the dominant elite , who firmly believe in their own ability to identify competence ( moss - racusin , molenda , & cramer , 2015 ) . raising awareness of bias is clearly not enough to mitigate its effects , may produce unintended ironic effects , and could even backfire by reinforcing stereotypes and prejudice ( apfelbaum , sommers , & norton , 2008 ; duguid & thomas - hunt , 2015 ; kulik , perry , & bourhis , 2000 ; macrae , bodenhausen , milne , & jetten , 1994 ; shelton , richeson , salvatore , & trawalter , 2005 ) . bias is notoriously hard if not impossible to eliminate and being confronted with one s biases can make decision makers angry , confused , and defensive ( burrell , 2016 ; j. l. howell & ratliff , 2016 ; pendry , driscoll , & field , 2007 ) . according to learning theory , raising awareness alone makes individuals conscious but incompetent . in order to build their competence , in this case literally developing the skills to mitigate the effects of bias on decision making , experiential learning is required ( w. c. howell & fleishman , 1982 ; raelin , 1997 ) . while gender bias generally predicts disadvantage for women relative to men , men people in general but professionals in particular share underlying normative expectations of careers as linear ( sabelis & schilling , 2013 ) , which implies that we value nonlinear , interrupted , or rerouted careers less than careers that reflect steady , smooth , upward mobility ( vinkenburg & weber , 2012 ) . the finding that men are more penalized for career breaks or other requests for flexibility than women ( coltrane , miller , dehaan , & stewart , 2013 ; vinkenburg , van engen , coffeng , & dikkers , 2012 ) , is clear evidence of bias . if criteria for promotion reflect the uninterrupted career trajectories traditionally followed by male breadwinners , anyone with care responsibilities loses the contest . it is important to realize that the degree of bias does not have to be large to have effects , especially over time . in from bias to exclusion , ( 2012 ) argue that if every career - related decision along the way is only a little bit biased in favor of one group versus another , the top of the hierarchy will remain segregated . if decision making is suboptimal in terms of filtering out the best candidates , favoring dominant group members is a likely result ( vinkenburg et al . , 2014 ) research evidence suggests that while the criteria used in selection and promotion decisions appear objective , their application may be less so . first , if the description of the criterion is abstract , allowing discretion in the operationalization of vague terms such as potential , talent , or excellence leaves room for bias ( festing , kornau , & schfer , 2014 ; robinson , fetters , riester , & bracco , 2009 ) . second , previously unspecified and perhaps irrelevant criteria such as social class or cultural capital may emerge during the process ( rivera , 2016 ; vinkenburg et al . , 2014 ) . third , decision makers tend to redefine the criteria as requiring the specific credentials that a candidate from the dominant group happens to have ( uhlmann & cohen , 2005 ) , effectively redefining merit . fourth , criteria as well as candidates strengths and weaknesses are selectively mentioned , downplayed , or stressed in committee deliberations ( ahlqvist , andersson , sderqvist , & tumpane , 2015 ; van den brink et al . , 2016 ) . finally , linguistic bias creeps into evaluative language , including using negations more frequently for nondominant group members ( e.g. , she does not have a bad cv ; beukeboom , finkenauer , & wigboldus , 2010 ; kaatz , magua , zimmerman , & carnes , 2015 ) . criteria , their construction and application appear to be an especially promising venue for designing diversity interventions and mitigating bias . ellemers ( 2014 ) argues that the first step is to recognize implicit bias in hiring and promotions , followed by the development of clear criteria for hiring , compensation , and promotion , instead of relying on subjective impressions [ or ] ambiguous prototypes bohnet ( 2016 ) recommends using comparative selection methods in promotion decisions , which means waiting until there is a pool of candidates which can then be evaluated per criterion rather than by candidate . in addition , strongly structuring the decision - making process removes the possible influence of irrelevant personal characteristics . the effects of intransitivity on the decision - making process can be reduced by discussing the meaning and relative weight of criteria prior to discussing candidates , and by leaving enough time at the end of the meeting to compare candidates explicitly in terms of the weight and value of their scores on each of the criteria . in practice , this often involves reopening the debate on the already established rank order , because of the recalibration of the importance of criteria ( vinkenburg et al . , 2014 ) . gatekeepers are decision makers , those in organizational positions of power who shape careers by selecting , promoting , and supporting organizational members ( bosley , arnold , & cohen , 2009 ; clerc & kels , 2013 ; van den brink & benschop , 2014 ) . in elite psfs , gatekeepers responsible for hiring procedures receive only minimal training in interviewing , use their personal theories of what constitutes merit and how best to judge it , and receive little feedback on the quality of their decisions meta - analyses of gender differences in performance evaluations clearly show that men are more favorably evaluated than women by their supervisors in field studies , in particular on ratings of promotability ( roth , purvis , & bobko , 2012 ) , especially when raters are men ( bowen , swim , & jacobs , 2000 ) , and more so when job complexity is high ( joshi et al . , 2015 ) . interestingly , sex differences in rewards were much larger than differences in performance evaluations , and differences in performance evaluations did not explain reward differences between men and women ( joshi et al . , 2015 ) . it appears that supervisors and other seniors who make career decisions hold an important key in disrupting the effect of bias on outcomes in up - or - out career systems . engaging power holders is essential for making diversity interventions successful ( benschop & van den brink , 2014 ; pendry et al . , 2007 ) , especially ( and paradoxically ) when the goal is to fundamentally alter power relations in organizations ( meyerson & kolb , 2000 ) . system interventions requires engaging organizations members up and down the hierarchy , men and women , to question their own and others deeply held assumptions about work , productivity and effectiveness including what constitutes and contributes to individual and organizational success block ( 2016 ) recommends cultivating awareness of diversity dynamics in organizations , combining bias awareness and power and privilege awareness . in order to expose and ultimately work through the paradox of meritocracy , i recommend to specifically engage gatekeepers in diversity interventions as they are power holders and important carriers of the belief in meritocracy . raising awareness of diversity dynamics , questioning implicit values , and working through paradox is a process that takes time and effort of a significant group of organization members in higher positions ( bleijenbergh , van engen , & vinkenburg , 2013 ) . castilla ( 2016 ) recommends implementing accountability and transparency in the pursuit of meritocracy decision makers are key in assigning or taking on these responsibilities . without the engagement of those with formal authority , diversity interventions may fizzle out ( dobbin & kalev , 2016 ; kalev , dobbin , & kelly , 2006 ) . i identify three main design specifications for systemic diversity interventions that can be drawn from the argumentation above ; namely engaging gatekeepers , mitigating bias , and optimizing decision making related to selection and promotion . the first refers to the target group , the second to the main lever , and the third to the relevant organizational or human resources processes at which the intervention is aimed . while the three intervention types described below share these three main design specifications , they are different in a few salient ways . the interventions differ in scope which is why i have ordered them from broad to narrow . interventions with a broad scope pertain to the entire institution or organization ; those with an intermediate scope pertain to organizational units ; and those with a narrow scope pertain to committees , councils , or panels responsible for career decisions . the interventions also differ in the degree to which they provide opportunities for rigorous field experimentation and/or established intervention techniques , including using control groups , piloting , pretesting , establishing a baseline , and posttesting or proper impact evaluation . the evidence - based movement would recommend such techniques but sometimes these get sacrificed in the struggle to gain access to an organization or population , the battle for scarce resources for a proposed intervention , or the one - off chance of jumping aboard an already moving train . while certainly a strong recommendation , these techniques should not stand in the way of designing an intervention using the three main specifications . the backbone of the description of each of the three interventions is a concrete example of this type of intervention , as published in a peer - reviewed journal article or publicly available report . each in its own way is built using the general specifications outlined above , which combined with their unique characteristics translate into design specifications for implementing these or similar interventions elsewhere ( see table 1 for an overview ) . any diversity intervention including the ones described here has much to gain from incorporating data - analytics or metrics ( williams , 2014 ) . the data to be analyzed might include ( longitudinal , individual ) career outcomes , representation , and progression rates , but also the impact evaluation of the intervention on several measures . data - analytics can serve as a starting point in diagnosing where bias occurs , and allow insight into intended and unintended intervention outcomes ( kaiser et al . , 2013 ) . measuring effects of interventions on various relevant indicators , including implicit measures and objective measures such as advancement rates could bring these ironies to light . additionally , all of the interventions below reflect design characteristics that follow from systematic reviews including meta - analyses on what works in diversity training ( benschop et al . , 2016 ; bezrukova , spell , perry , & jehn , 2016 ; kalinoski et al . , 2013 as also reported in table 1diversity interventions designed using these characteristics are conducted over a considerable period of time ; combine awareness and competence or skills building , use experiential learning , include various instruction methods , and are complemented by other diversity initiatives ( bezrukova et al . in addition , benschop et al . ( 2016 ) stress the importance of using an interactive approach and of addressing power relations in diversity interventions to help transform the structure and the culture of the organization . the peer - reviewed evidence on similar interventions in the corporate world is extremely limited ( benschop & van den brink , 2014 ; bezrukova et al . , 2016 ; kalinoski et al . , 2013 ) the very few examples of systemic diversity interventions from the corporate world ( to the best of my knowledge ) are based on action research using a dual agenda approach by focusing on work redesign , these interventions contribute to improved gender equity and workplace performance ( bailyn , rapoport , fletcher , & pruitt , 2002 ; charlesworth & baird , 2007 ; perlow & kelly , 2014 ) . as life integration but not on mitigating bias , i will not discuss these examples here . in academic settings , faculty members and peers act as gatekeepers by assessing merit and making career decisions about others regularly , including hiring , tenure and promotion , funding , and award nomination ( e.g. , bird , 2011)which makes this a prime location for interventions that engage gatekeepers , mitigate bias , and optimize decision making on careers . the first type of intervention can be described as large scale or broad scope , involving most if not all members of an organization . distinguishing features of the intervention are its considerable opportunity for field experimentation including long - term follow - up and control groups , and its emphasis on a combination of increasing bias awareness and building competence through experiential learning . program is a large scale systemic diversity intervention conducted at the university of wisconsin ( carnes et al . , using a pair - matched , single - blind , cluster - randomized , controlled intervention design , faculty members from 46 separate departments participated in a 2.5-hour gender bias habit - reducing training , with faculty members from 46 control departments waitlisted . bias literacy is about becoming conscious and competent in dealing with the biases that typically affect people decisions . during the workshop , research on the pervasiveness of stereotype - based gender bias in decision making and judgment the first module addresses the origins of bias as a habit , the second module promoted bias literacy by describing six kinds of stereotype - based gender bias , such as redefining credentials and stereotype priming , and the third module enhanced self - efficacy for overcoming gender bias by providing behavioral strategies , such as individuation , and by cautioning against counterproductive strategies such as a strong belief in one s ability to make objective judgments ( carnes et al . , 2015 ) . participants built positive outcome expectations by envisioning a link between their own actions and desired outcomes , as the facilitator reflected on benefits articulated by participants that can come from reducing implicit bias , and on the empowerment of aligning actions with personal beliefs to obtain a baseline premeasure , a short - term and a long - term postmeasure of effects of the intervention , participants were surveyed within 2 days before the intervention and at 3 days and again at 3 months postintervention . the results show significant differences in measures of ( explicit ) personal bias , external motivation to mitigate bias , and self - efficacy to engage in gender - equity - promoting behaviors ( carnes et al . , 2015 ) . in addition , significant increases in self - reported action to promote gender equity occurred at 3 months in departments where more than 25% of faculty had participated . such self - reported actions involved the degree to which participants said they were able to apply the behavioral strategies for reducing bias in decision making they had deliberately practiced during the workshops . moss - racusin et al . ( 2014 ) recommend combining self - report with behavioral observations from colleagues , students , and trained raters to strengthen the outcome measures . ideally , the intervention would be geared toward specific decision - making processes such as tenure or faculty hiring , rather than taking a more generalistic approach . in designing interventions of this type , data - analytics could be used to identify crucial career transitions where bias trumps merit - based assessment , or where certain criteria suddenly become more or less important in the process . participants could also be asked to bring in their own critical incidents , to be interactively discussed and resolved . in addition , the intervention could be strengthened by explicitly adding a system perspective to the curriculum ( adding to the duration of the workshop ) , for example , by explaining how a little bit of bias adds up to cumulative disadvantage and homogeneity in upward mobility systems ( martell et al . , 2012 ) , and how redesigning this type of intervention is especially successful in building not only participants awareness of bias but also their self - efficacy in being able to interrupt the effect of bias in future decision making . as has been argued elsewhere and shown meta - analytically , diversity self - efficacy plays a key role in facilitating diversity skill acquisition and implementation ( combs & luthans , 2007 ; kalinoski et al . , 2013 ) . active and especially experiential learning activities that enhance self - efficacy increase participants receptiveness to evidence of bias relative to more passive formats ( moss - racusin et al . , 2014 ) . through bias literacy s focus on promoting self - efficacy and behavioral intentions for future decision making , it moves beyond the bias awareness that is the common goal of many diversity interventions . participatory modeling or group model building is the only diversity intervention illustrated in this article that explicitly and purposefully takes a system perspective . using a method that has been developed for interventions in social systems , the overall goal is to engage problem owners from one organizational unit ( e.g. , department ) in building and playing with a system dynamics model to help them tackle an organizational issue ( lane , 2010 , as cited by bleijenbergh & van engen , 2015 , p. 423 ) . a complex problem is translated into a causal loop diagram of system components and feedback loops that shows the dynamics underlying the organizational issue . based on their experiences with this type of intervention in two separate dutch university settings , bleijenbergh and van engen ( 2015 ) describe the details of participatory modeling to support gender equality with gatekeepers such as department chairs , hr director , and dean . the intervention consists of three sessions ( a , b , and c ) of 3.5 hours each , with a 1-month interval to allow reflection and preparation . together with two facilitators and two gender experts , the participants strive to ( a ) identify and understand the underlying system dynamics of gender inequality in their own department , ( b ) to reach a shared problem definition and analysis , and ( c ) to identify and implement custom - made policies to promote change . the causal loop diagrams are literally built , adapted , and projected on the screen during the sessions , visualizing how feedback processes are interrelated and can stabilize or reinforce themselves . in figure 1 , a visualization of the causal loop diagram or models developed during one of these interventions by bleijenbergh and van engen ( 2015 ) can be found , mapping the percentage of women at various stages of the academic career ladder . participants identified the relative overrepresentation of men at the highest level as resulting from self - reinforcing feedback processes such as the masculinity of norms . interestingly , while the goal of the intervention was to promote gender equality , both generation and nationality emerged as additional sources of inequality , intersecting with gender . with an extremely diverse population of phd students and postdocs in terms of nationality , the continued overrepresentation of white dutch men with an undergraduate degree from the same university in senior faculty positions was striking . a major strong point of this type of intervention is related to the active involvement of gatekeepers . as a gender diversity expert participating in these sessions at one of the two universities , i personally experienced the strength of including problem owners in the modeling process to increase their commitment and facilitate implementation of interventions . in a personal account , my coauthor describes how confrontations that took place before and during the participatory modeling sessions between the research team and organizational stakeholders , while certainly challenging , ultimately supported the transformation of stakeholders into change agents ( bleijenbergh , 2016 ) . the masculinity of norms is a central element of the model built in both institutions . the bias that follows from the perceived lack of fit between women and the shared norm of the ideal academic who has no outside responsibilities negatively affects women s chances for tenure and promotion relative to men ( bleijenbergh et al . , 2013 ) . while participants recognized the masculinity of norms , they argued it would be difficult to change norms directly . however , by viewing norms as a crucial element of a larger system , the participants identified measures that could over time bend norms including introducing contract extensions for postdocs to compensate for parental leave . in a book chapter titled dare to care ( herschberg , vinkenburg , bleijenbergh , & van engen , 2014 ) , we describe in detail how the public negotiation of norms about career and care during the participatory modeling intervention resulted in a lively debate on what some participants considered normal and others did not . the dialogue as it spun out was literally transcribed into the chapter , as it proved to be a turning point in making change possible and in minimizing the impact of bias for all academics with care responsibilities ( not only mothers ! ) in promotion and tenure decisions . others have used a systems dynamics approach to uncover and investigate underlying feedback mechanisms that reinforce the overrepresentation of white men at the top of the hierarchy ( obrien et al . , 2015 ) , but participatory modeling is especially effective because gatekeepers identify particular rather than universal barriers and opportunities to increasing diversity in their own context . getting and keeping gatekeepers on board throughout the course of the intervention is a challenge , but if successful , the impact is palpable . bleijenbergh ( 2016 ) describes the very public actions to promote gender equality taken by initially quite resistant deans and other senior administrators after the invention . the rector ( i.e. , university president ) successfully defended one of the policies implemented in a discrimination case filed against the university by a male employee with the dutch national equal treatment commission . this intervention benefits from the use of data collected and analyzed prior to the first session by the gender experts , ranging from detailed longitudinal career metrics ( including pay differentials ) to interview or focus group data on how organizational members experience career opportunities , work family issues , and the atmosphere in the department . the evidence thus collected serves to challenge or confirm assumptions voiced by the participants during the sessions . while bias in merit assessment is not explicitly on the agenda , the perceived incompetence of women ( heilman , manzi , & braun , 2015 ) and ambiguity in hiring and promotion processes and criteria ( bird , 2011 ) are invariably a topic of discussion . implicit bias training to raise awareness and build competence is often high on the list of recommendations . bleijenbergh and van engen ( 2015 ) recommend tracking and measuring the implementation success of policies decided on , if needed by means of another round of participatory modeling . a more organic systemic intervention that puts less pressure on precious resources is participant observation during panel or committee meetings dedicated to reward allocation ( e.g. , research grants , prizes ) , performance evaluation , promotion or tenure decisions , and appointments . academic committees rely on peer review , performance evaluation , and merit assessment ( in terms of excellence , independence , track record , etc . ) as input for their deliberations and decisions . assuming a normal distribution of merit among candidates from different social groups , the urgency of mitigating bias in such decisions is relatively easy to establish if outcomes ( e.g. , success rates ) are unequal . in the context of psfs , joshi et al . ( 2015 ) recommend that trained neutral observers attend the so - called calibration meetings in which managers jointly discuss performance ratings and make bonus and promotion decisions about their direct reports . such observers may be able to direct discussion away from decision making based on stereotypes or biases toward performance - related information ( p. 1535 ) . ideally , the intervention would last throughout the complete lifecycle of a committee s main goal , be it hiring ( for one or multiple positions ) , annual performance evaluation or promotion rounds , semiannual or biannual selection panels or election councils , or similar . it would not demand any additional time from the committee members in terms of preparation , except for reserving some time at the end of each meeting or in an overall final session for feedback and reflection . the impact of the intervention could be measured in terms of the number of nondominant group members long - listed , short - listed , and selected or promoted , but also in terms of the awareness and competence of committee members in mitigating bias . eventually , if this kind of impact has been established , the task of addressing bias could be taken over by one of the committee members ( or even the chair ) . participant observation has been effectively applied at the swedish research council ( ahlqvist et al . , 2013 ; it requires the presence of a well - prepared , trained observer with status and voice who is allowed to give feedback on the content , process , and procedures of the committee meetings and decisions made . observers must be aware of bias and how it may affect the committee members and their decision making . observers may use several paper - and - pencil or app - supported tools to underscore their observations ; including counting the incidence and duration of time spent discussing various candidates or criteria , the use of evaluative language ( e.g. , masculine vs. feminine adjectives , positive vs. negative adjectives , negations ) , and the incidence and duration of talk by different committee members ( ahlqvist et al . , 2015 ) . if candidates are present during the meeting ( e.g. , interview , presentation ) , additional dimensions of the interaction and potential sources of bias may be observed . collecting and systematically analyzing the underlying documentation of the committee work , such as research proposals or personal statements , cvs , recommendation letters , performance reviews , and evaluation reports , could supplement observations ( kaatz et al . , 2015 ; madera , hebl , & martin , 2009 ) . due to the inherent intransitivity of the decision - making process , calibration meetings or final discussions during which several candidates are discussed and compared are highly informative in locating the emergent nature or differential application of criteria ( rivera , 2016 ; vinkenburg et al . , 2014 ) , and in charting the role of the chair in reducing cognitive load and allowing for joint evaluation or comparative selection ( bohnet , van geen , & bazerman , 2015 ; vinkenburg et al . , 2014 ) . observers can with their feedback expose and challenge normative expectations and paradoxes underlying the decision - making process , including the meritocracy myth or linear career bias . acting as change agents , dealing with resistance , and nudging gatekeepers toward optimization of the decision - making process , the observations and reflections from the participant observer and the responses from the committee may create a ripple effect that is noticeable throughout the system . the first type of intervention can be described as large scale or broad scope , involving most if not all members of an organization . distinguishing features of the intervention are its considerable opportunity for field experimentation including long - term follow - up and control groups , and its emphasis on a combination of increasing bias awareness and building competence through experiential learning . program is a large scale systemic diversity intervention conducted at the university of wisconsin ( carnes et al . , using a pair - matched , single - blind , cluster - randomized , controlled intervention design , faculty members from 46 separate departments participated in a 2.5-hour gender bias habit - reducing training , with faculty members from 46 control departments waitlisted . bias literacy is about becoming conscious and competent in dealing with the biases that typically affect people decisions . during the workshop , research on the pervasiveness of stereotype - based gender bias in decision making and judgment the first module addresses the origins of bias as a habit , the second module promoted bias literacy by describing six kinds of stereotype - based gender bias , such as redefining credentials and stereotype priming , and the third module enhanced self - efficacy for overcoming gender bias by providing behavioral strategies , such as individuation , and by cautioning against counterproductive strategies such as a strong belief in one s ability to make objective judgments ( carnes et al . , 2015 ) . participants built positive outcome expectations by envisioning a link between their own actions and desired outcomes , as the facilitator reflected on benefits articulated by participants that can come from reducing implicit bias , and on the empowerment of aligning actions with personal beliefs to obtain a baseline premeasure , a short - term and a long - term postmeasure of effects of the intervention , participants were surveyed within 2 days before the intervention and at 3 days and again at 3 months postintervention . the results show significant differences in measures of ( explicit ) personal bias , external motivation to mitigate bias , and self - efficacy to engage in gender - equity - promoting behaviors ( carnes et al . , 2015 ) . in addition , significant increases in self - reported action to promote gender equity occurred at 3 months in departments where more than 25% of faculty had participated . such self - reported actions involved the degree to which participants said they were able to apply the behavioral strategies for reducing bias in decision making they had deliberately practiced during the workshops . ( 2014 ) recommend combining self - report with behavioral observations from colleagues , students , and trained raters to strengthen the outcome measures . ideally , the intervention would be geared toward specific decision - making processes such as tenure or faculty hiring , rather than taking a more generalistic approach . in designing interventions of this type , data - analytics could be used to identify crucial career transitions where bias trumps merit - based assessment , or where certain criteria suddenly become more or less important in the process . participants could also be asked to bring in their own critical incidents , to be interactively discussed and resolved . in addition , the intervention could be strengthened by explicitly adding a system perspective to the curriculum ( adding to the duration of the workshop ) , for example , by explaining how a little bit of bias adds up to cumulative disadvantage and homogeneity in upward mobility systems ( martell et al . , 2012 ) , and how redesigning this type of intervention is especially successful in building not only participants awareness of bias but also their self - efficacy in being able to interrupt the effect of bias in future decision making . as has been argued elsewhere and shown meta - analytically , diversity self - efficacy plays a key role in facilitating diversity skill acquisition and implementation ( combs & luthans , 2007 ; kalinoski et al . , 2013 ) . active and especially experiential learning activities that enhance self - efficacy increase participants receptiveness to evidence of bias relative to more passive formats ( moss - racusin et al . , 2014 ) . through bias literacy s focus on promoting self - efficacy and behavioral intentions for future decision making , it moves beyond the bias awareness that is the common goal of many diversity interventions . participatory modeling or group model building is the only diversity intervention illustrated in this article that explicitly and purposefully takes a system perspective . using a method that has been developed for interventions in social systems , the overall goal is to engage problem owners from one organizational unit ( e.g. , department ) in building and playing with a system dynamics model to help them tackle an organizational issue ( lane , 2010 , as cited by bleijenbergh & van engen , 2015 , p. 423 ) . a complex problem is translated into a causal loop diagram of system components and feedback loops that shows the dynamics underlying the organizational issue . based on their experiences with this type of intervention in two separate dutch university settings , bleijenbergh and van engen ( 2015 ) describe the details of participatory modeling to support gender equality with gatekeepers such as department chairs , hr director , and dean . the intervention consists of three sessions ( a , b , and c ) of 3.5 hours each , with a 1-month interval to allow reflection and preparation . together with two facilitators and two gender experts , the participants strive to ( a ) identify and understand the underlying system dynamics of gender inequality in their own department , ( b ) to reach a shared problem definition and analysis , and ( c ) to identify and implement custom - made policies to promote change . the causal loop diagrams are literally built , adapted , and projected on the screen during the sessions , visualizing how feedback processes are interrelated and can stabilize or reinforce themselves . in figure 1 , a visualization of the causal loop diagram or models developed during one of these interventions by bleijenbergh and van engen ( 2015 ) can be found , mapping the percentage of women at various stages of the academic career ladder . participants identified the relative overrepresentation of men at the highest level as resulting from self - reinforcing feedback processes such as the masculinity of norms . interestingly , while the goal of the intervention was to promote gender equality , both generation and nationality emerged as additional sources of inequality , intersecting with gender . with an extremely diverse population of phd students and postdocs in terms of nationality , the continued overrepresentation of white dutch men with an undergraduate degree from the same university in senior faculty positions was striking . a major strong point of this type of intervention is related to the active involvement of gatekeepers . as a gender diversity expert participating in these sessions at one of the two universities , i personally experienced the strength of including problem owners in the modeling process to increase their commitment and facilitate implementation of interventions . in a personal account , my coauthor describes how confrontations that took place before and during the participatory modeling sessions between the research team and organizational stakeholders , while certainly challenging , ultimately supported the transformation of stakeholders into change agents ( bleijenbergh , 2016 ) . the masculinity of norms is a central element of the model built in both institutions . the bias that follows from the perceived lack of fit between women and the shared norm of the ideal academic who has no outside responsibilities negatively affects women s chances for tenure and promotion relative to men ( bleijenbergh et al . , 2013 ) . while participants recognized the masculinity of norms , they argued it would be difficult to change norms directly . however , by viewing norms as a crucial element of a larger system , the participants identified measures that could over time bend norms including introducing contract extensions for postdocs to compensate for parental leave . in a book chapter titled ( herschberg , vinkenburg , bleijenbergh , & van engen , 2014 ) , we describe in detail how the public negotiation of norms about career and care during the participatory modeling intervention resulted in a lively debate on what some participants considered normal and others did not . the dialogue as it spun out was literally transcribed into the chapter , as it proved to be a turning point in making change possible and in minimizing the impact of bias for all academics with care responsibilities ( not only mothers ! ) in promotion and tenure decisions . others have used a systems dynamics approach to uncover and investigate underlying feedback mechanisms that reinforce the overrepresentation of white men at the top of the hierarchy ( obrien et al . , 2015 ) , but participatory modeling is especially effective because gatekeepers identify particular rather than universal barriers and opportunities to increasing diversity in their own context . getting and keeping gatekeepers on board throughout the course of the intervention is a challenge , but if successful , the impact is palpable . bleijenbergh ( 2016 ) describes the very public actions to promote gender equality taken by initially quite resistant deans and other senior administrators after the invention . the rector ( i.e. , university president ) successfully defended one of the policies implemented in a discrimination case filed against the university by a male employee with the dutch national equal treatment commission . this intervention benefits from the use of data collected and analyzed prior to the first session by the gender experts , ranging from detailed longitudinal career metrics ( including pay differentials ) to interview or focus group data on how organizational members experience career opportunities , work family issues , and the atmosphere in the department . the evidence thus collected serves to challenge or confirm assumptions voiced by the participants during the sessions . while bias in merit assessment is not explicitly on the agenda , the perceived incompetence of women ( heilman , manzi , & braun , 2015 ) and ambiguity in hiring and promotion processes and criteria ( bird , 2011 ) are invariably a topic of discussion . implicit bias training to raise awareness and build competence is often high on the list of recommendations . bleijenbergh and van engen ( 2015 ) recommend tracking and measuring the implementation success of policies decided on , if needed by means of another round of participatory modeling . participatory modeling or group model building is the only diversity intervention illustrated in this article that explicitly and purposefully takes a system perspective . using a method that has been developed for interventions in social systems , the overall goal is to engage problem owners from one organizational unit ( e.g. , department ) in building and playing with a system dynamics model to help them tackle an organizational issue ( lane , 2010 , as cited by bleijenbergh & van engen , 2015 , p. 423 ) . a complex problem is translated into a causal loop diagram of system components and feedback loops that shows the dynamics underlying the organizational issue . based on their experiences with this type of intervention in two separate dutch university settings , bleijenbergh and van engen ( 2015 ) describe the details of participatory modeling to support gender equality with gatekeepers such as department chairs , hr director , and dean . the intervention consists of three sessions ( a , b , and c ) of 3.5 hours each , with a 1-month interval to allow reflection and preparation . together with two facilitators and two gender experts , the participants strive to ( a ) identify and understand the underlying system dynamics of gender inequality in their own department , ( b ) to reach a shared problem definition and analysis , and ( c ) to identify and implement custom - made policies to promote change . the causal loop diagrams are literally built , adapted , and projected on the screen during the sessions , visualizing how feedback processes are interrelated and can stabilize or reinforce themselves . in figure 1 , a visualization of the causal loop diagram or models developed during one of these interventions by bleijenbergh and van engen ( 2015 ) can be found , mapping the percentage of women at various stages of the academic career ladder . participants identified the relative overrepresentation of men at the highest level as resulting from self - reinforcing feedback processes such as the masculinity of norms . interestingly , while the goal of the intervention was to promote gender equality , both generation and nationality emerged as additional sources of inequality , intersecting with gender . with an extremely diverse population of phd students and postdocs in terms of nationality , the continued overrepresentation of white dutch men with an undergraduate degree from the same university in senior faculty positions was striking . a major strong point of this type of intervention is related to the active involvement of gatekeepers . as a gender diversity expert participating in these sessions at one of the two universities , i personally experienced the strength of including problem owners in the modeling process to increase their commitment and facilitate implementation of interventions . in a personal account , my coauthor describes how confrontations that took place before and during the participatory modeling sessions between the research team and organizational stakeholders , while certainly challenging , ultimately supported the transformation of stakeholders into change agents ( bleijenbergh , 2016 ) . the masculinity of norms is a central element of the model built in both institutions . the bias that follows from the perceived lack of fit between women and the shared norm of the ideal academic who has no outside responsibilities negatively affects women s chances for tenure and promotion relative to men ( bleijenbergh et al . , 2013 ) . while participants recognized the masculinity of norms , they argued it would be difficult to change norms directly . however , by viewing norms as a crucial element of a larger system , the participants identified measures that could over time bend norms including introducing contract extensions for postdocs to compensate for parental leave . in a book chapter titled dare to care ( herschberg , vinkenburg , bleijenbergh , & van engen , 2014 ) , we describe in detail how the public negotiation of norms about career and care during the participatory modeling intervention resulted in a lively debate on what some participants considered normal and others did not . the dialogue as it spun out was literally transcribed into the chapter , as it proved to be a turning point in making change possible and in minimizing the impact of bias for all academics with care responsibilities ( not only mothers ! ) in promotion and tenure decisions . others have used a systems dynamics approach to uncover and investigate underlying feedback mechanisms that reinforce the overrepresentation of white men at the top of the hierarchy ( obrien et al . , 2015 ) , but participatory modeling is especially effective because gatekeepers identify particular rather than universal barriers and opportunities to increasing diversity in their own context . getting and keeping gatekeepers on board throughout the course of the intervention is a challenge , but if successful , the impact is palpable . bleijenbergh ( 2016 ) describes the very public actions to promote gender equality taken by initially quite resistant deans and other senior administrators after the invention . the rector ( i.e. , university president ) successfully defended one of the policies implemented in a discrimination case filed against the university by a male employee with the dutch national equal treatment commission . this intervention benefits from the use of data collected and analyzed prior to the first session by the gender experts , ranging from detailed longitudinal career metrics ( including pay differentials ) to interview or focus group data on how organizational members experience career opportunities , work family issues , and the atmosphere in the department . the evidence thus collected serves to challenge or confirm assumptions voiced by the participants during the sessions . while bias in merit assessment is not explicitly on the agenda , the perceived incompetence of women ( heilman , manzi , & braun , 2015 ) and ambiguity in hiring and promotion processes and criteria ( bird , 2011 ) are invariably a topic of discussion . implicit bias training to raise awareness and build competence is often high on the list of recommendations . bleijenbergh and van engen ( 2015 ) recommend tracking and measuring the implementation success of policies decided on , if needed by means of another round of participatory modeling . a more organic systemic intervention that puts less pressure on precious resources is participant observation during panel or committee meetings dedicated to reward allocation ( e.g. , research grants , prizes ) , performance evaluation , promotion or tenure decisions , and appointments . academic committees rely on peer review , performance evaluation , and merit assessment ( in terms of excellence , independence , track record , etc . ) as input for their deliberations and decisions . assuming a normal distribution of merit among candidates from different social groups , the urgency of mitigating bias in such decisions is relatively easy to establish if outcomes ( e.g. , success rates ) are unequal . in the context of psfs , joshi et al . ( 2015 ) recommend that trained neutral observers attend the so - called calibration meetings in which managers jointly discuss performance ratings and make bonus and promotion decisions about their direct reports . such observers may be able to direct discussion away from decision making based on stereotypes or biases toward performance - related information ( p. 1535 ) . ideally , the intervention would last throughout the complete lifecycle of a committee s main goal , be it hiring ( for one or multiple positions ) , annual performance evaluation or promotion rounds , semiannual or biannual selection panels or election councils , or similar . it would not demand any additional time from the committee members in terms of preparation , except for reserving some time at the end of each meeting or in an overall final session for feedback and reflection . the impact of the intervention could be measured in terms of the number of nondominant group members long - listed , short - listed , and selected or promoted , but also in terms of the awareness and competence of committee members in mitigating bias . eventually , if this kind of impact has been established , the task of addressing bias could be taken over by one of the committee members ( or even the chair ) . participant observation has been effectively applied at the swedish research council ( ahlqvist et al . , 2013 ; ahlqvist et al . , 2015 ) . it requires the presence of a well - prepared , trained observer with status and voice who is allowed to give feedback on the content , process , and procedures of the committee meetings and decisions made . observers must be aware of bias and how it may affect the committee members and their decision making . observers may use several paper - and - pencil or app - supported tools to underscore their observations ; including counting the incidence and duration of time spent discussing various candidates or criteria , the use of evaluative language ( e.g. , masculine vs. feminine adjectives , positive vs. negative adjectives , negations ) , and the incidence and duration of talk by different committee members ( ahlqvist et al . , 2015 ) . if candidates are present during the meeting ( e.g. , interview , presentation ) , additional dimensions of the interaction and potential sources of bias may be observed . collecting and systematically analyzing the underlying documentation of the committee work , such as research proposals or personal statements , cvs , recommendation letters , performance reviews , and evaluation reports , could supplement observations ( kaatz et al . , 2015 ; madera , hebl , & martin , 2009 ) . due to the inherent intransitivity of the decision - making process , calibration meetings or final discussions during which several candidates are discussed and compared are highly informative in locating the emergent nature or differential application of criteria ( rivera , 2016 ; vinkenburg et al . , 2014 ) , and in charting the role of the chair in reducing cognitive load and allowing for joint evaluation or comparative selection ( bohnet , van geen , & bazerman , 2015 ; vinkenburg et al . , 2014 ) . observers can with their feedback expose and challenge normative expectations and paradoxes underlying the decision - making process , including the meritocracy myth or linear career bias . acting as change agents , dealing with resistance , and nudging gatekeepers toward optimization of the decision - making process , the observations and reflections from the participant observer and the responses from the committee may create a ripple effect that is noticeable throughout the system . with these three examples of systemic diversity interventions in academic settings , i have highlighted the design specifications for interventions effective in promoting diversity in upward mobility systems . by optimizing decision making , mitigating bias , and engaging gatekeepers , each of the three interventions will improve promotion and advancement rates of nondominant group members and thus reduce the overrepresentation of white men at the top of the pyramid . in contrast to many other diversity interventions , the interventions described here do challenge and change existing merit assessment , performance evaluation , and reward allocation practices and address the resistance that both dominant and nondominant organizational members may experience when these practices are exposed . all three examples of the interventions provided here share a distinctive action research characteristic , namely that researchers as experts work together with organizational members in creating organizational change and generating robust , actionable knowledge ( coghlan , 2011 ) . but even without a distinct research goal or the active involvement of researchers , interventions build using the design specifications outlined here and provided in table 1 , should encourage action learning , through reflection and ( for some participants at least ) reflexivity ( raelin , 1997 ) . follow - up is needed to ensure the measurement of long - term results and the sustained transformation of ongoing decision - making processes . taking a systems perspective , by looking at career decisions in particular , each of these interventions aims to debias the system rather than to debias the mind - set ( bohnet , 2016 ) . in doing so , in all three interventions the underlying paradox of meritocracy is exposed and worked through to promote equal representation at all levels of the hierarchy . without a formal method to compare the effectiveness of these interventions , the bias literacy program finds its main strength in bias mitigation , as it shows how to move beyond the stereotype - induced presumed incompetence of nondominant group members being evaluated . in its current form , it is perhaps too generalistic and does not cover enough ground in terms of system dynamics . participatory modeling or group model building finds its main strength in truly engaging gatekeepers as problem owners in the identification and solution for specific problems within their own setting . whether bias mitigation becomes part of the solution depends strongly on whether participants identify this as an important factor and possible solution in the model . participant observation , finally , finds its main strength in optimizing decision making as it takes place , thus directly improving the operationalization and application of criteria for evaluation , selection , and promotion . without explicitly linking the intervention and its consequences to the wider organization and its decision - making habits , learning and change may be limited to individual committee members . which of these interventions fits the diversity issue a policy maker or diversity officer would like to tackle , depends on the situation . but the basic design specifications of engaging gatekeepers and optimizing decision making by mitigating bias can be applied in many organizational settings where people decisions are made and careers develop over time . the most natural habitat for these interventions are upward mobility career systems with their fixed steps or routes and formal promotion criteria such as psfs ( e.g. , kumra , 2014 ) , but other organizational settings in which performance evaluations serve as input for promotion and remuneration decisions would benefit as well ( e.g. , van den brink et al . , 2016 ) . as many diversity interventions seem to fail , and as efforts to promote diversity at the top of the organizational hierarchy are especially unsuccessful , we need more evidence on what does work and how to do it . with this collage of successful systemic diversity interventions and their matching design specifications , i hope to have inspired both researchers and practitioners in designing and studying the effects of diversity interventions both inside and outside career systems that value upward mobility as well as social equality .

in this contribution to the journal of applied behavioral science special issue on understanding diversity dynamics in systems : social equality as an organization change issue , i develop and describe design specifications for systemic diversity interventions in upward mobility career systems , aimed at optimizing decision making through mitigating bias by engaging gatekeepers . these interventions address the paradox of meritocracy that underlies the surprising lack of diversity at the top of the career pyramid in these systems . i ground the design specifications in the limited empirical evidence on what works in systemic interventions . specifically , i describe examples from interventions in academic settings , including a bias literacy program , participatory modeling , and participant observation . the design specifications , paired with inspirational examples of successful interventions , should assist diversity officers and consultants in designing and implementing interventions to promote the advancement to and representation of nondominant group members at the top of the organizational hierarchy .

Taking a Systems Perspective Mitigating Bias in Selection and Promotion Decisions Engaging Gatekeepers Design Specifications for Diversity Interventions Broad Scope Intervention: Bias Literacy Program Intermediate Scope Intervention: Participatory Modeling Narrow Scope Intervention: Participant Observation Discussion and Conclusion

the paradox of meritocracy is alive and well under conditions where careers are primarily evaluated in terms of upward mobility ( vinkenburg & weber , 2012 ) . in order to sustain the pyramidal shape of the hierarchy , those not advancing to the next career level according to the given parameters are counseled to leave generally , an up - or - out system strongly suggests meritocracy through its reliance on formal procedures and transparent criteria . , billable hours , impact factor ) , are not decisive in the process , but only serve as a threshold for being considered , are systematically overestimated for dominant group members , or even dropped from the deliberations altogether ( van den brink , holgersson , linghag , & de , 2016 ; vinkenburg , jansen , dries , & pepermans , 2014 ) . decision making on promotion to the highest levels of organizational hierarchies , due to structural conditions and situational components , can be characterized as especially imperfect ( vinkenburg et al . under such conditions , and regardless of actual merit , the likelihood of upward mobility is higher for dominant group members than for others . the consequence is the typical image of almost exclusively white men at the top of the hierarchy . in a career system that thus creates and maintains outcome disparities between members of different social groups , and where the paradox of meritocracy feeds into a limited awareness of biased decision making ( block , 2016 ) , breaking the cycle is complicated . while many organizations that embrace an up - or - out career system recruit and select a heterogeneous or diverse pool of entry - level candidates , after 12 to 15 years typically a disproportionate number of dominant group members will have made it to the highest level . nondominant group members will be absent at this level , as the career system does not allow up - or - stay . taking a systems perspective , martell , emrich , and robison - cox ( 2012 ) conceptualize the process by which micro - level forces such as bias in performance assessment and macro - level forces such as demographic composition operate together to ( re)produce gender segregation at the top of the organizational hierarchy . in upward mobility systems , promotion signals such as early success and velocity , as well as promotion models shaped as tournaments and gatekeeping exacerbate segregation ( martell et al . organizational members increasingly adapt to and adopt behavioral and interactional rules for reasons of efficiency and uncertainty reduction , and over time the unwritten rules of the game ( scott - morgan , 1994 ; sydow , schreygg , & koch , 2009 ) become so normalized that members are locked - in and no longer see alternative , broader scopes of action . as an example , the rule to spend friday in the office instead of at the client site ( invented for efficiency reasons ) becomes a way to increase visibility with gatekeepers , and emerges as a criterion for promotion , effectively excluding those who do not spend friday in the office for religious or care - related reasons . as a system tends to reproduce and reinforce itself , targeted interventions common diversity interventions in organizations such as training , mentoring , and networking are criticized because they fail to transform organizational structures and cultures , because they aim to fix other employees rather than fix the system , and because they do not target dominant group members ( benschop , holgersson , van den brink , & wahl , 2016 ; bird , 2011 ; ely & meyerson , 2000 ) . designing systemic diversity interventions with transformative power , such as the examples described later on in this article , requires basic knowledge of system dynamics , an approach that is useful in understanding the behavior of complex systems over time . when drawing a causal loop diagram representing diversity in a career system , stocks represent the relative representation of members of various social groups at different levels in the hierarchy , flows represent the relative advancement rates from one level in the hierarchy to the next , and feedback loops represent self - reinforcing positive or negative feedback processes in the diagram ( bleijenbergh & van engen , 2015 ) . 2015 ) use system dynamics , including stocks and flows , to predict the rate of change in workforce composition at different levels of the hierarchy depending on the rate at which different categories of employees enter , are promoted , and leave the organization . the main challenge in designing an effective diversity intervention lies in the identification of the main lever for change . bias in the strictest definition of the term is a cognitive distortion and is evidenced in decision making . i will show how bias plays a role in reproducing the overrepresentation of a homogeneous group at the top of organizations by looking at cognitive distortions in general and gender bias in particular . while racioethnic bias is another common and tenacious kind of bias , i do not discuss it here , because underlying stereotypes of various racioethnic categories are first more particular to the societal and organizational context than gender stereotypes ( roberson & block , 2001 ) , second are more conflated with stereotypes about immigration and religion than gender stereotypes ( vinkenburg , 2014 ) , third are more ambivalent in terms of content ( fiske , 2012 ) than gender stereotypes , and fourth are less well described in the literature in terms of career consequences ( ossenkop , vinkenburg , jansen , & ghorashi , 2015a ) than gender stereotypes . in up - or - out career systems , where white men are relatively overrepresented at the top of the hierarchy , the biased assessment of merit is in fact more a matter of favoritism ( bias for white men ) than of discrimination ( bias against all others ) . in from bias to exclusion , ( 2012 ) argue that if every career - related decision along the way is only a little bit biased in favor of one group versus another , the top of the hierarchy will remain segregated . first , if the description of the criterion is abstract , allowing discretion in the operationalization of vague terms such as potential , talent , or excellence leaves room for bias ( festing , kornau , & schfer , 2014 ; robinson , fetters , riester , & bracco , 2009 ) . finally , linguistic bias creeps into evaluative language , including using negations more frequently for nondominant group members ( e.g. the effects of intransitivity on the decision - making process can be reduced by discussing the meaning and relative weight of criteria prior to discussing candidates , and by leaving enough time at the end of the meeting to compare candidates explicitly in terms of the weight and value of their scores on each of the criteria . system interventions requires engaging organizations members up and down the hierarchy , men and women , to question their own and others deeply held assumptions about work , productivity and effectiveness including what constitutes and contributes to individual and organizational success block ( 2016 ) recommends cultivating awareness of diversity dynamics in organizations , combining bias awareness and power and privilege awareness . in order to expose and ultimately work through the paradox of meritocracy , i recommend to specifically engage gatekeepers in diversity interventions as they are power holders and important carriers of the belief in meritocracy . raising awareness of diversity dynamics , questioning implicit values , and working through paradox is a process that takes time and effort of a significant group of organization members in higher positions ( bleijenbergh , van engen , & vinkenburg , 2013 ) . i identify three main design specifications for systemic diversity interventions that can be drawn from the argumentation above ; namely engaging gatekeepers , mitigating bias , and optimizing decision making related to selection and promotion . the first refers to the target group , the second to the main lever , and the third to the relevant organizational or human resources processes at which the intervention is aimed . the interventions also differ in the degree to which they provide opportunities for rigorous field experimentation and/or established intervention techniques , including using control groups , piloting , pretesting , establishing a baseline , and posttesting or proper impact evaluation . the evidence - based movement would recommend such techniques but sometimes these get sacrificed in the struggle to gain access to an organization or population , the battle for scarce resources for a proposed intervention , or the one - off chance of jumping aboard an already moving train . additionally , all of the interventions below reflect design characteristics that follow from systematic reviews including meta - analyses on what works in diversity training ( benschop et al . ( 2016 ) stress the importance of using an interactive approach and of addressing power relations in diversity interventions to help transform the structure and the culture of the organization . the peer - reviewed evidence on similar interventions in the corporate world is extremely limited ( benschop & van den brink , 2014 ; bezrukova et al . , 2013 ) the very few examples of systemic diversity interventions from the corporate world ( to the best of my knowledge ) are based on action research using a dual agenda approach by focusing on work redesign , these interventions contribute to improved gender equity and workplace performance ( bailyn , rapoport , fletcher , & pruitt , 2002 ; charlesworth & baird , 2007 ; perlow & kelly , 2014 ) . in academic settings , faculty members and peers act as gatekeepers by assessing merit and making career decisions about others regularly , including hiring , tenure and promotion , funding , and award nomination ( e.g. program is a large scale systemic diversity intervention conducted at the university of wisconsin ( carnes et al . during the workshop , research on the pervasiveness of stereotype - based gender bias in decision making and judgment the first module addresses the origins of bias as a habit , the second module promoted bias literacy by describing six kinds of stereotype - based gender bias , such as redefining credentials and stereotype priming , and the third module enhanced self - efficacy for overcoming gender bias by providing behavioral strategies , such as individuation , and by cautioning against counterproductive strategies such as a strong belief in one s ability to make objective judgments ( carnes et al . in designing interventions of this type , data - analytics could be used to identify crucial career transitions where bias trumps merit - based assessment , or where certain criteria suddenly become more or less important in the process . in addition , the intervention could be strengthened by explicitly adding a system perspective to the curriculum ( adding to the duration of the workshop ) , for example , by explaining how a little bit of bias adds up to cumulative disadvantage and homogeneity in upward mobility systems ( martell et al . through bias literacy s focus on promoting self - efficacy and behavioral intentions for future decision making , it moves beyond the bias awareness that is the common goal of many diversity interventions . based on their experiences with this type of intervention in two separate dutch university settings , bleijenbergh and van engen ( 2015 ) describe the details of participatory modeling to support gender equality with gatekeepers such as department chairs , hr director , and dean . interestingly , while the goal of the intervention was to promote gender equality , both generation and nationality emerged as additional sources of inequality , intersecting with gender . as a gender diversity expert participating in these sessions at one of the two universities , i personally experienced the strength of including problem owners in the modeling process to increase their commitment and facilitate implementation of interventions . others have used a systems dynamics approach to uncover and investigate underlying feedback mechanisms that reinforce the overrepresentation of white men at the top of the hierarchy ( obrien et al . this intervention benefits from the use of data collected and analyzed prior to the first session by the gender experts , ranging from detailed longitudinal career metrics ( including pay differentials ) to interview or focus group data on how organizational members experience career opportunities , work family issues , and the atmosphere in the department . the impact of the intervention could be measured in terms of the number of nondominant group members long - listed , short - listed , and selected or promoted , but also in terms of the awareness and competence of committee members in mitigating bias . , 2013 ; it requires the presence of a well - prepared , trained observer with status and voice who is allowed to give feedback on the content , process , and procedures of the committee meetings and decisions made . due to the inherent intransitivity of the decision - making process , calibration meetings or final discussions during which several candidates are discussed and compared are highly informative in locating the emergent nature or differential application of criteria ( rivera , 2016 ; vinkenburg et al . acting as change agents , dealing with resistance , and nudging gatekeepers toward optimization of the decision - making process , the observations and reflections from the participant observer and the responses from the committee may create a ripple effect that is noticeable throughout the system . during the workshop , research on the pervasiveness of stereotype - based gender bias in decision making and judgment the first module addresses the origins of bias as a habit , the second module promoted bias literacy by describing six kinds of stereotype - based gender bias , such as redefining credentials and stereotype priming , and the third module enhanced self - efficacy for overcoming gender bias by providing behavioral strategies , such as individuation , and by cautioning against counterproductive strategies such as a strong belief in one s ability to make objective judgments ( carnes et al . in addition , the intervention could be strengthened by explicitly adding a system perspective to the curriculum ( adding to the duration of the workshop ) , for example , by explaining how a little bit of bias adds up to cumulative disadvantage and homogeneity in upward mobility systems ( martell et al . through bias literacy s focus on promoting self - efficacy and behavioral intentions for future decision making , it moves beyond the bias awareness that is the common goal of many diversity interventions . participatory modeling or group model building is the only diversity intervention illustrated in this article that explicitly and purposefully takes a system perspective . using a method that has been developed for interventions in social systems , the overall goal is to engage problem owners from one organizational unit ( e.g. based on their experiences with this type of intervention in two separate dutch university settings , bleijenbergh and van engen ( 2015 ) describe the details of participatory modeling to support gender equality with gatekeepers such as department chairs , hr director , and dean . together with two facilitators and two gender experts , the participants strive to ( a ) identify and understand the underlying system dynamics of gender inequality in their own department , ( b ) to reach a shared problem definition and analysis , and ( c ) to identify and implement custom - made policies to promote change . in figure 1 , a visualization of the causal loop diagram or models developed during one of these interventions by bleijenbergh and van engen ( 2015 ) can be found , mapping the percentage of women at various stages of the academic career ladder . as a gender diversity expert participating in these sessions at one of the two universities , i personally experienced the strength of including problem owners in the modeling process to increase their commitment and facilitate implementation of interventions . others have used a systems dynamics approach to uncover and investigate underlying feedback mechanisms that reinforce the overrepresentation of white men at the top of the hierarchy ( obrien et al . this intervention benefits from the use of data collected and analyzed prior to the first session by the gender experts , ranging from detailed longitudinal career metrics ( including pay differentials ) to interview or focus group data on how organizational members experience career opportunities , work family issues , and the atmosphere in the department . participatory modeling or group model building is the only diversity intervention illustrated in this article that explicitly and purposefully takes a system perspective . using a method that has been developed for interventions in social systems , the overall goal is to engage problem owners from one organizational unit ( e.g. based on their experiences with this type of intervention in two separate dutch university settings , bleijenbergh and van engen ( 2015 ) describe the details of participatory modeling to support gender equality with gatekeepers such as department chairs , hr director , and dean . together with two facilitators and two gender experts , the participants strive to ( a ) identify and understand the underlying system dynamics of gender inequality in their own department , ( b ) to reach a shared problem definition and analysis , and ( c ) to identify and implement custom - made policies to promote change . interestingly , while the goal of the intervention was to promote gender equality , both generation and nationality emerged as additional sources of inequality , intersecting with gender . as a gender diversity expert participating in these sessions at one of the two universities , i personally experienced the strength of including problem owners in the modeling process to increase their commitment and facilitate implementation of interventions . others have used a systems dynamics approach to uncover and investigate underlying feedback mechanisms that reinforce the overrepresentation of white men at the top of the hierarchy ( obrien et al . this intervention benefits from the use of data collected and analyzed prior to the first session by the gender experts , ranging from detailed longitudinal career metrics ( including pay differentials ) to interview or focus group data on how organizational members experience career opportunities , work family issues , and the atmosphere in the department . the impact of the intervention could be measured in terms of the number of nondominant group members long - listed , short - listed , and selected or promoted , but also in terms of the awareness and competence of committee members in mitigating bias . it requires the presence of a well - prepared , trained observer with status and voice who is allowed to give feedback on the content , process , and procedures of the committee meetings and decisions made . collecting and systematically analyzing the underlying documentation of the committee work , such as research proposals or personal statements , cvs , recommendation letters , performance reviews , and evaluation reports , could supplement observations ( kaatz et al . with these three examples of systemic diversity interventions in academic settings , i have highlighted the design specifications for interventions effective in promoting diversity in upward mobility systems . by optimizing decision making , mitigating bias , and engaging gatekeepers , each of the three interventions will improve promotion and advancement rates of nondominant group members and thus reduce the overrepresentation of white men at the top of the pyramid . in contrast to many other diversity interventions , the interventions described here do challenge and change existing merit assessment , performance evaluation , and reward allocation practices and address the resistance that both dominant and nondominant organizational members may experience when these practices are exposed . but even without a distinct research goal or the active involvement of researchers , interventions build using the design specifications outlined here and provided in table 1 , should encourage action learning , through reflection and ( for some participants at least ) reflexivity ( raelin , 1997 ) . in doing so , in all three interventions the underlying paradox of meritocracy is exposed and worked through to promote equal representation at all levels of the hierarchy . without a formal method to compare the effectiveness of these interventions , the bias literacy program finds its main strength in bias mitigation , as it shows how to move beyond the stereotype - induced presumed incompetence of nondominant group members being evaluated . participatory modeling or group model building finds its main strength in truly engaging gatekeepers as problem owners in the identification and solution for specific problems within their own setting . whether bias mitigation becomes part of the solution depends strongly on whether participants identify this as an important factor and possible solution in the model . participant observation , finally , finds its main strength in optimizing decision making as it takes place , thus directly improving the operationalization and application of criteria for evaluation , selection , and promotion . but the basic design specifications of engaging gatekeepers and optimizing decision making by mitigating bias can be applied in many organizational settings where people decisions are made and careers develop over time . the most natural habitat for these interventions are upward mobility career systems with their fixed steps or routes and formal promotion criteria such as psfs ( e.g. as many diversity interventions seem to fail , and as efforts to promote diversity at the top of the organizational hierarchy are especially unsuccessful , we need more evidence on what does work and how to do it . with this collage of successful systemic diversity interventions and their matching design specifications , i hope to have inspired both researchers and practitioners in designing and studying the effects of diversity interventions both inside and outside career systems that value upward mobility as well as social equality .

the success of the human genome project has clearly shown that the human genome encodes about 35,000 genes and over half of them are poorly understood proteins . a number of these proteins are thought to be related to pathological disorders and other biological phenomena , and thus , are potentially useful as therapeutic agents and as targets for pharmaceutical development . therefore , focus in life science research has currently shifted to newer fields , such as proteomics and structural genomics , in which the function and structure of proteins are analyzed en masse . such analyses of large number of proteins , including the newly identified proteins , are expected to contribute to the identification of proteins of therapeutic importance in various diseases . in recent years indeed , attempts are currently under progress to develop a wide range of therapeutic proteins for treating various diseases including cancer , hepatitis and autoimmune conditions[1 - 4 ] . unfortunately , clinical applications of many of these proteins are limited because of their unexpectedly poor therapeutic effects . often these proteins are degraded by various proteases in vivo and rapidly removed from the circulatory system . consequently , frequent administration of an excessively high dose of a protein is required to obtain its desired therapeutic effect in vivo , leading to a disturbance in the homeostasis and unexpected side effects . additionally , bioactive proteins , such as cytokines , generally show pleiotropic actions through a number of receptors in vivo , making it difficult to elicit the desired effect without simultaneously triggering undesirable secondary effects . from this standpoint , creation of novel technologies that overcome the problems peculiar to bioactive proteins is essential for the advancement of pharmaco - proteomic - based drug development . these technologies are suitable as drug delivery systems ( ddss ) , aiming to maximize the therapeutic potency of proteins . our laboratory aims to develop novel dds techniques for overcoming the problems of protein therapy : ( i ) establishment of a novel polymer - conjugation system to dramatically improve in vivo stability and selectively of bioactive proteins ( polymeric dds ) and ( ii ) development of a powerful system to rapidly create functional mutant proteins ( muteins ) with enhanced receptor affinity and receptor specificity using a phage display technique ( biological dds ) . we are currently in the process of combining both approaches to create a protein - drug innovation system to further promote pharmaco - proteomic - based drug development . in this review , we will describe dds - based technology to create functional mutants for advanced medical applications using the tumor necrosis factor - alpha ( tnf ) as an example and the usefulness of site - specific polymer - conjugation of proteins . one way to circumvent the problems of protein therapy is to conjugate them with polyethylene glycol ( peg ) and other water - soluble polymeric modifiers ( figure 1 ) . the covalent conjugation of proteins with peg ( pegylation ) increases their molecular size and steric hindrance , both of which depend on the properties of the peg attached to the protein . in addition , because the polymers cover the protein surface , attack by a protease is generally blocked due to the steric hindrance , thus prolonging the half - life of the protein in vivo . the prolonged circulating lifetime in blood induces the enhanced permeability and retention ( epr ) effect , which is based on the leaky nature of the tumor blood vessels , resulting in increased delivery of the conjugates to the tumor tissue . because of all these advantages , it is possible to use the bioactive protein at a decreased dose . however , this approach is limited by the frequent substantial loss of protein specific activity associated with polymer - conjugation . bioconjugation commonly targets the -amino group of lysine residues and/or the n - terminal -amino group of the protein because they are highly reactive and the bionconjugation reaction is mild enough to minimize disruption of the protein structure . because lysine residues often assume important roles in the formation of multi - dimensional structures and in bonding between ligands and receptors , introduction of polymers at these sites can potentially reduce the biological activity of the protein . indeed , pegylated interferon , which has raised hopes as a potential cure for hepatitis c , can only be produced as a heterogeneous mixture with 10 - 30% of the anticipated activity . tumor necrosis factor- ( tnf- ) , an antitumor cytokine , has numerous bioactivities including direct cytotoxicity against tumor cells , activation of immune antitumor response , and selective impairment of tumor - blood vessels . thus , tnf- has been considered as a novel antitumor agent[13 - 15 ] . however , as a systemic antitumor agent , very high doses of tnf- were required to obtain sufficient clinical responses , because tnf- is rapidly cleared from the circulation and also because it becomes widely distributed to various tissues following intravenous administration . as a result , tnf- with pleiotropic in vivo actions exhibited unexpected toxic side - effects , typified by pyrexia and hypertension . systemic application of tnf- was , therefore , abandoned despite intratumoral administration of tnf- showing significant antitumor effects in phase i studies . similar in vivo drawbacks are also found in the clinical applications of other bioactive proteins . keeping these problems in mind , we have devised ways to improve the polymer - conjugation system using the tnf- as a model protein[18 - 23 ] . in pegylation of tnf- , the specific activity of pegylated tnf- decreased with the degree of peg - modification ( i.e. , peg modification rate ) . additionally , when the peg modification rates are same , the bioactivity of pegylated tnf- decreased with an increase in the molecular size of the attached peg . thus , when bioactive proteins , such as tnf- , are able to stably express their activities after binding to polymeric macromolecules , proper attention should be paid to minimize the loss in activity arising from the inhibition of their binding to the receptor molecules due to the steric hindrance posed by the polymeric modifier . by assessing the relationship between the molecular weight of peg attached to tnf- , degree of modification of peg - modified tnf- , and their in vivo antitumor potency , we succeeded in markedly and selectively enhancing the antitumor potency of pegylated - tnf- over that of the native tnf-. our results suggest that comprehensive analysis of the relationship between the degree of modification by polymer , molecular size , and specific activity could lead to the development of bioconjugated proteins with increased therapeutic value and decreased side effects . these results suggest that pegylation is a pragmatic approach for developing successful therapies with drugs , such as enzymes and antitumor agents . we believe that the technique of bioconjugating drugs to a polymeric carrier will indeed be an important technique for expanding the clinical applications of therapeutic proteins . in this context it is noteworthy that in recent years several research laboratories around the world have created similar polymer - conjugated bioactive proteins . as a result , pegylated granulocyte - colony stimulating factor ( peg - g - csf ; peg filgrastim ) , pegylated interferon - alpha ( peg - ifn ; pegasys , peg - intron ) , pegylated asparaginase ( peg - asp ; oncasper ) , pegylated adenosine deaminase ( peg - ada ; adagen ) and polystyrene - co - maleic acid - conjugated neocarzinostatin ( smancs ) have been developed , all of which showed marked improvement in therapeutic efficacy in comparison to their corresponding native forms[24 - 28 ] . as described above , pegylation is limited by the frequent substantial loss of protein specific activity associated with polymer - conjugation . in addition , such bioconjugated proteins could also consist of positional isomers of polymeric modifiers at various sites , each one of which may have distinct activity and other characteristics . random modification could produce a heterogeneous mixture of conjugated proteins , consisting of different number of modifier molecules bound to various sites of the protein . as a result , functional properties of the bioconjugated proteins ( such as activation , in vivo behavior , stability ) are compromised , and thus , they could exhibit inconsistent therapeutic effects . therefore , to improve the polymer - conjugation technique in the post - genome era , the pharmaceutical technology would require a method that maintains the present efficiency of polymer modification while simultaneously making the site - specific modification possible . however , introduction of cysteine residues led to protein misfolding and aggregation , resulting in unexpected loss of activity . furthermore , even if it were possible to produce a cysteine mutant that retained activity , the poor efficiency of polymer conjugation to the thiol group made it impossible to obtain the desired product . to overcome this problem of pegylation , we have developed a strategy for site - specific pegylation of tnf- ( figure 2 ) . because a deletion mutant of tnf- lacking eight residues at the n terminus retains full bioactivity , we surmised that the n terminus of tnf- is not important for function and might therefore be a good target for pegylation . however , tnf- contains six internal lysine residues and the amino groups of all six of them are also targets for pegylation . site - directed mutagenesis analysis have shown that lys11 and lys90 are vital for the bioactivity of tnf-. nevertheless , if one could construct fully bioactive tnf- in which all the lysine residues were replaced with other amino acids , site - specific pegylation of the n - terminus could then be carried out . this pegylated mutant tnf- would be expected to have excellent molecular uniformity and retain high bioactivity . site - specific pegylation . to construct fully bioactive tnf- in which all the lysine residues were replaced with other amino acids , we employed the " molecular evolution strategy " developed in our laboratory to artificially create functional muteins using our phage display system . phage libraries displaying polypeptides , such as naive antibodies or random peptides , have been extensively used for identifying specific molecules with high affinity for a target ligand[34 - 36 ] . the advantages of a phage display system are easy preparation of a library consisting of structural variants of a polypeptide as diverse as over one hundred million and isolation of several targeted ligand - binding molecules from this library in few weeks . there are , however , few studies in which the phage display technique has been used to create therapeutically useful structural variants of a bioactive protein , such as a mutein with stronger bioactivity and longer plasma half - life . to create a lysine - deficient mutant tnf- , a phage library displaying mutant tnf- lacking any lysine residue was prepared , and it consisted of < 1 10independent structural variants . after two rounds of biopanning against tnf - receptor , a lysine - deficient mutant tnf- , mtnf - k90r , with an in vitro bioactivity that was 6-fold stronger than that of the wild - type tnf- ( wtnf- ) was obtained , despite reports that some of the lysine residues of tnf- were essential for its bioactivity . this mtnf - k90r had < 10-fold higher in vivo antitumor potency and 1.3-fold lower in vivo toxicity compared to those of wtnf-. therefore , the therapeutic window of mtnf - k90r was extended by < 13-fold more than that of the wtnf-. whereas multiple peg molecules attached randomly to various sites of wtnf- , only a single peg molecule attached selectively to the n - terminus of mtnf - k90r . the site - specific mono - pegylated mtnf - k90r ( sp - peg - mtnf - k90r ) exhibited 60% specific activity of that of the mtnf - k90r , whereas the randomly mono - pegylated wtnf- ( ran - peg - wtnf- ) exhibited only 6% specific activity of that of the wtnf- ( table . surprisingly , the sp - peg - mtnf - k90r showed higher in vitro bioactivity than the wtnf-. thus , these results suggest that by using this site - specific pegylation method , we have been able to circumvent the problems associated with the random pegylation method . the sp - peg - mtnf - k90r exhibited an antitumor potency that was 3-fold , > 30-fold , and > 30-fold higher than the antitumor potency exhibited by mtnf - k90r , wtnf- and ran - peg - wtnf- , respectively . in vivo toxicity of sp - peg - mtnf - k90r was < 1.5-fold , 2.0-fold , and 0.6-fold lower than the in vivo toxicity exhibited by mtnf - k90r , wtnf- , and ran - peg - wtnf- , respectively . thus , the therapeutic window of sp - peg - mtnf - k90r expanded by > 5-fold , 60-fold , and 18-fold than the therapeutic window of mtnf - k90r , wtnf- , and ran - peg - wtnf- , respectively . these results clearly demonstrate the advantages of creating muteins and developing methods for site - specific pegylation to promote pharmacoproteomic - based protein - drug discovery and development . in vitro bioactivity of mono - pegylated forms of tnf-s the specific activity of the mono - pegylated forms of tnf- was measured by a cytotoxic assay using lm cells in the presence of actinomycin d. ec50 is the concentration of various pegylated tnf-s capable of killing 50% of the cells . site - specific polymer - conjugation is possible even for a protein whose n - terminal region plays an essential role in activation by making use of the differences between the reaction patterns of -amino groups and -amino groups , and this could be achieved by first creating a functional lysine - deficient mutant and then introducing a new lysine residue in an area that is not connected with activation . in order to deliver a bioconjugated drug to the targeted tissue , the conjugate must demonstrate desirable pharmacokinetic characteristics , such as plasma clearance and tissue distribution . it is well known that the in vivo pharmacokinetics of polymer - conjugated drugs , such as bioactive proteins , could be markedly influenced by the properties ( such as electric charge and hydrophilic / hydrophobic balance ) of the polymeric carriers attached to the drug surface . any increase in the therapeutic effect of a drug bioconjugated with a polymeric modifier could be attributed to the pharmacokinetics of the bioconjugated drug . therefore , prior to selecting a polymeric modifier for bioconjugation , it is very important to take into consideration the influence of physicochemical characteristics on the pharmacokinetics of the polymer . for example , we have demonstrated that for increased retention in blood , polyvinylpyrrolidone ( pvp ) is overwhelmingly superior to peg as a polymer carrier . these series of advances have also led us to successfully synthesize polyvinylpyrrolidone - co - dimethyl maleic anhydride ( pvd ) , a renal - targeting polymer carrier with ph - sensitive controlled release capability . pvd attaches to the amino group of a protein at ph 8 and these pvd - protein conjugates gradually releases the proteins at ph 7 ( figure 3 ) . since the diseased tissues ( including tissues with inflammation , cancer , etc generally have lower ph levels than the healthy ones , this means that pvd as a drug / protein carrier would usually respond to ph levels and release the bound drug / protein , but only in the diseased tissues . amazingly , 80% of pvd administered intravenously to mice accumulated in the kidney in only a few hours and 40% of pvd remained in the kidney for 4-days ( figure 3 ) . it neither showed any sign of cytotoxicity nor did it cause any tissue damage in the kidney or elsewhere even when administered at a high dose . in addition , the pvd conjugate of anti - inflammatory protein superoxide dismutase ( sod ) was found to be highly stable in vivo and it accumulated at high levels in the kidney following an intravenous injection , bringing out the possibility that this conjugate may have significant therapeutic value in treating kidney inflammation . there is no cure for renal disease , which is a serious problem on the rise world - wide , and very few preventive strategies are available against this disease . bioactive proteins , such as sod and il-10 , were thought to be able to prevent the progression of renal disease , but their therapeutic potencies were too low because they were poorly distributed in the kidney . however , so far , there is no available report on delivery of drugs specifically to the kidney . development of a renal delivery system that selectively carries drugs to the kidney is a promising way of limiting the tissue distribution of drugs and controlling the drug - associated toxicity . therefore , pvd may be a new and useful renal - targeting drug carrier , and pvd - sod conjugate may find clinical applications in the effective treatment of various renal diseases . one way to circumvent the problems of protein therapy is to conjugate them with polyethylene glycol ( peg ) and other water - soluble polymeric modifiers ( figure 1 ) . the covalent conjugation of proteins with peg ( pegylation ) increases their molecular size and steric hindrance , both of which depend on the properties of the peg attached to the protein . in addition , because the polymers cover the protein surface , attack by a protease is generally blocked due to the steric hindrance , thus prolonging the half - life of the protein in vivo . the prolonged circulating lifetime in blood induces the enhanced permeability and retention ( epr ) effect , which is based on the leaky nature of the tumor blood vessels , resulting in increased delivery of the conjugates to the tumor tissue . because of all these advantages , it is possible to use the bioactive protein at a decreased dose however , this approach is limited by the frequent substantial loss of protein specific activity associated with polymer - conjugation . bioconjugation commonly targets the -amino group of lysine residues and/or the n - terminal -amino group of the protein because they are highly reactive and the bionconjugation reaction is mild enough to minimize disruption of the protein structure . because lysine residues often assume important roles in the formation of multi - dimensional structures and in bonding between ligands and receptors , introduction of polymers at these sites can potentially reduce the biological activity of the protein . indeed , pegylated interferon , which has raised hopes as a potential cure for hepatitis c , can only be produced as a heterogeneous mixture with 10 - 30% of the anticipated activity . tumor necrosis factor- ( tnf- ) , an antitumor cytokine , has numerous bioactivities including direct cytotoxicity against tumor cells , activation of immune antitumor response , and selective impairment of tumor - blood vessels . thus , tnf- has been considered as a novel antitumor agent[13 - 15 ] . however , as a systemic antitumor agent , very high doses of tnf- were required to obtain sufficient clinical responses , because tnf- is rapidly cleared from the circulation and also because it becomes widely distributed to various tissues following intravenous administration . as a result , tnf- with pleiotropic in vivo actions exhibited unexpected toxic side - effects , typified by pyrexia and hypertension . systemic application of tnf- was , therefore , abandoned despite intratumoral administration of tnf- showing significant antitumor effects in phase i studies . similar in vivo drawbacks are also found in the clinical applications of other bioactive proteins . keeping these problems in mind , we have devised ways to improve the polymer - conjugation system using the tnf- as a model protein[18 - 23 ] . in pegylation of tnf- , the specific activity of pegylated tnf- decreased with the degree of peg - modification ( i.e. , peg modification rate ) . additionally , when the peg modification rates are same , the bioactivity of pegylated tnf- decreased with an increase in the molecular size of the attached peg . thus , when bioactive proteins , such as tnf- , are able to stably express their activities after binding to polymeric macromolecules , proper attention should be paid to minimize the loss in activity arising from the inhibition of their binding to the receptor molecules due to the steric hindrance posed by the polymeric modifier . by assessing the relationship between the molecular weight of peg attached to tnf- , degree of modification of peg - modified tnf- , and their in vivo antitumor potency , we succeeded in markedly and selectively enhancing the antitumor potency of pegylated - tnf- over that of the native tnf-. our results suggest that comprehensive analysis of the relationship between the degree of modification by polymer , molecular size , and specific activity could lead to the development of bioconjugated proteins with increased therapeutic value and decreased side effects these results suggest that pegylation is a pragmatic approach for developing successful therapies with drugs , such as enzymes and antitumor agents . we believe that the technique of bioconjugating drugs to a polymeric carrier will indeed be an important technique for expanding the clinical applications of therapeutic proteins . in this context it is noteworthy that in recent years several research laboratories around the world have created similar polymer - conjugated bioactive proteins . as a result , pegylated granulocyte - colony stimulating factor ( peg - g - csf ; peg filgrastim ) , pegylated interferon - alpha ( peg - ifn ; pegasys , peg - intron ) , pegylated asparaginase ( peg - asp ; oncasper ) , pegylated adenosine deaminase ( peg - ada ; adagen ) and polystyrene - co - maleic acid - conjugated neocarzinostatin ( smancs ) have been developed , all of which showed marked improvement in therapeutic efficacy in comparison to their corresponding native forms[24 - 28 ] . as described above , pegylation is limited by the frequent substantial loss of protein specific activity associated with polymer - conjugation . in addition , such bioconjugated proteins could also consist of positional isomers of polymeric modifiers at various sites , each one of which may have distinct activity and other characteristics . random modification could produce a heterogeneous mixture of conjugated proteins , consisting of different number of modifier molecules bound to various sites of the protein . as a result , functional properties of the bioconjugated proteins ( such as activation , in vivo behavior , stability ) are compromised , and thus , they could exhibit inconsistent therapeutic effects . therefore , to improve the polymer - conjugation technique in the post - genome era , the pharmaceutical technology would require a method that maintains the present efficiency of polymer modification while simultaneously making the site - specific modification possible . for example , mutant proteins with free cysteine residues were created for modification . however , introduction of cysteine residues led to protein misfolding and aggregation , resulting in unexpected loss of activity . furthermore , even if it were possible to produce a cysteine mutant that retained activity , the poor efficiency of polymer conjugation to the thiol group made it impossible to obtain the desired product . to overcome this problem of pegylation , we have developed a strategy for site - specific pegylation of tnf- ( figure 2 ) . because a deletion mutant of tnf- lacking eight residues at the n terminus retains full bioactivity , we surmised that the n terminus of tnf- is not important for function and might therefore be a good target for pegylation . however , tnf- contains six internal lysine residues and the amino groups of all six of them are also targets for pegylation . site - directed mutagenesis analysis have shown that lys11 and lys90 are vital for the bioactivity of tnf-. nevertheless , if one could construct fully bioactive tnf- in which all the lysine residues were replaced with other amino acids , site - specific pegylation of the n - terminus could then be carried out . this pegylated mutant tnf- would be expected to have excellent molecular uniformity and retain high bioactivity . site - specific pegylation . to construct fully bioactive tnf- in which all the lysine residues were replaced with other amino acids , we employed the " molecular evolution strategy " developed in our laboratory to artificially create functional muteins using our phage display system . phage libraries displaying polypeptides , such as naive antibodies or random peptides , have been extensively used for identifying specific molecules with high affinity for a target ligand[34 - 36 ] . the advantages of a phage display system are easy preparation of a library consisting of structural variants of a polypeptide as diverse as over one hundred million and isolation of several targeted ligand - binding molecules from this library in few weeks . there are , however , few studies in which the phage display technique has been used to create therapeutically useful structural variants of a bioactive protein , such as a mutein with stronger bioactivity and longer plasma half - life . to create a lysine - deficient mutant tnf- , a phage library displaying mutant tnf- lacking any lysine residue was prepared , and it consisted of < 1 10independent structural variants . after two rounds of biopanning against tnf - receptor , a lysine - deficient mutant tnf- , mtnf - k90r , with an in vitro bioactivity that was 6-fold stronger than that of the wild - type tnf- ( wtnf- ) was obtained , despite reports that some of the lysine residues of tnf- were essential for its bioactivity . this mtnf - k90r had < 10-fold higher in vivo antitumor potency and 1.3-fold lower in vivo toxicity compared to those of wtnf-. therefore , the therapeutic window of mtnf - k90r was extended by < 13-fold more than that of the wtnf-. whereas multiple peg molecules attached randomly to various sites of wtnf- , only a single peg molecule attached selectively to the n - terminus of mtnf - k90r . the site - specific mono - pegylated mtnf - k90r ( sp - peg - mtnf - k90r ) exhibited 60% specific activity of that of the mtnf - k90r , whereas the randomly mono - pegylated wtnf- ( ran - peg - wtnf- ) exhibited only 6% specific activity of that of the wtnf- ( table . 1 ) . surprisingly , the sp - peg - mtnf - k90r showed higher in vitro bioactivity than the wtnf-. thus , these results suggest that by using this site - specific pegylation method , we have been able to circumvent the problems associated with the random pegylation method . the sp - peg - mtnf - k90r exhibited an antitumor potency that was 3-fold , > 30-fold , and > 30-fold higher than the antitumor potency exhibited by mtnf - k90r , wtnf- and ran - peg - wtnf- , respectively . in vivo toxicity of sp - peg - mtnf - k90r was < 1.5-fold , 2.0-fold , and 0.6-fold lower than the in vivo toxicity exhibited by mtnf - k90r , wtnf- , and ran - peg - wtnf- , respectively . thus , the therapeutic window of sp - peg - mtnf - k90r expanded by > 5-fold , 60-fold , and 18-fold than the therapeutic window of mtnf - k90r , wtnf- , and ran - peg - wtnf- , respectively . these results clearly demonstrate the advantages of creating muteins and developing methods for site - specific pegylation to promote pharmacoproteomic - based protein - drug discovery and development . in vitro bioactivity of mono - pegylated forms of tnf-s the specific activity of the mono - pegylated forms of tnf- was measured by a cytotoxic assay using lm cells in the presence of actinomycin d. ec50 is the concentration of various pegylated tnf-s capable of killing 50% of the cells . site - specific polymer - conjugation is possible even for a protein whose n - terminal region plays an essential role in activation by making use of the differences between the reaction patterns of -amino groups and -amino groups , and this could be achieved by first creating a functional lysine - deficient mutant and then introducing a new lysine residue in an area that is not connected with activation . in order to deliver a bioconjugated drug to the targeted tissue , the conjugate must demonstrate desirable pharmacokinetic characteristics , such as plasma clearance and tissue distribution . it is well known that the in vivo pharmacokinetics of polymer - conjugated drugs , such as bioactive proteins , could be markedly influenced by the properties ( such as electric charge and hydrophilic / hydrophobic balance ) of the polymeric carriers attached to the drug surface . any increase in the therapeutic effect of a drug bioconjugated with a polymeric modifier could be attributed to the pharmacokinetics of the bioconjugated drug . therefore , prior to selecting a polymeric modifier for bioconjugation , it is very important to take into consideration the influence of physicochemical characteristics on the pharmacokinetics of the polymer . for example , we have demonstrated that for increased retention in blood , polyvinylpyrrolidone ( pvp ) is overwhelmingly superior to peg as a polymer carrier . these series of advances have also led us to successfully synthesize polyvinylpyrrolidone - co - dimethyl maleic anhydride ( pvd ) , a renal - targeting polymer carrier with ph - sensitive controlled release capability . pvd attaches to the amino group of a protein at ph 8 and these pvd - protein conjugates gradually releases the proteins at ph 7 ( figure 3 ) . since the diseased tissues ( including tissues with inflammation , cancer , etc . ) generally have lower ph levels than the healthy ones , this means that pvd as a drug / protein carrier would usually respond to ph levels and release the bound drug / protein , but only in the diseased tissues . amazingly , 80% of pvd administered intravenously to mice accumulated in the kidney in only a few hours and 40% of pvd remained in the kidney for 4-days ( figure 3 ) . it neither showed any sign of cytotoxicity nor did it cause any tissue damage in the kidney or elsewhere even when administered at a high dose . in addition , the pvd conjugate of anti - inflammatory protein superoxide dismutase ( sod ) was found to be highly stable in vivo and it accumulated at high levels in the kidney following an intravenous injection , bringing out the possibility that this conjugate may have significant therapeutic value in treating kidney inflammation . there is no cure for renal disease , which is a serious problem on the rise world - wide , and very few preventive strategies are available against this disease . bioactive proteins , such as sod and il-10 , were thought to be able to prevent the progression of renal disease , but their therapeutic potencies were too low because they were poorly distributed in the kidney . however , so far , there is no available report on delivery of drugs specifically to the kidney . development of a renal delivery system that selectively carries drugs to the kidney is a promising way of limiting the tissue distribution of drugs and controlling the drug - associated toxicity . therefore , pvd may be a new and useful renal - targeting drug carrier , and pvd - sod conjugate may find clinical applications in the effective treatment of various renal diseases . in this review , we have demonstrated the usefulness of dds - based technologies to selectively enhance the desirable therapeutic activities of bioactive proteins without increasing their side effects . thus , we suggest that , to further promote the clinical applications of bioactive proteins , it is necessary for the protein - drug innovation system to combine 1 ) a method for creating mutant proteins ( muteins ) with desired properties and 2 ) a bioconjugation method that generates conjugated products with optimal properties , such as targeting capability . we believe that this protein - drug innovation system will be a valuable tool for the development of novel protein - based therapies . this study was supported in part by grants - in - aid for scientific research from the ministry of education , culture , sports , science and technology of japan , and from the japan society for the promotion of science ( jsps ) . this study was also supported in part by health labour sciences research grants from the ministry of health , labor and welfare of japan ; by health sciences research grants for research on publicly essential drugs and medical devices from the japan health sciences foundation ; by a global environment research fund from minister of the environment ; and by a the knowledge cluster initiative ; and by the nagai foundation tokyo ; and by the takeda science foundation .

because of the shifted focus in life science research from genome analyses to genetic and protein function analyses , we now know functions of numerous proteins . these analyses , including those of newly identified proteins , are expected to contribute to the identification of proteins of therapeutic value in various diseases . consequently , pharmacoproteomic - based drug discovery and development of protein therapies attracted a great deal of attention in recent years . clinical applications of most of these proteins are , however , limited because of their unexpectedly low therapeutic effects , resulting from the proteolytic degradation in vivo followed by rapid removal from the circulatory system . therefore , frequent administration of excessively high dose of a protein is required to observe its therapeutic effect in vivo . this often results in impaired homeostasis in vivo and leads to severe adverse effects . to overcome these problems , we have devised a method for chemical modification of proteins with polyethylene glycol ( pegylation ) and other water - soluble polymers . in addition , we have established a method for creating functional mutant proteins ( muteins ) with desired properties , and developed a site - specific polymer - conjugation method to further improve their therapeutic potency . in this review , we are introducing our original protein - drug innovation system mentioned above .

Introduction Bioconjugation as a polymeric DDS A new method for site-specific PEGylation Functionalized polymer carriers as DDS Conclusions Competing interests Authors' contributions Acknowledgements

a number of these proteins are thought to be related to pathological disorders and other biological phenomena , and thus , are potentially useful as therapeutic agents and as targets for pharmaceutical development . therefore , focus in life science research has currently shifted to newer fields , such as proteomics and structural genomics , in which the function and structure of proteins are analyzed en masse . such analyses of large number of proteins , including the newly identified proteins , are expected to contribute to the identification of proteins of therapeutic importance in various diseases . in recent years indeed , attempts are currently under progress to develop a wide range of therapeutic proteins for treating various diseases including cancer , hepatitis and autoimmune conditions[1 - 4 ] . unfortunately , clinical applications of many of these proteins are limited because of their unexpectedly poor therapeutic effects . often these proteins are degraded by various proteases in vivo and rapidly removed from the circulatory system . consequently , frequent administration of an excessively high dose of a protein is required to obtain its desired therapeutic effect in vivo , leading to a disturbance in the homeostasis and unexpected side effects . additionally , bioactive proteins , such as cytokines , generally show pleiotropic actions through a number of receptors in vivo , making it difficult to elicit the desired effect without simultaneously triggering undesirable secondary effects . from this standpoint , creation of novel technologies that overcome the problems peculiar to bioactive proteins is essential for the advancement of pharmaco - proteomic - based drug development . these technologies are suitable as drug delivery systems ( ddss ) , aiming to maximize the therapeutic potency of proteins . our laboratory aims to develop novel dds techniques for overcoming the problems of protein therapy : ( i ) establishment of a novel polymer - conjugation system to dramatically improve in vivo stability and selectively of bioactive proteins ( polymeric dds ) and ( ii ) development of a powerful system to rapidly create functional mutant proteins ( muteins ) with enhanced receptor affinity and receptor specificity using a phage display technique ( biological dds ) . we are currently in the process of combining both approaches to create a protein - drug innovation system to further promote pharmaco - proteomic - based drug development . in this review , we will describe dds - based technology to create functional mutants for advanced medical applications using the tumor necrosis factor - alpha ( tnf ) as an example and the usefulness of site - specific polymer - conjugation of proteins . one way to circumvent the problems of protein therapy is to conjugate them with polyethylene glycol ( peg ) and other water - soluble polymeric modifiers ( figure 1 ) . the covalent conjugation of proteins with peg ( pegylation ) increases their molecular size and steric hindrance , both of which depend on the properties of the peg attached to the protein . in addition , because the polymers cover the protein surface , attack by a protease is generally blocked due to the steric hindrance , thus prolonging the half - life of the protein in vivo . the prolonged circulating lifetime in blood induces the enhanced permeability and retention ( epr ) effect , which is based on the leaky nature of the tumor blood vessels , resulting in increased delivery of the conjugates to the tumor tissue . however , this approach is limited by the frequent substantial loss of protein specific activity associated with polymer - conjugation . however , as a systemic antitumor agent , very high doses of tnf- were required to obtain sufficient clinical responses , because tnf- is rapidly cleared from the circulation and also because it becomes widely distributed to various tissues following intravenous administration . as a result , tnf- with pleiotropic in vivo actions exhibited unexpected toxic side - effects , typified by pyrexia and hypertension . systemic application of tnf- was , therefore , abandoned despite intratumoral administration of tnf- showing significant antitumor effects in phase i studies . similar in vivo drawbacks are also found in the clinical applications of other bioactive proteins . keeping these problems in mind , we have devised ways to improve the polymer - conjugation system using the tnf- as a model protein[18 - 23 ] . thus , when bioactive proteins , such as tnf- , are able to stably express their activities after binding to polymeric macromolecules , proper attention should be paid to minimize the loss in activity arising from the inhibition of their binding to the receptor molecules due to the steric hindrance posed by the polymeric modifier . by assessing the relationship between the molecular weight of peg attached to tnf- , degree of modification of peg - modified tnf- , and their in vivo antitumor potency , we succeeded in markedly and selectively enhancing the antitumor potency of pegylated - tnf- over that of the native tnf-. our results suggest that comprehensive analysis of the relationship between the degree of modification by polymer , molecular size , and specific activity could lead to the development of bioconjugated proteins with increased therapeutic value and decreased side effects . we believe that the technique of bioconjugating drugs to a polymeric carrier will indeed be an important technique for expanding the clinical applications of therapeutic proteins . in this context it is noteworthy that in recent years several research laboratories around the world have created similar polymer - conjugated bioactive proteins . as described above , pegylation is limited by the frequent substantial loss of protein specific activity associated with polymer - conjugation . in addition , such bioconjugated proteins could also consist of positional isomers of polymeric modifiers at various sites , each one of which may have distinct activity and other characteristics . random modification could produce a heterogeneous mixture of conjugated proteins , consisting of different number of modifier molecules bound to various sites of the protein . as a result , functional properties of the bioconjugated proteins ( such as activation , in vivo behavior , stability ) are compromised , and thus , they could exhibit inconsistent therapeutic effects . therefore , to improve the polymer - conjugation technique in the post - genome era , the pharmaceutical technology would require a method that maintains the present efficiency of polymer modification while simultaneously making the site - specific modification possible . however , introduction of cysteine residues led to protein misfolding and aggregation , resulting in unexpected loss of activity . to overcome this problem of pegylation , we have developed a strategy for site - specific pegylation of tnf- ( figure 2 ) . nevertheless , if one could construct fully bioactive tnf- in which all the lysine residues were replaced with other amino acids , site - specific pegylation of the n - terminus could then be carried out . there are , however , few studies in which the phage display technique has been used to create therapeutically useful structural variants of a bioactive protein , such as a mutein with stronger bioactivity and longer plasma half - life . this mtnf - k90r had < 10-fold higher in vivo antitumor potency and 1.3-fold lower in vivo toxicity compared to those of wtnf-. therefore , the therapeutic window of mtnf - k90r was extended by < 13-fold more than that of the wtnf-. the site - specific mono - pegylated mtnf - k90r ( sp - peg - mtnf - k90r ) exhibited 60% specific activity of that of the mtnf - k90r , whereas the randomly mono - pegylated wtnf- ( ran - peg - wtnf- ) exhibited only 6% specific activity of that of the wtnf- ( table . thus , these results suggest that by using this site - specific pegylation method , we have been able to circumvent the problems associated with the random pegylation method . in vivo toxicity of sp - peg - mtnf - k90r was < 1.5-fold , 2.0-fold , and 0.6-fold lower than the in vivo toxicity exhibited by mtnf - k90r , wtnf- , and ran - peg - wtnf- , respectively . these results clearly demonstrate the advantages of creating muteins and developing methods for site - specific pegylation to promote pharmacoproteomic - based protein - drug discovery and development . site - specific polymer - conjugation is possible even for a protein whose n - terminal region plays an essential role in activation by making use of the differences between the reaction patterns of -amino groups and -amino groups , and this could be achieved by first creating a functional lysine - deficient mutant and then introducing a new lysine residue in an area that is not connected with activation . it is well known that the in vivo pharmacokinetics of polymer - conjugated drugs , such as bioactive proteins , could be markedly influenced by the properties ( such as electric charge and hydrophilic / hydrophobic balance ) of the polymeric carriers attached to the drug surface . any increase in the therapeutic effect of a drug bioconjugated with a polymeric modifier could be attributed to the pharmacokinetics of the bioconjugated drug . therefore , prior to selecting a polymeric modifier for bioconjugation , it is very important to take into consideration the influence of physicochemical characteristics on the pharmacokinetics of the polymer . for example , we have demonstrated that for increased retention in blood , polyvinylpyrrolidone ( pvp ) is overwhelmingly superior to peg as a polymer carrier . pvd attaches to the amino group of a protein at ph 8 and these pvd - protein conjugates gradually releases the proteins at ph 7 ( figure 3 ) . in addition , the pvd conjugate of anti - inflammatory protein superoxide dismutase ( sod ) was found to be highly stable in vivo and it accumulated at high levels in the kidney following an intravenous injection , bringing out the possibility that this conjugate may have significant therapeutic value in treating kidney inflammation . bioactive proteins , such as sod and il-10 , were thought to be able to prevent the progression of renal disease , but their therapeutic potencies were too low because they were poorly distributed in the kidney . however , so far , there is no available report on delivery of drugs specifically to the kidney . development of a renal delivery system that selectively carries drugs to the kidney is a promising way of limiting the tissue distribution of drugs and controlling the drug - associated toxicity . therefore , pvd may be a new and useful renal - targeting drug carrier , and pvd - sod conjugate may find clinical applications in the effective treatment of various renal diseases . one way to circumvent the problems of protein therapy is to conjugate them with polyethylene glycol ( peg ) and other water - soluble polymeric modifiers ( figure 1 ) . the covalent conjugation of proteins with peg ( pegylation ) increases their molecular size and steric hindrance , both of which depend on the properties of the peg attached to the protein . in addition , because the polymers cover the protein surface , attack by a protease is generally blocked due to the steric hindrance , thus prolonging the half - life of the protein in vivo . the prolonged circulating lifetime in blood induces the enhanced permeability and retention ( epr ) effect , which is based on the leaky nature of the tumor blood vessels , resulting in increased delivery of the conjugates to the tumor tissue . because of all these advantages , it is possible to use the bioactive protein at a decreased dose however , this approach is limited by the frequent substantial loss of protein specific activity associated with polymer - conjugation . however , as a systemic antitumor agent , very high doses of tnf- were required to obtain sufficient clinical responses , because tnf- is rapidly cleared from the circulation and also because it becomes widely distributed to various tissues following intravenous administration . as a result , tnf- with pleiotropic in vivo actions exhibited unexpected toxic side - effects , typified by pyrexia and hypertension . systemic application of tnf- was , therefore , abandoned despite intratumoral administration of tnf- showing significant antitumor effects in phase i studies . similar in vivo drawbacks are also found in the clinical applications of other bioactive proteins . keeping these problems in mind , we have devised ways to improve the polymer - conjugation system using the tnf- as a model protein[18 - 23 ] . thus , when bioactive proteins , such as tnf- , are able to stably express their activities after binding to polymeric macromolecules , proper attention should be paid to minimize the loss in activity arising from the inhibition of their binding to the receptor molecules due to the steric hindrance posed by the polymeric modifier . by assessing the relationship between the molecular weight of peg attached to tnf- , degree of modification of peg - modified tnf- , and their in vivo antitumor potency , we succeeded in markedly and selectively enhancing the antitumor potency of pegylated - tnf- over that of the native tnf-. our results suggest that comprehensive analysis of the relationship between the degree of modification by polymer , molecular size , and specific activity could lead to the development of bioconjugated proteins with increased therapeutic value and decreased side effects these results suggest that pegylation is a pragmatic approach for developing successful therapies with drugs , such as enzymes and antitumor agents . we believe that the technique of bioconjugating drugs to a polymeric carrier will indeed be an important technique for expanding the clinical applications of therapeutic proteins . in this context it is noteworthy that in recent years several research laboratories around the world have created similar polymer - conjugated bioactive proteins . as described above , pegylation is limited by the frequent substantial loss of protein specific activity associated with polymer - conjugation . in addition , such bioconjugated proteins could also consist of positional isomers of polymeric modifiers at various sites , each one of which may have distinct activity and other characteristics . random modification could produce a heterogeneous mixture of conjugated proteins , consisting of different number of modifier molecules bound to various sites of the protein . as a result , functional properties of the bioconjugated proteins ( such as activation , in vivo behavior , stability ) are compromised , and thus , they could exhibit inconsistent therapeutic effects . therefore , to improve the polymer - conjugation technique in the post - genome era , the pharmaceutical technology would require a method that maintains the present efficiency of polymer modification while simultaneously making the site - specific modification possible . to overcome this problem of pegylation , we have developed a strategy for site - specific pegylation of tnf- ( figure 2 ) . nevertheless , if one could construct fully bioactive tnf- in which all the lysine residues were replaced with other amino acids , site - specific pegylation of the n - terminus could then be carried out . site - specific pegylation . there are , however , few studies in which the phage display technique has been used to create therapeutically useful structural variants of a bioactive protein , such as a mutein with stronger bioactivity and longer plasma half - life . the site - specific mono - pegylated mtnf - k90r ( sp - peg - mtnf - k90r ) exhibited 60% specific activity of that of the mtnf - k90r , whereas the randomly mono - pegylated wtnf- ( ran - peg - wtnf- ) exhibited only 6% specific activity of that of the wtnf- ( table . thus , these results suggest that by using this site - specific pegylation method , we have been able to circumvent the problems associated with the random pegylation method . in vivo toxicity of sp - peg - mtnf - k90r was < 1.5-fold , 2.0-fold , and 0.6-fold lower than the in vivo toxicity exhibited by mtnf - k90r , wtnf- , and ran - peg - wtnf- , respectively . these results clearly demonstrate the advantages of creating muteins and developing methods for site - specific pegylation to promote pharmacoproteomic - based protein - drug discovery and development . site - specific polymer - conjugation is possible even for a protein whose n - terminal region plays an essential role in activation by making use of the differences between the reaction patterns of -amino groups and -amino groups , and this could be achieved by first creating a functional lysine - deficient mutant and then introducing a new lysine residue in an area that is not connected with activation . it is well known that the in vivo pharmacokinetics of polymer - conjugated drugs , such as bioactive proteins , could be markedly influenced by the properties ( such as electric charge and hydrophilic / hydrophobic balance ) of the polymeric carriers attached to the drug surface . any increase in the therapeutic effect of a drug bioconjugated with a polymeric modifier could be attributed to the pharmacokinetics of the bioconjugated drug . therefore , prior to selecting a polymeric modifier for bioconjugation , it is very important to take into consideration the influence of physicochemical characteristics on the pharmacokinetics of the polymer . for example , we have demonstrated that for increased retention in blood , polyvinylpyrrolidone ( pvp ) is overwhelmingly superior to peg as a polymer carrier . pvd attaches to the amino group of a protein at ph 8 and these pvd - protein conjugates gradually releases the proteins at ph 7 ( figure 3 ) . in addition , the pvd conjugate of anti - inflammatory protein superoxide dismutase ( sod ) was found to be highly stable in vivo and it accumulated at high levels in the kidney following an intravenous injection , bringing out the possibility that this conjugate may have significant therapeutic value in treating kidney inflammation . bioactive proteins , such as sod and il-10 , were thought to be able to prevent the progression of renal disease , but their therapeutic potencies were too low because they were poorly distributed in the kidney . however , so far , there is no available report on delivery of drugs specifically to the kidney . development of a renal delivery system that selectively carries drugs to the kidney is a promising way of limiting the tissue distribution of drugs and controlling the drug - associated toxicity . therefore , pvd may be a new and useful renal - targeting drug carrier , and pvd - sod conjugate may find clinical applications in the effective treatment of various renal diseases . in this review , we have demonstrated the usefulness of dds - based technologies to selectively enhance the desirable therapeutic activities of bioactive proteins without increasing their side effects . thus , we suggest that , to further promote the clinical applications of bioactive proteins , it is necessary for the protein - drug innovation system to combine 1 ) a method for creating mutant proteins ( muteins ) with desired properties and 2 ) a bioconjugation method that generates conjugated products with optimal properties , such as targeting capability . we believe that this protein - drug innovation system will be a valuable tool for the development of novel protein - based therapies . this study was supported in part by grants - in - aid for scientific research from the ministry of education , culture , sports , science and technology of japan , and from the japan society for the promotion of science ( jsps ) . this study was also supported in part by health labour sciences research grants from the ministry of health , labor and welfare of japan ; by health sciences research grants for research on publicly essential drugs and medical devices from the japan health sciences foundation ; by a global environment research fund from minister of the environment ; and by a the knowledge cluster initiative ; and by the nagai foundation tokyo ; and by the takeda science foundation .

the subplate forms a transient layer in the developing cerebral cortex and consists of migratory and postmigratory neurons , dendrites , axons , growth cones , synapses and glial cells . the subplate is located between the intermediate zone and the cortical plate , which during further development differentiates into the neocortical layers ii to vi ( figure 1a ) . kostovic and molliver were the first who identified the subplate as a distinct layer in the embryonic human cerebral cortex ( kostovic and molliver , 1974 ) . a subplate can be identified in all mammals although its relative thickness , developmental profile and persistence in adulthood vary among species . anatomical data indicate an evolutionary difference in the ontogenetic fate of the subplate . in the rat and other rodents many subplate cells survive into adulthood forming layer vib or vii ( aboitiz and montiel , 2007 ) . prominent species differences also exist in the relative thickness of the subplate , which increased during evolution . in humans the subplate develops to approximately six times the thickness of the cortical plate around 29 weeks of gestation ( mrzljak et al . , 1990 ) , whereas in rodents it remains a relatively thin layer during development ( uylings et al . , 1990 ) . the majority of the subplate cells are born early before the first cortical plate neurons ( luskin and shatz , 1985 ; valverde et al . , 1989 ) . in rodents subplate neurons may be also generated later in development ( hevner et al . , 2004 ) . a substantial proportion of subplate cells are not born in the ventricular neuroepithelium , but instead originate in the medial ganglionic eminence and follow a tangential migratory route to their positions in the developing cortex ( lavdas et al . , 1999 ) . subplate cells represent a rather heterogeneous neuronal population according to their morphology , neurotransmitter identity and connectivity ( for review allendoerfer and shatz , 1994 ) . ( a ) prenatal development of the human cerebral cortex as suggested by bystron et al . ( 2008 ) . drawing from pasko rakic . photograph shows coronal brain section stained with cresyl violet of a gestational week 18 human neocortex . approximate embryonic day ( e ) and gestational week ( gw ) are given for each developmental stage . cp , cortical plate ; iz , intermediate zone ; mz , marginal zone ; pp , preplate ; sp , subplate ; svz , subventricular zone ; ( sg ) , subpial granular layer ( part of mz ) ; vz , ventricular zone . ( b ) morphological properties of biocytin - stained subplate neurons in newborn rat cerebral cortex . ( b1 ) postnatal day ( p ) three horizontal bitufted cell with large fusiform soma and primary dendrites oriented parallel to the pial surface . ( b3 ) p3 multipolar cell with extensive dendritic arborization within sp and layers v / vi . ( b4 ) p2 inverted pyramidal neuron with triangular soma and apical dendrite oriented towards white matter . scale bar in ( b4 ) corresponds to ( b1b4 ) and pial surface is located toward the top in all photomicrographs of ( b ) . reproduced and modified with permission from ( bystron et al . , 2008 ) ( a ) , ( kostovic et al . , 2002 ) [ photograph in ( a ) ] and from ( hanganu et al . , 2002 ) ( b ) . subplate cells play important roles in the structural and functional organization of the cerebral cortex and in early necortical plasticity . axons arising from subplate neurons pioneer the corticofugal pathway and have been proposed to form a cellular scaffold for guiding thalamocortical axons ( mcconnell et al . 1990 ) ( for review allendoerfer and shatz , 1994 ; molnr , 1998 ) . thalamic axons ( lund and mustari , 1977 ; rakic , 1977 , 1983 ) and early deletion of subplate neurons in kitten visual cortex prevents the segregation of thalamocortical axons within layer iv and the formation of ocular dominance columns ( ghosh and shatz , 1992 ; kanold et al . , 2003 ) . furthermore , subplate cells regulate the maturation of gabaergic synaptic transmission and establish the balance between excitation and inhibition in the developing neocortical network ( kanold and shatz , 2006 ) . subplate neurons reveal a large variety of morphologies ( figure 1b ) ( hanganu et al . , 2002 ) . inverted pyramidal - like and horizontal cells as well as polymorphic neurons with different shapes and spiny or smooth dendrites have been classified as subplate neurons ( kostovic and rakic , 1980 ; wahle et al . , 1987 ; valverde et al . , 1989 ; kostovic and rakic , 1990 ) due to their earlier generation and more mature developmental stage , subplate neurons show a relatively extensive dendritic tree when compared to the more immature pyramidal neurons of the cortical plate ( mrzljak et al . , 1992 ) . descending dendrites from subplate neurons may invade the underlying intermediate zone and ascending dendrites may extend into the cortical plate ( del ro et al . , 2000 ) . subplate neurons reveal markers for gaba or glutamate and may co - express various peptides ( chun et al . , 1987 ; the morphological and neurochemical heterogeneity also explains , why various attempts failed to identify a specific marker for subplate neurons ( wahle et al . , 1994 ) . ( 2009 ) succeeded in identifying a number of novel markers for murine subplate cells , which may soon allow the definition of different subpopulations of subplate neurons . subplate cells participate in local and long - distance axonal connections indicating that these neurons may function as local circuit as well as projection neurons . they show a dense axonal arborization within the subplate , but also project to the marginal zone / layer i ( clancy and cauller , 1999 ) and to the cortical plate , where they form axonal collaterals within layer iv ( friauf et al . , the majority of the subplate neurons projecting into the cortical plate reside in the upper half of the subplate and provide a glutamatergic synaptic input to the developing cortical plate , including the layer iv neurons ( friauf et al . , 1990 ; long - distance axons from subplate neurons invade the thalamus during early stages of corticogenesis and form an axonal scaffold for the establishment of cortical efferent and afferent projections ( mcconnell et al . , 1989 , 1994 ; kim et al . , 1991 ; de carlos and o'leary , 1992 ) . beside these local and long - distance projections arising from glutamatergic subplate neurons , gabaergic subplate cells also project to both neighbouring and more distant neocortical regions and form a corticocortical synaptic network ( tomioka et al . , 2005 ; higo et al . , 2007 ) . since gaba may act as an excitatory neurotransmitter during early cortical development ( for review ben - ari et al . , 2007 ) , the postsynaptic action of gabaergic subplate neurons may be also depolarizing ( figure 6 in hanganu et al . , 2002 ) . ultrastructural studies of subplate cells in various species have demonstrated symmetrical as well as asymmetrical synapses with relatively mature properties ( kostovic and rakic , 1980 , 1990 ; chun and shatz , 1988 ; herrmann et al . , 1994 ) , indicating that subplate neurons receive gabaergic as well as glutamatergic synaptic inputs . as suggested by kostovic and rakic ( 1980 ) , glutamatergic inputs onto subplate neurons may arise from the thalamus and other neocortical areas , whereas gabaergic synaptic inputs may originate from gabaergic interneurons in the subplate . thalamocortical synaptic contacts with spines and shafts of subplate neuron dendrites have been demonstrated in the neonatal ferret ( herrmann et al . , 1994 ) and a dense network of corticocortical fibers have been reported in the subplate of the embryonic mouse ( crandall and caviness , 1984 ) . n - methyl - d - aspartate ( nmda ) , -amino-3-hydroxy-5-methylisoxazole-4-propionic acid ( ampa ) and kainate receptors and the essential subunits for their receptor function have been demonstrated in the subplate of various species , suggesting the presence of functional glutamatergic synapses in subplate neurons ( herrmann , 1996 ; aoki , 1997 ; catalano et al . , gabaa receptors ( huntley et al . , 1990 ) and gabaa receptor subunits ( meinecke and rakic , 1992 ) in the subplate indicate that functional gabaergic synaptic inputs should also be present in subplate neurons . these morphological , ultrastructural and immunohistochemical data are complemented by functional studies on the properties of the subplate and single subplate cells in different mammalian species . in contrast to the heterogeneity in morphological and chemical appearance , electrophysiological recordings from single subplate neurons in rodents demonstrate rather homogeneous functional properties . whole - cell patch - clamp recordings from subplate cells in newborn rodent neocortical slices revealed resting membrane potentials and input resistances in the range of 55 mv and 11.2 g , respectively ( luhmann et al . , hirsch and luhmann , 2008 ) . in response to sustained depolarization by intracellular current injection , subplate neurons are capable of firing overshooting and repetitive action potentials at frequencies exceeding 40 hz ( figures 2c and 3a ) . similar results were obtained from subplate neurons in acute neocortical slices harvested postmortem from human fetal brain at gestational week 1622 ( moore et al . , 2009 ) . ( a ) intracellular biocytin staining of one subplate neuron ( yellow arrow ) in the p3 mouse neocortex results in columnar labeling of several dye - coupled cells in the subplate and in the cortical plate.(b ) photograph of two simultaneously recorded p1 subplate neurons , which were both stained with biocytin . ( c ) paired current - clamp recordings of the cells shown in ( b ) . black traces were obtained from the cell marked by black arrow in ( b ) and red traces are from the cell marked by red arrow . ( c1 ) injection of a hyperpolarizing or suprathreshold depolarizing current pulse into the cell marked by black arrow causes voltage deflections in the gap junction coupled neuron . ( c2 ) vice versa , injection of a hyperpolarizing or suprathreshold depolarizing current pulse into the cell marked by red arrow causes voltage deflections in the other neuron . cortical surface in ( a ) and ( b ) is up , action potentials in ( c ) are truncated . the hyperpolarizing responses in the lower traces are averages of four recordings . reproduced and modified with permission from ( dupont et al . , 2006 ) . ( a ) typical action potential firing pattern of a subplate neuron in the cerebral cortex of a p1 rat . ( b ) excitatory postsynaptic currents ( epscs ) recorded in a p3 subplate cell at a holding potential of 70 mv and elicited by selective electrical stimulation of the thalamocortical input ( see c ) . synaptic responses were obtained under control conditions in normal extracellular bathing solution and after application of 10 m cnqx to block ampa / kainate receptors . the inset shows the unimodal latency distribution of 50 thalamocortical epscs recorded from one spn . ( c ) photomicrograph of biocytin - stained thalamocortical projections in a 400-m thick coronal slice from a p1 rat . asterisk shows extracellular injection site of biocytin crystal and white circles mark position of bipolar electrode to selectively activate the thalamocortical input . ( d ) schematic diagram illustrating the distribution of postsynaptic receptors on a subplate neuron for monosynaptic inputs arising from the thalamus ( thal . ) , the cortical plate ( cp ) including layers v / vi and within the subplate ( sp ) . note that synaptic inputs from other subplate neurons can lead to activation of depolarizing gabaa receptors and nmda receptors activated at negative membrane potentials . reproduced and modified with permission from ( luhmann et al . , 2000 ) ( a ) ( hanganu et al . , 2002 ) ( b d ) . in comparison to other neurons in the immature cerebral cortex , subplate cells also reveal the most mature properties in action potential characteristics and in the biophysical properties of voltage - dependent sodium and calcium currents . these observations have been made in developing rodent ( luhmann et al . , 2000 ) as well as in human cerebral cortex ( moore et al . , 2009 ) , indicating that these relatively mature functional properties enable subplate cells to transmit afferent neuronal activity faithfully to the developing cortical plate . intracellular labeling of single subplate cells with fluorescent dyes or biocytin revealed an extensive neuronal network of dye - coupled neurons in the subplate and cortical plate ( figure 2a ) . in newborn rats , on average about nine neurons are dye - coupled to a single subplate cell and these gap junction coupled networks are often organized in a columnar manner ( dupont et al . , 2006 ) . whole - cell patch - clamp recordings from pairs of dye - coupled subplate neurons ( figure 2b ) allowed a more detailed electrophysiological characterization of the electrical synapses connecting subplate neurons ( figure 2c ) . the average coupling conductance amounted to about 1.2 ns ( dupont et al . , 2006 ) . these data indicate that subplate cells are strongly coupled via electrical synapses and form a functional columnar syncytium with neurons located in the cortical plate . it is tempting to speculate that this early columnar organization results from the radial , column - like neuronal migration of newly generated neurons into the developing neocortex ( noctor et al . , 2004 ) , which is also controlled by gap junctional coupling ( elias et al . , 2007 ) electrophysiological and optical imaging recordings further support the hypothesis that subplate neurons are well integrated in the developing cerebral cortex . in vitro intracellular recordings and current - source density analyses in late embryonic and early postnatal kitten visual cortex demonstrated that subplate neurons receive functional excitatory synaptic inputs from axons that course in the developing white matter ( friauf et al . subplate cells in newborn rat somatosensory cortical slices reveal a substantial amount of spontaneous postsynaptic currents ( spscs ) with different kinetics and pharmacological profile demonstrating that subplate neurons receive functional synaptic inputs mediated by ampa , nmda and gabaa receptors ( hanganu et al . , 2001 ) . a more detailed analysis of the synaptic inputs onto subplate cells could be obtained by electrical stimulation of specific afferent axonal projections ( figures 3b , c ) ( staiger et al . , 1999 ) . brief electrical stimulation of the thalamocortical projection elicits in immature rat and cat subplate neurons a fast and reliable excitatory postsynaptic potential / current ( epsp / c ) , which can blocked by the ampa / kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione ( cnqx ) ( friauf et al . , 1990 ; hanganu et al . , elicits a cnqx - insensitive component , which can be blocked by the nmda receptor antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ( cpp ) , demonstrating that the thalamic input also activates functional nmda receptors on subplate cells ( hanganu et al . , 2002 ) . these electrophysiological observations from single subplate neurons are further supported by current source - density analyses performed in late embryonic and early postnatal kitten ( friauf and shatz , 1991 ) and rat cerebral cortex ( molnr et al . , 2003 ) , demonstrating that subplate cells in these species receive a functional thalamic input at earliest stages of neocortical development . recording of optical images with voltage - sensitive dyes in neocortical slices of prenatal rats have shown that there is a few days delay between the arrival of thalamocortical axons at the subplate at e16 and the appearance of functional thalamocortical synaptic transmission at e19 ( higashi et al . , 2002 ) . beside a glutamatergic thalamocortical input with relatively mature functional properties ( short delay , fast kinetics , reliable responses ) , subplate cells in newborn rodents receive additional intracortical synaptic inputs from various presynaptic sources ( figure 3d ) . local glutamatergic synaptic inputs arise from pyramidal neurons in the cortical plate and from glutamatergic subplate cells , which both activate postsynaptic ampa / kainate and nmda receptors ( hanganu et al . , this intra - subplate glutamatergic input differs from the thalamocortical input . whereas the intra - subplate input reveals a pronounced facilitation when repetitively activated at 1040 hz , the thalamocortical input is rather stable or suppressed at these stimulation frequencies ( figure 4 ) ( hirsch and luhmann , 2008 ) . the intra - subplate synaptic input can sustain high stimulation frequencies and may boost the thalamocortical input , thereby enhancing activity in the developing neocortical circuit ( hirsch and luhmann , 2008 ) . anatomical studies in rodent and human immature cerebral cortex indicate that glutamatergic synaptic inputs most likely also arise from other neocortical sources via corticocortical connections ( ivy and killackey , 1981 ; kostovic and jovanov - milosevic , 2006 ) , but the functional properties of these long - distance synaptic inputs onto subplate cells are currently unknown . input - specific differences in temporal summation of nmda receptor - mediated epsps recorded in subplate neurons of the newborn mouse cerebral cortex . recordings were performed in the presence of cnqx and bicuculline methiodide to isolate nmda receptor - mediated epsps . ( a ) five successive stimuli to the thalamic ( left ) or subplate ( right ) input reveals prominent temporal summation and repetitive action potentials ( truncated ) of the subplate input at frequencies between 10 and 40 hz . ( b ) synaptic response to 20-hz subplate stimulation at higher resolution [ same trace as in ( a ) ] . ( c ) quotient between nmda receptor - epsp integral and control integral ( determined at 0.03 hz ) for 13 subplate neurons . reproduced with permission from hirsch and luhmann ( 2008 ) . subplate cells in rodents receive a gabaergic synaptic input from neighbouring gabaergic neurons located in the subplate and probably also in the cortical plate ( figure 3d ) ( hanganu et al . , 2002 ) . however , as in other immature brain structures , this gabaergic input most likely has a pure excitatory postsynaptic effect ( figure 6 in hanganu et al . an excitatory effect has been demonstrated in subplate cells also for glycine and taurine ( kilb et al . , 2008 ) . subplate neurons are not only well integrated in the developing cortical circuit , but during certain developmental periods they also receive a very selective input from neuromodulatory brain structures . both in primates ( for review ( rakic , 1995 ) as well as in rodents ( calarco and robertson , 1995 ; mechawar and descarries , 2001 ) the subplate is specifically innervated by cholinergic fibers arising from the basal forebrain and from monoaminergic inputs . the activation of postsynaptic nicotinic acetylcholine receptors elicits in subplate cells a marked depolarization which is largely mediated by the activation of alpha4/beta2 receptors ( hanganu and luhmann , 2004 ) . the responsiveness to activation of muscarinic acetylcholine receptors ( machr ) is more complex and reveals a remarkable oscillatory discharge mode of subplate cells ( hanganu et al . , 2009 ) . application of muscarine to neocortical slices from newborn rats induces in subplate cells repetitive burst discharges with burst frequencies in the range of 20 hz ( figure 5a ) ( hanganu et al . , 2009 ) . similar burst patterns can be recorded from subplate neurons when the tissue concentration of acetylcholine is raised by application of the cholinesterase inhibitor neostigmine ( figure 5b ) . these electrophysiological data are further supported by calcium imaging experiments , which demonstrate that activation of muscarinic receptors induces repetitive ca - transients which are highly coordinated within the subplate ( figure 1 in hanganu et al . , 2009 ) . subplate cells , which express the m1m5 subunits of the machr , are instantly and massively excited upon activation of postsynaptic muscarinic receptors and switch into an oscillatory burst firing mode ( figure 5 ) . since subplate cells are densely coupled via electrical ( figure 2 ) and chemical synapses ( figure 3d ) , these cellular oscillations are synchronized and amplified within the intra - subplate network . gap junctional coupling , depolarizing gaba actions and a tonic non - synaptic gaba release contribute to the generation and maintenance of the cholinergic network oscillations ( figure 9 in hanganu et al . , 2009 ) . subplate neurons in newborn rat cerebral cortex reveal repetitive burst discharges upon activation of postsynaptic cholinergic receptors . ( a ) current - clamp whole - cell recording from a p3 subplate cell at a holding potential of 70 mv . note the long - lasting burst discharge after a single 30-s long bath application of muscarine ( marked by a black bar ) . frequency power spectrum of the action potential discharge within one representative burst from the trace above . ( b ) current - clamp whole - cell recording from a p2 subplate neuron at a holding potential of 70 mv displaying the effects of 10 m neostigmine , which blocks endogenous acetylcholine esterase and thereby increases the concentration of acetylcholine in the neocortical slice . frequency power spectrum of the action potential discharge within burst from the upper trace in ( b ) . the crucial role of the subplate in generating cholinergic network oscillations is supported by experiments on thick ( 8001000 m ) neocortical slices from newborn rodents . only slices with an intact subplate reveal network oscillations upon activation of muscarinic receptors with carbachol ( dupont et al . , 2006 ) . when the subplate is removed , slices do not show any carbachol - induced network oscillations ( figure 6 ) . furthermore , selective electrical stimulation of the subplate in thick neocortical slices from newborn mice using a multi - electrode array ( mea ) also evokes large - scale network oscillations ( figure 7 ) ( sun and luhmann , 2007 ) . in a recent study on mouse neocortical slice cultures , moody and coworkers suggested that the subplate may act as a pacemaker region to generate propagating waves of spontaneous activity in the neonatal cerebral cortex ( lischalk et al . , 2009 ) . , 2006 ; sun and luhmann , 2007 ) or by the sensory input , i.e. the retina ( hanganu et al . , 2006 ) or the whiskers ( yang et al . , 2009 ) . at early developmental stages , the sensory input from the periphery firstly reaches the subplate and is subsequently transmitted to the cortex ( friauf and shatz , 1991 ) . during this developmental period , role of the subplate in triggering carbachol - induced network oscillations in the neonatal mouse cerebral cortex . ( a ) field potential response to carbachol application in a coronal 800-m thick cortical slab preparation with an intact subplate as shown in nissl stained section below . ( b ) lack of carbachol - induced activity in a cortical slab preparation , in which the subplate was eliminated . local electrical stimulation of the subplate elicits propagating network oscillations in the developing cerebral cortical network of the newborn mouse . ( a ) photograph of p3 mouse nissl - stained coronal slice with cortical layers and position of 60-channel multi - electrode array ( mea ) . ( b ) mea recordings from the neocortical slice shown in ( a ) with site of bipolar stimulation in the subplate ( 80 a , 200-s duration , 50 hz , 10 times ) . spacing between electrodes ( c ) recording at electrode number 84 [ dotted red rectangle in ( a ) ] shown at higher resolution with corresponding wavelet analysis and fourier spectrum . reproduced with permission from sun and luhmann ( 2007 ) . electrophysiological and calcium imaging recordings in acute neocortical slices from newborn rodents ( hanganu et al . , 2009 ) as well as calcium imaging data obtained in neocortical cell cultures ( voigt et al . , 2001 ) indicate that gabaergic subplate cells play a central role in generating this neuronal network activity . voigt et al . ( 2001 ) estimated that a minimal number of two gabaergic subplate neurons per square millimeter are required for the occurrence of synchronous network activity . the unique structural and functional properties enable subplate cells to receive , synchronize and amplify the afferent and intrinsic synaptic inputs . it is tempting to speculate on the downstream effects of these early network oscillations in cortical maturation ? it has been demonstrated that brain derived neurotrophic factor ( bdnf ) is released from synaptically localized secretory granules following burst stimulation at 2050 hz ( balkowiec and katz , 2000 ; hartmann et al . , 2001 ) ( for review lessmann et al . , 2003 ) . if subplate - driven oscillations contribute to the local secretion of bdnf , the released bdnf may strengthen the synaptic connectivity between neurons in these early ensembles . beside strengthening synaptic connections via local bdnf release , we recently demonstrated that the rate of apoptosis in organotypic slice cultures of the neonatal mouse cerebral cortex is regulated by electrical activity patterns , which resemble in many aspects the subplate - driven activity ( heck et al . , 2008 ) . this activity - dependent regulation of neuronal apoptosis was at least partly mediated by activation of the bdnf receptor trkb , indicating that subplate - driven network oscillations may also influence programmed cell death via activity - dependent bdnf release . a large proportion of newly generated gabaergic interneurons arising from the medial ganglionic eminence and pyramdial neurons from the ventricular zone must migrate through the subplate on their way to the developing cortical plate ( kriegstein and noctor , 2004 ) . gabaergic and glutamatergic subplate neurons partly release their neurotransmitter in a paracrine , non - synaptic manner ( hanganu et al . , 2009 ) , thereby regulating the neuronal migration pattern ( reiprich et al . , 2005 ; manent et al . , 2005 , 2006 ; . neurotransmitter release in the subplate may rise substantially during oscillatory network activity , thereby activating low affinity or extrasynaptic receptors causing alterations in neuronal migration ( denter et al . , 2009 ) . on the basis of the currently available data we suggest the following model ( figure 8) : during early cortical development , in most mammals before birth , subplate cells with relatively mature structural properties ( elaborated dendritic tree , complex axonal projections , mature symmetrical and asymmetrical synapses ) receive a functional glutamatergic synaptic input from specific thalamic nuclei and a selective input from neuromodulatory systems ( e.g. cholinergic inputs from the basal forebrain ) . in rodents , subplate neurons are densely interconnected via electrical and chemical synapses and upon activation of muscarinic ( and probably also other metabotropic receptors ( wagner and luhmann , 2006 ) discharge in repetitive 20 hz bursts ( lower inset in figure 8) . gabaergic subplate neurons releasing gaba in a synaptic ( 1 in figure 8) and tonic non - synaptic manner ( 2 in figure 8) facilitate the generation of oscillatory network activity ( hanganu et al . , 2009 ) . intra - subplate connections arising from glutamatergic subplate cells may boost the subplate activity in the 1040 hz frequency range ( hirsch and luhmann , 2008 ) . subplate cells are dye - coupled to cortical plate neurons in a columnar manner ( dupont et al . , 2006 ) and faithfully transmit synchronized 1020 hz network oscillations generated and amplified in the subplate to the cortical plate and marginal zone ( upper inset in figure 8) ( dupont et al . , 2006 ; wagner and luhmann , 2006 ; the subplate receives a transient and selective synaptic input from neuromodulatory systems , from the sensory periphery via specific thalamic nuclei and via corticocortical fibers from other neocortical areas . subplate cells are capable to fire repetitive action potentials ( figure 3a ) and upon activation of cholinergic postsynaptic receptors discharge in repetitive bursts ( lower inset and figure 5 ) . gabaergic subplate neurons release gaba in an activity - dependent ( 1 ) and tonic ( 2 ) manner thereby inducing a postsynaptic excitatory response . subplate neurons are not only coupled via these excitatory synaptic circuits , but also via gap junctions ( figure 2 ) . chemical and electrical synapses contribute to the generation of subplate - driven oscillatory activity which is transmitted to the gap junctional coupled network in the cortical plate ( figure 2a ) and marginal zone and induces 1020 hz neocortical network oscillations ( upper inset ) . elimination or interruption of the subplate prevents the generation of the subplate - driven synchronized activity patterns and disturbs the maturation of the columnar architecture ( ghosh and shatz , 1992 ; kanold et al . , 2003 ; kanold and shatz , 2006 ) . under normal conditions and with further development , subplate cells disappear by apoptosis ( ferrer et al . , 1990 ; price et al . , 1997 ; arias et al . , 2002 ) or transform into white matter interstitial cells ( valverde and facal - valverde , 1988 ; valverde et al . , 1995 ) or layer vib ( layer vii ) neurons ( woo et al . , 1991 ; reep , 2000 ) in the normal mature cerebral cortex , subplate neurons have lost their capabilities to receive and amplify incoming neuronal activity and to generate synchronized network oscillations . however , as already suggested by jones ( 1995 ) , disturbances in the pattern of programmed cell death in the subplate may cause a failure to establish normal patterns of connections in the overlying cerebral cortex , leading to long - term neurological deficits such as schizophrenia . abnormal placement of ( surviving subplate ? ) neurons in the white matter and atypical circuits have been observed in the prefrontal cortex of schizophrenic patients ( akbarian et al . , 1996 ; it has been further suggested that subplate - like neurons may persist in cortical dysplasia and contribute to the manifestation of pharmacoresistant epilepsy in adults ( cepeda et al . , 2007 ) . interestingly in the resectioned human tissue gaba application induced depolarizing postsynaptic responses and spontaneous gabaergic synaptic potentials even elicited action potentials ( cepeda et al . , 2007 ) . in summary , experimental data indicate that subplate cells , most likely gabaergic subplate neurons , play an important role in the generation of early synchronized network activity and in the normal development of the neocortical network and columnar architecture . clinical evidence suggests that subplate cells in the mature cortex contribute to the manifestation of abnormal neuronal circuits , pathological activities and long - term neurological deficits . the authors declare that the research presented in this paper was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

due to their unique structural and functional properties , subplate cells are ideally suited to function as important amplifying units within the developing neocortical circuit . subplate neurons have extensive dendritic and axonal ramifications and relatively mature functional properties , i.e. their action potential firing can exceed frequencies of 40 hz . at earliest stages of corticogenesis subplate cells receive functional synaptic inputs from the thalamus and from other cortical and non - cortical sources . glutamatergic and depolarizing gabaergic inputs arise from cortical neurons and neuromodulatory inputs arise from the basal forebrain and other sources . activation of postsynaptic metabotropic receptors , i.e. muscarinic receptors , elicits in subplate neurons oscillatory burst discharges which are transmitted via electrical and chemical synapses to neighbouring subplate cells and to immature neurons in the cortical plate . the tonic non - synaptic release of gaba from gabaergic subplate cells facilitates the generation of burst discharges . these cellular bursts are amplified by prominent gap junction coupling in the subplate and cortical plate , thereby eliciting 1020 hz oscillations in a local columnar network . thus , we propose that neuronal networks are organized at earliest stages in a gap junction coupled columnar syncytium . we postulate that the subplate does not only serve as a transient relay station for afferent inputs , but rather as an active element amplifying the afferent and intracortical activity .

Introduction Structural Properties of Subplate Neurons Functional Properties of Subplate Neurons Subplate Neurons are Well Integrated in the Developing Neocortical Network Oscillatory Properties of Subplate Cells GABAergic Subplate Cells Drive Early Cortical Networks Conflict of Interest Statement

the subplate forms a transient layer in the developing cerebral cortex and consists of migratory and postmigratory neurons , dendrites , axons , growth cones , synapses and glial cells . the subplate is located between the intermediate zone and the cortical plate , which during further development differentiates into the neocortical layers ii to vi ( figure 1a ) . kostovic and molliver were the first who identified the subplate as a distinct layer in the embryonic human cerebral cortex ( kostovic and molliver , 1974 ) . in the rat and other rodents many subplate cells survive into adulthood forming layer vib or vii ( aboitiz and montiel , 2007 ) . prominent species differences also exist in the relative thickness of the subplate , which increased during evolution . in humans the subplate develops to approximately six times the thickness of the cortical plate around 29 weeks of gestation ( mrzljak et al . the majority of the subplate cells are born early before the first cortical plate neurons ( luskin and shatz , 1985 ; valverde et al . a substantial proportion of subplate cells are not born in the ventricular neuroepithelium , but instead originate in the medial ganglionic eminence and follow a tangential migratory route to their positions in the developing cortex ( lavdas et al . subplate cells represent a rather heterogeneous neuronal population according to their morphology , neurotransmitter identity and connectivity ( for review allendoerfer and shatz , 1994 ) . subplate cells play important roles in the structural and functional organization of the cerebral cortex and in early necortical plasticity . thalamic axons ( lund and mustari , 1977 ; rakic , 1977 , 1983 ) and early deletion of subplate neurons in kitten visual cortex prevents the segregation of thalamocortical axons within layer iv and the formation of ocular dominance columns ( ghosh and shatz , 1992 ; kanold et al . furthermore , subplate cells regulate the maturation of gabaergic synaptic transmission and establish the balance between excitation and inhibition in the developing neocortical network ( kanold and shatz , 2006 ) . , 1989 ; kostovic and rakic , 1990 ) due to their earlier generation and more mature developmental stage , subplate neurons show a relatively extensive dendritic tree when compared to the more immature pyramidal neurons of the cortical plate ( mrzljak et al . descending dendrites from subplate neurons may invade the underlying intermediate zone and ascending dendrites may extend into the cortical plate ( del ro et al . ( 2009 ) succeeded in identifying a number of novel markers for murine subplate cells , which may soon allow the definition of different subpopulations of subplate neurons . they show a dense axonal arborization within the subplate , but also project to the marginal zone / layer i ( clancy and cauller , 1999 ) and to the cortical plate , where they form axonal collaterals within layer iv ( friauf et al . , the majority of the subplate neurons projecting into the cortical plate reside in the upper half of the subplate and provide a glutamatergic synaptic input to the developing cortical plate , including the layer iv neurons ( friauf et al . , 1990 ; long - distance axons from subplate neurons invade the thalamus during early stages of corticogenesis and form an axonal scaffold for the establishment of cortical efferent and afferent projections ( mcconnell et al . beside these local and long - distance projections arising from glutamatergic subplate neurons , gabaergic subplate cells also project to both neighbouring and more distant neocortical regions and form a corticocortical synaptic network ( tomioka et al . , 2007 ) , the postsynaptic action of gabaergic subplate neurons may be also depolarizing ( figure 6 in hanganu et al . ultrastructural studies of subplate cells in various species have demonstrated symmetrical as well as asymmetrical synapses with relatively mature properties ( kostovic and rakic , 1980 , 1990 ; chun and shatz , 1988 ; herrmann et al . , 1994 ) , indicating that subplate neurons receive gabaergic as well as glutamatergic synaptic inputs . as suggested by kostovic and rakic ( 1980 ) , glutamatergic inputs onto subplate neurons may arise from the thalamus and other neocortical areas , whereas gabaergic synaptic inputs may originate from gabaergic interneurons in the subplate . n - methyl - d - aspartate ( nmda ) , -amino-3-hydroxy-5-methylisoxazole-4-propionic acid ( ampa ) and kainate receptors and the essential subunits for their receptor function have been demonstrated in the subplate of various species , suggesting the presence of functional glutamatergic synapses in subplate neurons ( herrmann , 1996 ; aoki , 1997 ; catalano et al . , 1990 ) and gabaa receptor subunits ( meinecke and rakic , 1992 ) in the subplate indicate that functional gabaergic synaptic inputs should also be present in subplate neurons . these morphological , ultrastructural and immunohistochemical data are complemented by functional studies on the properties of the subplate and single subplate cells in different mammalian species . in contrast to the heterogeneity in morphological and chemical appearance , electrophysiological recordings from single subplate neurons in rodents demonstrate rather homogeneous functional properties . whole - cell patch - clamp recordings from subplate cells in newborn rodent neocortical slices revealed resting membrane potentials and input resistances in the range of 55 mv and 11.2 g , respectively ( luhmann et al . in response to sustained depolarization by intracellular current injection , subplate neurons are capable of firing overshooting and repetitive action potentials at frequencies exceeding 40 hz ( figures 2c and 3a ) . similar results were obtained from subplate neurons in acute neocortical slices harvested postmortem from human fetal brain at gestational week 1622 ( moore et al . ( a ) intracellular biocytin staining of one subplate neuron ( yellow arrow ) in the p3 mouse neocortex results in columnar labeling of several dye - coupled cells in the subplate and in the cortical plate. ( c1 ) injection of a hyperpolarizing or suprathreshold depolarizing current pulse into the cell marked by black arrow causes voltage deflections in the gap junction coupled neuron . ( a ) typical action potential firing pattern of a subplate neuron in the cerebral cortex of a p1 rat . ( d ) schematic diagram illustrating the distribution of postsynaptic receptors on a subplate neuron for monosynaptic inputs arising from the thalamus ( thal . ) , the cortical plate ( cp ) including layers v / vi and within the subplate ( sp ) . note that synaptic inputs from other subplate neurons can lead to activation of depolarizing gabaa receptors and nmda receptors activated at negative membrane potentials . in comparison to other neurons in the immature cerebral cortex , subplate cells also reveal the most mature properties in action potential characteristics and in the biophysical properties of voltage - dependent sodium and calcium currents . , 2009 ) , indicating that these relatively mature functional properties enable subplate cells to transmit afferent neuronal activity faithfully to the developing cortical plate . intracellular labeling of single subplate cells with fluorescent dyes or biocytin revealed an extensive neuronal network of dye - coupled neurons in the subplate and cortical plate ( figure 2a ) . in newborn rats , on average about nine neurons are dye - coupled to a single subplate cell and these gap junction coupled networks are often organized in a columnar manner ( dupont et al . these data indicate that subplate cells are strongly coupled via electrical synapses and form a functional columnar syncytium with neurons located in the cortical plate . , 2007 ) electrophysiological and optical imaging recordings further support the hypothesis that subplate neurons are well integrated in the developing cerebral cortex . in vitro intracellular recordings and current - source density analyses in late embryonic and early postnatal kitten visual cortex demonstrated that subplate neurons receive functional excitatory synaptic inputs from axons that course in the developing white matter ( friauf et al . subplate cells in newborn rat somatosensory cortical slices reveal a substantial amount of spontaneous postsynaptic currents ( spscs ) with different kinetics and pharmacological profile demonstrating that subplate neurons receive functional synaptic inputs mediated by ampa , nmda and gabaa receptors ( hanganu et al . a more detailed analysis of the synaptic inputs onto subplate cells could be obtained by electrical stimulation of specific afferent axonal projections ( figures 3b , c ) ( staiger et al . brief electrical stimulation of the thalamocortical projection elicits in immature rat and cat subplate neurons a fast and reliable excitatory postsynaptic potential / current ( epsp / c ) , which can blocked by the ampa / kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione ( cnqx ) ( friauf et al . , elicits a cnqx - insensitive component , which can be blocked by the nmda receptor antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ( cpp ) , demonstrating that the thalamic input also activates functional nmda receptors on subplate cells ( hanganu et al . , 2003 ) , demonstrating that subplate cells in these species receive a functional thalamic input at earliest stages of neocortical development . beside a glutamatergic thalamocortical input with relatively mature functional properties ( short delay , fast kinetics , reliable responses ) , subplate cells in newborn rodents receive additional intracortical synaptic inputs from various presynaptic sources ( figure 3d ) . local glutamatergic synaptic inputs arise from pyramidal neurons in the cortical plate and from glutamatergic subplate cells , which both activate postsynaptic ampa / kainate and nmda receptors ( hanganu et al . the intra - subplate synaptic input can sustain high stimulation frequencies and may boost the thalamocortical input , thereby enhancing activity in the developing neocortical circuit ( hirsch and luhmann , 2008 ) . anatomical studies in rodent and human immature cerebral cortex indicate that glutamatergic synaptic inputs most likely also arise from other neocortical sources via corticocortical connections ( ivy and killackey , 1981 ; kostovic and jovanov - milosevic , 2006 ) , but the functional properties of these long - distance synaptic inputs onto subplate cells are currently unknown . input - specific differences in temporal summation of nmda receptor - mediated epsps recorded in subplate neurons of the newborn mouse cerebral cortex . ( a ) five successive stimuli to the thalamic ( left ) or subplate ( right ) input reveals prominent temporal summation and repetitive action potentials ( truncated ) of the subplate input at frequencies between 10 and 40 hz . subplate cells in rodents receive a gabaergic synaptic input from neighbouring gabaergic neurons located in the subplate and probably also in the cortical plate ( figure 3d ) ( hanganu et al . subplate neurons are not only well integrated in the developing cortical circuit , but during certain developmental periods they also receive a very selective input from neuromodulatory brain structures . both in primates ( for review ( rakic , 1995 ) as well as in rodents ( calarco and robertson , 1995 ; mechawar and descarries , 2001 ) the subplate is specifically innervated by cholinergic fibers arising from the basal forebrain and from monoaminergic inputs . the activation of postsynaptic nicotinic acetylcholine receptors elicits in subplate cells a marked depolarization which is largely mediated by the activation of alpha4/beta2 receptors ( hanganu and luhmann , 2004 ) . the responsiveness to activation of muscarinic acetylcholine receptors ( machr ) is more complex and reveals a remarkable oscillatory discharge mode of subplate cells ( hanganu et al . application of muscarine to neocortical slices from newborn rats induces in subplate cells repetitive burst discharges with burst frequencies in the range of 20 hz ( figure 5a ) ( hanganu et al . these electrophysiological data are further supported by calcium imaging experiments , which demonstrate that activation of muscarinic receptors induces repetitive ca - transients which are highly coordinated within the subplate ( figure 1 in hanganu et al . subplate cells , which express the m1m5 subunits of the machr , are instantly and massively excited upon activation of postsynaptic muscarinic receptors and switch into an oscillatory burst firing mode ( figure 5 ) . since subplate cells are densely coupled via electrical ( figure 2 ) and chemical synapses ( figure 3d ) , these cellular oscillations are synchronized and amplified within the intra - subplate network . gap junctional coupling , depolarizing gaba actions and a tonic non - synaptic gaba release contribute to the generation and maintenance of the cholinergic network oscillations ( figure 9 in hanganu et al . subplate neurons in newborn rat cerebral cortex reveal repetitive burst discharges upon activation of postsynaptic cholinergic receptors . frequency power spectrum of the action potential discharge within burst from the upper trace in ( b ) . only slices with an intact subplate reveal network oscillations upon activation of muscarinic receptors with carbachol ( dupont et al . in a recent study on mouse neocortical slice cultures , moody and coworkers suggested that the subplate may act as a pacemaker region to generate propagating waves of spontaneous activity in the neonatal cerebral cortex ( lischalk et al . at early developmental stages , the sensory input from the periphery firstly reaches the subplate and is subsequently transmitted to the cortex ( friauf and shatz , 1991 ) . during this developmental period , role of the subplate in triggering carbachol - induced network oscillations in the neonatal mouse cerebral cortex . ( b ) lack of carbachol - induced activity in a cortical slab preparation , in which the subplate was eliminated . local electrical stimulation of the subplate elicits propagating network oscillations in the developing cerebral cortical network of the newborn mouse . ( b ) mea recordings from the neocortical slice shown in ( a ) with site of bipolar stimulation in the subplate ( 80 a , 200-s duration , 50 hz , 10 times ) . ( 2001 ) estimated that a minimal number of two gabaergic subplate neurons per square millimeter are required for the occurrence of synchronous network activity . the unique structural and functional properties enable subplate cells to receive , synchronize and amplify the afferent and intrinsic synaptic inputs . beside strengthening synaptic connections via local bdnf release , we recently demonstrated that the rate of apoptosis in organotypic slice cultures of the neonatal mouse cerebral cortex is regulated by electrical activity patterns , which resemble in many aspects the subplate - driven activity ( heck et al . a large proportion of newly generated gabaergic interneurons arising from the medial ganglionic eminence and pyramdial neurons from the ventricular zone must migrate through the subplate on their way to the developing cortical plate ( kriegstein and noctor , 2004 ) . gabaergic and glutamatergic subplate neurons partly release their neurotransmitter in a paracrine , non - synaptic manner ( hanganu et al . neurotransmitter release in the subplate may rise substantially during oscillatory network activity , thereby activating low affinity or extrasynaptic receptors causing alterations in neuronal migration ( denter et al . on the basis of the currently available data we suggest the following model ( figure 8) : during early cortical development , in most mammals before birth , subplate cells with relatively mature structural properties ( elaborated dendritic tree , complex axonal projections , mature symmetrical and asymmetrical synapses ) receive a functional glutamatergic synaptic input from specific thalamic nuclei and a selective input from neuromodulatory systems ( e.g. cholinergic inputs from the basal forebrain ) . in rodents , subplate neurons are densely interconnected via electrical and chemical synapses and upon activation of muscarinic ( and probably also other metabotropic receptors ( wagner and luhmann , 2006 ) discharge in repetitive 20 hz bursts ( lower inset in figure 8) . gabaergic subplate neurons releasing gaba in a synaptic ( 1 in figure 8) and tonic non - synaptic manner ( 2 in figure 8) facilitate the generation of oscillatory network activity ( hanganu et al . intra - subplate connections arising from glutamatergic subplate cells may boost the subplate activity in the 1040 hz frequency range ( hirsch and luhmann , 2008 ) . subplate cells are dye - coupled to cortical plate neurons in a columnar manner ( dupont et al . , 2006 ) and faithfully transmit synchronized 1020 hz network oscillations generated and amplified in the subplate to the cortical plate and marginal zone ( upper inset in figure 8) ( dupont et al . , 2006 ; wagner and luhmann , 2006 ; the subplate receives a transient and selective synaptic input from neuromodulatory systems , from the sensory periphery via specific thalamic nuclei and via corticocortical fibers from other neocortical areas . subplate cells are capable to fire repetitive action potentials ( figure 3a ) and upon activation of cholinergic postsynaptic receptors discharge in repetitive bursts ( lower inset and figure 5 ) . gabaergic subplate neurons release gaba in an activity - dependent ( 1 ) and tonic ( 2 ) manner thereby inducing a postsynaptic excitatory response . subplate neurons are not only coupled via these excitatory synaptic circuits , but also via gap junctions ( figure 2 ) . chemical and electrical synapses contribute to the generation of subplate - driven oscillatory activity which is transmitted to the gap junctional coupled network in the cortical plate ( figure 2a ) and marginal zone and induces 1020 hz neocortical network oscillations ( upper inset ) . elimination or interruption of the subplate prevents the generation of the subplate - driven synchronized activity patterns and disturbs the maturation of the columnar architecture ( ghosh and shatz , 1992 ; kanold et al . under normal conditions and with further development , subplate cells disappear by apoptosis ( ferrer et al . , 1991 ; reep , 2000 ) in the normal mature cerebral cortex , subplate neurons have lost their capabilities to receive and amplify incoming neuronal activity and to generate synchronized network oscillations . neurons in the white matter and atypical circuits have been observed in the prefrontal cortex of schizophrenic patients ( akbarian et al . in summary , experimental data indicate that subplate cells , most likely gabaergic subplate neurons , play an important role in the generation of early synchronized network activity and in the normal development of the neocortical network and columnar architecture . clinical evidence suggests that subplate cells in the mature cortex contribute to the manifestation of abnormal neuronal circuits , pathological activities and long - term neurological deficits . the authors declare that the research presented in this paper was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

the relationship of the pharynx and associated structures such as the tongue and hyoid bone with dentofacial pattern has been intensively researched . several studies have tried to correlate patients with normal nasorespiratory functions with different malocclusions and airway dimensions . ceylan and oktay reported no statistically significant difference between the groups based on the anb angle and nasopharyngeal size , but they observed a negative correlation between the anb angle and oropharyngeal size . the belief that the hyoid bone and tongue may be correlated with the mandibular position and morphology has led to a consideration of various skeletal types . gobeille and bowman , cuozzo and bowman reported variations in the position of the hyoid bone relative to the mandible within a skeletal class i context , which led to the speculation that variability in the hyoid position is partly determined by mandibular posture . according to brodie ( 1950 ) , the position of the hyoid bone relative to the cranial base and the mandible has been of interest specifically as an indicator of tongue posture and function , and has an important role in maintaining the airway and upright natural head position . clinicians generally agree on a morphogenetic role of the tongue ; thus , any alteration or change in the spatial relationships of the hyoid bone , tongue , and airway have wide functional significance and reciprocal consequences . according to biorgue , the influence of the tongue on the morphology of the dental arches and on the occlusion depends not only on the lingual volume but also on its posture and mobility . thurow proposed that the geniohyoid muscle functions to adjust the anterioposterior position of the hyoid and to maintain the airway patency throughout the various movements of the craniofacial complex . the position of the hyoid bone on the genial tubercle level will increase the efficiency of the muscle in pulling the tongue forward and maintaining the airway . a more inferior position of the hyoid bone with lower tongue posture places the geniohyoid muscle at a mechanical disadvantage by its angulation . this may increase the mandibular load because of the need to elevate the tongue , as well as create a stronger opening force on the mandible , which can be of significance in the development and establishment of the dentofacial pattern and function . as of yet , there are only few studies relating the position of the pharyngeal airway , the hyoid bone and tongue to different skeletal patterns using the anb angle . the present study aims to evaluate the pharyngeal airway dimension , tongue and hyoid position by the use of lateral cephalograms in subjects having anb > 4 and anb < 4 . this study was undertaken to evaluate the pharyngeal airway dimension , tongue and hyoid position by the use of lateral cephalograms in subjects with normal nasorespiratory functions having anb > 4 and anb < 4. a total of 61 subjects ( class i , n = 29 ; class ii , n = 32 ) in an age range of 11 - 19 years ( mean ) were evaluated . the study was approved by the ethics committee of career post graduate institute of dental sciences and hospital . the sagittal skeletal differentiation of subjects with angle 's class i and class ii division i molar relation was achieved with measurement of the anb angle . they possessed all permanent teeth up to second molars ; there was no burn / wound scar in the head and neck region . a lead foil was placed on the tip of the tongue with glass ionomer cement ( gic ) mixture and a barium paste coating was done on the tonguethe radiographs obtained were traced using lead acetate paper and 4h pencil , and various landmarks for evaluation of the tongue , hyoid bone , and pharynx were identified and marked [ figure 1 ] anb analysis was done for the sagittal apical base relationship on the study samples . a lead foil was placed on the tip of the tongue with glass ionomer cement ( gic ) mixture and a barium paste coating was done on the tongue the radiographs obtained were traced using lead acetate paper and 4h pencil , and various landmarks for evaluation of the tongue , hyoid bone , and pharynx were identified and marked [ figure 1 ] anb analysis was done for the sagittal apical base relationship on the study samples . the x and y coordinates of the deepest point of the epiglottis ( e ) and the center of the lead disc on the tongue tip ( tt ) were identified . linear distance along the perpendicular bisector of the e - tt line to the dorsum of the tongue ; tt / lop ( mm ) = tongue tip relative to lower occlusal plane . linear distance between tt and the lop ( a line between the midpoint of the occlusal surface of the mandibular molar and incisor tip ) measured perpendicular to the lop ; tt - l1/lop ( mm ) = tongue tip relative to lower incisor tip . linear distance between tt and a perpendicular line to lop through the incisor tip . the frankfort horizontal plane and a line perpendicular to it passing through sella the horizontal measurements made were : yl ; at - aph : distance of the most anterior point of the hyoid bone ( aph ) from the most anterior point of the atlas ( at ) , y2 ; s - aph : distance of aph from the center of sella turcica ( s ) , y3 ; pog - aph : distance of the anterior point of hyoid bone ( aph ) to pogonion ( pog ) ( measured indirectly through projections of both points on the line perpendicular to fh from s ) , y4 ; pog - aph : distance measured directly from aph to pog , y5 ; a - aph : distance of aph to point a ( measured through projections of both points on the line perpendicular to fh from s ) , y6 ; n - aph : distance of aph to nasion ( n ) ( measured through projections of both points on the line perpendicular to fh from s ) . the vertical measurements made were : y7 ; pph - fh : distance of the posterior point of hyoid ( pph ) to fh , y8 ; aph - fh : distance of aph to fh , y9 ; gop - aph : distance of aph to a line drawn from gonion parallel ( gop ) to fh , y10 ; gop - pph : distance of pph to a line drawn from gop to fh . the angular measurements taken were : y11 ; lah - mp : angle formed by the lah and mandibular plane , y12 ; lah - fop : angle formed by the lah and the functional occlusal plane ( fop ) , y13 ; lah - pp : angle formed by the lah and the palatal plane ( pp ) , y14 ; lah- ban : angle formed by the lah and the basion - nasion ( ban ) plane , y15 ; lah - fh : angle formed by the lah and the fh , y16 ; lah - pbr : angle formed by the lah and the posterior border of the ramus of the mandible ( pbr ) . for the evaluation of the upper airway , the following measurements were made : the length of the ( presellar ) anterior cranial base ( s - n ) , the length of the postsellar part of the posterior cranial base ( ba - s ) , the total or effective cranial base length ( ba - n ) , the length of the palate ( floor of the nasal cavity ) . the distance between pns and ans . the distance from ba to pns . the effective length of the maxilla ( tmj to ans ) . the upper anterior facial height ( n and ans ) , the distance from aa to hy , the distance from so to in , the distance from hy to rgn , upper pharynx , lower pharynx , ho - ad1 , ad2-ptm , ba - ad1 , ad1-ptm , e - pns , soft palate midpoint - pharnyx , soft palate end - pharynx , 3cv - pharynx , e - pharynx , eb - pharynx . the saddle angle included between the lines joining ba to s and s to n ( ba - s - n ) . the angle between the palatal plane ( pns - ans ) and the anterior cranial base ( s - n ) . the areas of the tongue ; upper , middle , and lower pharynx ; soft palate ; and oral cavity were calculated using imagej software ( freely available online ) . imagej is an image - processing program that can calculate the area and pixel value statistics of user - defined selections . the outlines of the soft tissues were traced by means of the freehand preselection tool and the computer mouse . all the data obtained were subjected to statistical analysis and summarized as mean sd . the groups were compared by independent student 's t - test and the significance of parametric t - test was also confirmed by nonparametric mann - whitney u test . diagnostic accuracy of different linear and angular measurements was done by receiver operating characteristics ( roc ) curve analysis . a two - sided ( = 2 ) p value the x and y coordinates of the deepest point of the epiglottis ( e ) and the center of the lead disc on the tongue tip ( tt ) were identified . linear distance along the perpendicular bisector of the e - tt line to the dorsum of the tongue ; tt / lop ( mm ) = tongue tip relative to lower occlusal plane . linear distance between tt and the lop ( a line between the midpoint of the occlusal surface of the mandibular molar and incisor tip ) measured perpendicular to the lop ; tt - l1/lop ( mm ) = tongue tip relative to lower incisor tip . the frankfort horizontal plane and a line perpendicular to it passing through sella were taken as reference plane to evaluate the hyoid bone . the horizontal measurements made were : yl ; at - aph : distance of the most anterior point of the hyoid bone ( aph ) from the most anterior point of the atlas ( at ) , y2 ; s - aph : distance of aph from the center of sella turcica ( s ) , y3 ; pog - aph : distance of the anterior point of hyoid bone ( aph ) to pogonion ( pog ) ( measured indirectly through projections of both points on the line perpendicular to fh from s ) , y4 ; pog - aph : distance measured directly from aph to pog , y5 ; a - aph : distance of aph to point a ( measured through projections of both points on the line perpendicular to fh from s ) , y6 ; n - aph : distance of aph to nasion ( n ) ( measured through projections of both points on the line perpendicular to fh from s ) . the vertical measurements made were : y7 ; pph - fh : distance of the posterior point of hyoid ( pph ) to fh , y8 ; aph - fh : distance of aph to fh , y9 ; gop - aph : distance of aph to a line drawn from gonion parallel ( gop ) to fh , y10 ; gop - pph : distance of pph to a line drawn from gop to fh . the angular measurements taken were : y11 ; lah - mp : angle formed by the lah and mandibular plane , y12 ; lah - fop : angle formed by the lah and the functional occlusal plane ( fop ) , y13 ; lah - pp : angle formed by the lah and the palatal plane ( pp ) , y14 ; lah- ban : angle formed by the lah and the basion - nasion ( ban ) plane , y15 ; lah - fh : angle formed by the lah and the fh , y16 ; lah - pbr : angle formed by the lah and the posterior border of the ramus of the mandible ( pbr ) . for the evaluation of the upper airway , the following measurements were made : the length of the ( presellar ) anterior cranial base ( s - n ) , the length of the postsellar part of the posterior cranial base ( ba - s ) , the total or effective cranial base length ( ba - n ) , the length of the palate ( floor of the nasal cavity ) . the distance between pns and ans . the upper anterior facial height ( n and ans ) , the distance from aa to hy , the distance from so to in , the distance from hy to rgn , upper pharynx , lower pharynx , ho - ad1 , ad2-ptm , ba - ad1 , ad1-ptm , e - pns , soft palate midpoint - pharnyx , soft palate end - pharynx , 3cv - pharynx , e - pharynx , eb - pharynx . the saddle angle included between the lines joining ba to s and s to n ( ba - s - n ) . the angle between the palatal plane ( pns - ans ) and the anterior cranial base ( s - n ) . the areas of the tongue ; upper , middle , and lower pharynx ; soft palate ; and oral cavity were calculated using imagej software ( freely available online ) . imagej is an image - processing program that can calculate the area and pixel value statistics of user - defined selections . the outlines of the soft tissues were traced by means of the freehand preselection tool and the computer mouse . all the data obtained were subjected to statistical analysis and summarized as mean sd . the groups were compared by independent student 's t - test and the significance of parametric t - test was also confirmed by nonparametric mann - whitney u test . diagnostic accuracy of different linear and angular measurements was done by receiver operating characteristics ( roc ) curve analysis . a two - sided ( = 2 ) p value less than 0.05 ( p < 0.05 ) was considered statistically significant . the saddle angle included between the lines joining ba to s and s to n ( ba - s - n ) . the angle between the palatal plane ( pns - ans ) and the anterior cranial base ( s - n ) . the areas of the tongue ; upper , middle , and lower pharynx ; soft palate ; and oral cavity were calculated using imagej software ( freely available online ) . imagej is an image - processing program that can calculate the area and pixel value statistics of user - defined selections . the outlines of the soft tissues were traced by means of the freehand preselection tool and the computer mouse . all the data obtained were subjected to statistical analysis and summarized as mean sd . the groups were compared by independent student 's t - test and the significance of parametric t - test was also confirmed by nonparametric mann - whitney u test . diagnostic accuracy of different linear and angular measurements was done by receiver operating characteristics ( roc ) curve analysis . a two - sided ( = 2 ) p value less than 0.05 ( p < 0.05 ) was considered statistically significant . the present cephalometric study determines and compares linear and angular parameters of the pharyngeal airway , the tongue and hyoid bone in angle 's class ii division i malocclusion subject with anb > 4 with those with anb < 4 . a total of 61 symptomatic ( class i , n = 29 ; class ii , n = 32 ) age - matched patients of either sex were recruited and evaluated . the comparative linear and angular parameters of the pharyngeal airway , tongue , and hyoid bone in subjects with the anb angle more than 4 are summarized below respectively . females : the pharynx linear and angular parameters of two groups of females are summarized in table 1 , and the t - test revealed a significant difference between the two groups ( p < 0.05 or p < 0.001 ) and lower ho - pns and up in group 1 as compared to group 2 ; however , other parameters ( p > 0.05 ) between the two groups were found to be statistically similar [ table 1 ] . linear and angular parameters ( meansd ) of class i and class ii division i females similarly , in the two groups the difference in the pharynx angular parameters of females was statistically significant [ table 1 ] . the mean pharynx - linear parameters of males of the two groups and the t - test revealed significantly different ( p < 0.05 ) and lower aa - h4 and ad2-ptm while significantly ( p < 0.05 ) higher e - p in class ii division i as compared to class i ; however , other parameters did not differ ( p > 0.05 ) between the two groups , i.e. they were found to be statistically similar [ table 2 ] . similarly , comparing the mean pharynx angular parameters of males of class ii , the t - test revealed that they were similar ( p > 0.05 ) to the pharynx angular parameters of males between the two classes , i.e. they did not differ statistically [ table 2 ] . pharynx linear and angular parameter ( meansd ) of class i and class ii division i males the mean pharynx - linear parameters of the two groups ( females and males ) and the t - test revealed significantly ( p < 0.05 or p < 0.01 ) different and lower mean s - n , s - pns , ho - pns , aa - hy , hy - rgn , up , lp , and ad2-ptm in class ii division i subjects as compared to class i ; however , it showed no significant difference between the two groups [ table 3 ] . the overall mean pharynx - angular parameters of the two groups were similar ( p > 0.05 ) . pharynx linear and angular parameters ( meansd ) of two groups linear parameters of the tongue in the females of the two groups showed no significant difference and the t - test revealed significantly lower ( p < 0.05 ) tgl while higher ( p < 0.01 ) tt / lop was significantly higher ( p < 0.01 ) in class ii division i as compared to class i ; however , other parameters between the two groups were found to be statistically the same [ table 4 ] . tongue linear parameters ( meansd ) of class i and class ii division i females the mean male tongue linear parameters of class ii and t - test revealed significantly different ( p < 0.05 ) and higher tt / lop in class ii division i as compared to class i ; however , the difference between the two groups was not statistically significant ( p < 0.05 ) [ table 5 ] . tongue linear parameters ( mean sd ) of class i and class ii division i males the overall ( male and female ) mean tongue linear parameters of the two groups revealed higher tt / lop in class ii division i as compared to class i ; however , the difference in other parameters between the two groups was not statistically significant [ table 6 ] . tongue linear parameter levels ( meansd ) of two groups females : the hyoid bone linear and angular parameters of class i and class ii division i females are summarized in table 7 . comparing the mean hyoid bone linear parameters of females of two groups showed significantly different ( p < 0.05 ) and lower mean pog - aph in class ii division i as compared to class i ; however , other parameters did not differ significantly between the two groups [ table 7 ] . similarly , comparing the mean hyoid bone angular parameters of females of the two groups and the t - test revealed similar ( p > 0.05 ) hyoid bone angular parameters between the two groups [ table 7 ] . hyoid bone linear and angular parameters ( meansd ) of class i and class ii division i females the mean hyoid bone linear parameters of males of two groups and the t - test revealed significantly lower pog - aph ( p < 0.01 ) while significantly higher ( p < 0.05 ) a - aph and n - aph in class ii division i as compared to class i. other parameters between the two groups were found to be statistically similar [ table 8 ] . similarly , the t - test revealed ( p > 0.05 ) hyoid bone angular parameters between the two groups that did not differ significantly ( p > 0.05 ) . i and class ii division i males comparing the overall mean hyoid bone linear parameters of the two groups and the t - test revealed significantly lower aa - aph , s - aph , pog - aph , pog - aph , and pph - fh while significantly higher ( p < 0.05 or p < 0.01 ) a - aph , n - aph , and gop - pph in class ii division i as compared to class i ( p < 0.05 ) ; however , aph - fh and gop - pph did not differ between the two groups ( p > 0.05 ) [ table 9 ] . similarly , comparing the overall mean hyoid bone angular parameters of the two groups and the t - test revealed that hyoid bone angular parameters between the two groups did not differ statistically ( p > 0.05 ) [ table 10 ] . hyoid bone linear and angular parameter levels ( mean sd ) of two groups area of different parameters ( meansd ) of class i and class ii division i females the mean area of different parameters of females of the two groups and the t - test revealed that there was no statistically significant difference between the two groups . the mean area of different parameters of males of the two groups and the t - test revealed statistically significant difference ( p < 0.05 ) and higher cervical pharynx of class ii division i as compared to class i ; however , the difference in area of other parameters was not statistically significant ( p > 0.05 ) [ table 11 ] . area of different parameters ( meansd ) of class i and class ii division i malocclusion males the overall ( females and males ) area of different parameters of the two groups ( class i and class ii division i ) are summarized in table 12 . comparing the overall mean area of different parameters of the two groups and the t - test revealed statistically significant ( p < 0.05 ) difference and lower upper pharynx in class ii division i as compared to class i ; however , area of the other parameters did not differ ( p > 0.05 ) and were found to be statistically similar [ table 12 ] . females : the pharynx linear and angular parameters of two groups of females are summarized in table 1 , and the t - test revealed a significant difference between the two groups ( p < 0.05 or p < 0.001 ) and lower ho - pns and up in group 1 as compared to group 2 ; however , other parameters ( p > 0.05 ) between the two groups were found to be statistically similar [ table 1 ] . linear and angular parameters ( meansd ) of class i and class ii division i females similarly , in the two groups the difference in the pharynx angular parameters of females was statistically significant [ table 1 ] . the mean pharynx - linear parameters of males of the two groups and the t - test revealed significantly different ( p < 0.05 ) and lower aa - h4 and ad2-ptm while significantly ( p < 0.05 ) higher e - p in class ii division i as compared to class i ; however , other parameters did not differ ( p > 0.05 ) between the two groups , i.e. they were found to be statistically similar [ table 2 ] . similarly , comparing the mean pharynx angular parameters of males of class ii , the t - test revealed that they were similar ( p > 0.05 ) to the pharynx angular parameters of males between the two classes , i.e. they did not differ statistically [ table 2 ] . pharynx linear and angular parameter ( meansd ) of class i and class ii division i males the mean pharynx - linear parameters of the two groups ( females and males ) and the t - test revealed significantly ( p < 0.05 or p < 0.01 ) different and lower mean s - n , s - pns , ho - pns , aa - hy , hy - rgn , up , lp , and ad2-ptm in class ii division i subjects as compared to class i ; however , it showed no significant difference between the two groups [ table 3 ] . the overall mean pharynx - angular parameters of the two groups were similar ( p > 0.05 ) . linear parameters of the tongue in the females of the two groups showed no significant difference and the t - test revealed significantly lower ( p < 0.05 ) tgl while higher ( p < 0.01 ) tt / lop was significantly higher ( p < 0.01 ) in class ii division i as compared to class i ; however , other parameters between the two groups were found to be statistically the same [ table 4 ] . tongue linear parameters ( meansd ) of class i and class ii division i females the mean male tongue linear parameters of class ii and t - test revealed significantly different ( p < 0.05 ) and higher tt / lop in class ii division i as compared to class i ; however , the difference between the two groups was not statistically significant ( p < 0.05 ) [ table 5 ] . tongue linear parameters ( mean sd ) of class i and class ii division i males the overall ( male and female ) mean tongue linear parameters of the two groups revealed higher tt / lop in class ii division i as compared to class i ; however , the difference in other parameters between the two groups was not statistically significant [ table 6 ] . tongue linear parameter levels ( meansd ) of two groups females : the hyoid bone linear and angular parameters of class i and class ii division i females are summarized in table 7 . comparing the mean hyoid bone linear parameters of females of two groups showed significantly different ( p < 0.05 ) and lower mean pog - aph in class ii division i as compared to class i ; however , other parameters did not differ significantly between the two groups [ table 7 ] . similarly , comparing the mean hyoid bone angular parameters of females of the two groups and the t - test revealed similar ( p > 0.05 ) hyoid bone angular parameters between the two groups [ table 7 ] . hyoid bone linear and angular parameters ( meansd ) of class i and class ii division i females the mean hyoid bone linear parameters of males of two groups and the t - test revealed significantly lower pog - aph ( p < 0.01 ) while significantly higher ( p < 0.05 ) a - aph and n - aph in class ii division i as compared to class i. other parameters between the two groups were found to be statistically similar [ table 8 ] . similarly , the t - test revealed ( p > 0.05 ) hyoid bone angular parameters between the two groups that did not differ significantly ( p > 0.05 ) . hyoid bone linear and angular parameters ( meansd ) of class i and class ii division i males comparing the overall mean hyoid bone linear parameters of the two groups and the t - test revealed significantly lower aa - aph , s - aph , pog - aph , pog - aph , and pph - fh while significantly higher ( p < 0.05 or p < 0.01 ) a - aph , n - aph , and gop - pph in class ii division i as compared to class i ( p < 0.05 ) ; however , aph - fh and gop - pph did not differ between the two groups ( p > 0.05 ) [ table 9 ] . similarly , comparing the overall mean hyoid bone angular parameters of the two groups and the t - test revealed that hyoid bone angular parameters between the two groups did not differ statistically ( p > 0.05 ) [ table 10 ] . hyoid bone linear and angular parameter levels ( mean sd ) of two groups area of different parameters ( meansd ) of class i and class ii division i females the mean area of different parameters of females of the two groups and the t - test revealed that there was no statistically significant difference between the two groups . the mean area of different parameters of males of the two groups and the t - test revealed statistically significant difference ( p < 0.05 ) and higher cervical pharynx of class ii division i as compared to class i ; however , the difference in area of other parameters was not statistically significant ( p > 0.05 ) [ table 11 ] . area of different parameters ( meansd ) of class i and class ii division i malocclusion males the overall ( females and males ) area of different parameters of the two groups ( class i and class ii division i ) are summarized in table 12 . comparing the overall mean area of different parameters of the two groups and the t - test revealed statistically significant ( p < 0.05 ) difference and lower upper pharynx in class ii division i as compared to class i ; however , area of the other parameters did not differ ( p > 0.05 ) and were found to be statistically similar [ table 12 ] . area of different parameters ( meansd ) of two groups the present study compares linear and angular parameters of the pharyngeal airway , tongue , and hyoid bone between class i and class ii division i subjects based on the anb angle . roc analysis for the anb angle revealed it to be a significant predictor for skeletal sagittal differentiation into class i and class ii division i , as a criterion value of 4 in both females ( sensitivity : 100% , 95% class i = 81.3 - 100 ; specificity : 85.71% , 95% ci = 42.2 - 97.6 ) and males ( sensitivity : 100% , 95% ci = 76.7 - 100 ; specificity : 81.82% , 95% ci = 59.7 - 94 ) . overall the anb angle showed 100% sensitivity ( 95% ci = 89 - 100 ) and 82.76% specificity ( 95% ci = 64.2 - 94.1 ) with 86.5% positive predictive value for class ii and 100% negative class i predictive value . these results conform to predictive value of the anb angle at 4 for differentiating into class i and class ii division i skeletal types for the lucknow population . in this cephalometric study , comparison between class i and class ii division i females revealed similar angular pharyngeal parameters ( p > 0.05 ) [ table 1 ] and significantly different and lower ( p < 0.05 or p < 0.001 ) pharyngeal linear parameters of ho - pns and up in class ii division i as compared to class i [ table 1 ] . ceylan and oktay found no statistically significant difference between the groups based on the anb angle and nasopharyngeal size , but they found a negative correlation between the anb angle and oropharyngeal size . statistical comparison between class i and class ii division i males revealed similar ( p > 0.05 ) pharyngeal angular parameters [ table 2 ] . the linear pharyngeal parameters , aa- hy and ad2- ptm , were significantly lower ( p < 0.05 ) in class ii division i as compared to class i and the linear parameter , e - p was significantly higher ( p < 0.05 ) in class ii division i as compared to class i ; however , other linear pharyngeal parameters did not differ significantly ( p > 0.05 ) [ table 2 ] . comparing the overall ( males + females ) mean pharynx angular parameters of the two groups and the t - test revealed similar ( p > 0.05 ) pharygneal angular parameters [ table 3 ] . comparison of overall pharyngeal linear parameters revealed significantly lower ( p > 0.05 or p < 0.01 ) s - n , s - pns , ho - pns , aa - hy , hy- rgn , up , lp , and ad2- ptm in class ii division i subjects as compared to class i ; however , other parameters did not differ significantly ( p > 0.05 ) between the two groups [ table 3 ] . although significant correlations of certain linear parameters are found in this study between anterioposterior skeletal relationship and airway dimensions , the correlations are low , which is in agreement with that reported by kerr . out of a total 25 linear measurements for the pharynx , significantly lower aa - hy measurement in class ii division i males is an indicator of superiorly placed hyoid than that of class i males and can probably explain higher e - p linear values in class ii division i males in comparison to class i males . a reduced vertical ( ho - pns ) and sagittal dimension ( up ) of the nasopharynx in class ii division i females as compared to class i females is contrary to the findings of some of the previous studies and needs to be interpreted in the light of the studies that have reported no sexual dimorphism of pharyngeal dimensions in relation to dentoskeletal patterns . this may also be a reflection of the fact that the bony nasopharygeal dimension is a relatively independent variable in relation to other dimensions of facial complex . these divergent views can be explained in part by the fact that variables used to measure pharyngeal airway in the previous studies differed from those used in this investigation , which makes the comparison more difficult . the change in anterioposterior position of the mandible on the hyoid bone position and the pharyngeal airway space is well documented . although cephalometrics have been the preferred research technique , slight variation in the head position in the cephalostat , postural position of the spine , and the state of function have significant effect on the hyoid bone position . the fact that the hyoid bone is supported by the soft tissue and thus lacks hard tissue reference adds to its relative instability . regarding the setting of the head position , nakamura evaluated the head position while changing it within the range of 10 and reported that the influence of change in the head position was negligible when the change was within 5 . furthermore , fujitan et al . reported that the position of the tongue and the hyoid bone could be ascertained in a cephalogram taken with the head within the natural position between -5 and + 5 . the position and relationship of the hyoid bone were described by ioannis p adamidis and meriopi n spyropoulos in 1992 . the mean angular measures of the hyoid bone in both males and females did not differ statistically ( p > 0.05 ) between class i and class ii division i groups [ tables 7 , 8 ] . a significantly lower mean pog - aph in class ii division i females ( p < 0.05 ) as compared to class i was the only linear measurement to be statistically significant [ table 7 ] . the linear parameters to differ in the two classes of males that were significantly lower ( p < 0.01 ) pog - aph and higher ( p < 0.05 ) a - aph and n - aph statistically significant values in class ii division i males as compared to class i males [ table 8 ] . these values signify a more anteriorly placed hyoid with respect to the pogonion in both males and females of the class ii division i group and a more inferiorly placed hyoid in class ii division i males . the overall mean angular values also were not statistically different in both class i and class ii division i subjects [ table 9 ] . the linear values in the total subjects that were statistically lower ( p < 0.05 or p < 0.01 ) include s - aph , aa - aph , pog- aph and pph - fh and parameters that were statistically higher include a - aph , n - nph , and gop - pph in class ii division i as compared to class i subjects . the greater value of distances from point a , point n and gonion to the anterior part of the hyoid may be interpreted as downward displacement of the hyoid in overall class ii division i subjects as compared to class i subjects . the statistically similar angular values in males , females , and overall subjects are indicative of no anterioposterior displacement of the hyoid in any of the classes studied . tongue posture and function are of interest with regard to their relationship to malocclusion and speech defects . abnormalities of either posture and/or function could possibly contribute to the development of malocclusion and vice versa . it is possible that malocclusion and speech defects could be the causes for the abnormal posture and function of the tongue . the influence of tongue on the morphology of dental arches and on the occlusion depends not only on the lingual volume but also on its posture and on its mobility . in this study , the tip of the tongue was made radiologically evident with attachment of lead foil to it . the position and relation of the tongue were described by some of the parameters as described by allan a. lowe et al . in 1997 . in females , the linear parameter of tl was statistically lower ( p < 0.05 ) in the class ii division i group as compared to the class i group . the linear parameter of the perpendicular distance from the tt / lop had statistically higher positive values ( p < 0.01 ) in class ii division i group as compared to class i group [ table 4 ] . these values are significantly indicative of a shorter tongue length and inferiorly positioned tongue in female class ii division i subjects as compared to class i female subjects . in males , the t - test revealed a significantly higher ( p < 0.05 ) positive values for the perpendicular distance from the tt / lop in class ii division i males as compared to class i males . however , other parameters did not differ significantly ( p > 0.05 ) between class i and class ii division i males . these statistics are indicative of an inferiorly positioned tongue in class ii division i males as compared to class i males . the overall tongue linear measurements did not differ statistically except for the linear measurement of tt / lop , which was significantly positively higher ( p < 0.01 ) in the class ii division i group as compared to class i group . the inference for the tongue position and relation from these statistics are indicative of an inferiorly positioned tongue tip in relation to the lower occlusal plane in class ii division i subjects compared to class i subjects . when the tongue is forced to assume an altered position , adaptation occurs in an attempt to return to the structural and functional relationships which the patient has accepted as normal . in the pharynx , comparing the mean area of different parameters in males as well as females and overall between the two groups of class i and class ii division i subjects and the t - test revealed a similar ( p < 0.05 ) area of all parameters , i.e. they did not differ statistically except for significantly different ( p < 0.05 ) and higher hypopharyngeal area in class ii division i males as compared to class i males [ table 11 ] and significantly different ( p < 0.05 ) and lower nasopharyngeal area in total class ii division i subjects as compared to class i subjects . the higher hypopharyngeal area and the earlier finding of increased e - p linear distance in male class ii division i subjects is contrary to the findings of other studies that have reported a decrease in the dimensions of hypopharyngeal area in class ii division i subjects . this decreased nasopharyngeal area in total can be corroborated to some extent by the observation of smaller volumes of nasopharynx for class ii division i group as compared to class i group in a previous study . since significant correlations could not be identified with direct comparisons of airway areas and tongue areas , there remains a possibility for future studies to evaluate area ratio variables to verify the importance of relative interaction among the different components of the pharyngeal airway and oral airway . the result should be viewed in the light of the fact that only anterioposterior dimensions were taken into consideration . the importance of understanding the spatial interactions between the pharyngeal airway and the surrounding structures for diagnosing and treating patients with class ii division i dentofacial patterns is emphasized in this study . generally speaking , there was no difference in the pharyngeal airway anterioposterior dimension and also in the position and relationship of the hyoid bone and tongue , between class i and class ii division i patients . these findings are consistent with those studies in which the anterioposterior dimension of the upper airway is usually maintained by adaptation of both the tongue and hyoid bone . the vertical and transverse dimensions of the complex three - dimensional ( 3-d ) anatomical structures need to be evaluated and correlated to arrive at a definitive conclusion . the factors brought forth in this article provide an insight with regard to the interdependence between the geometric arrangement of the pharynx , hyoid , and tongue . given the possible clinical importance of their neuromuscular adaptations , more quantitative research on their adaptations is recommended . with the advent of 3-d computerized tomography ( ct ) scans , this diagnostic modality is recommended for better evaluation in orthodontic patients for potential assessments of 3-d craniofacial structures , like the pharynx , hyoid bone , and also the tongue position between different facial types .

objective : this study was undertaken to cephalometrically evaluate the pharyngeal airway dimension , tongue and hyoid position in subjects with normal nasorespiratory functions having different dentofacial patterns ( a - point - nasion - b - point [ anb ] > 40 and anb < 40 ) and to find if a correlation existed.materials and methods : class i and class ii division i patients were selected randomly . lateral head cephalograms were taken in normal head position within a lead foil attached to the tongue tip and a barium coating on the dorsal surface of tongue . the lateral cephalograms obtained were traced using lead acetate paper and measurements were taken . different analyses were done for the pharyngeal airways , hyoid bone , and tongue.results:the anb angle is a significant predictor for class i and class ii division i malocclusion , and the mean anb angle of class ii division i was different and higher . the overall mean pharynx and hyoid parameters were different and lower in class ii division i patients than in class i patients . the mean tongue parameter almost remained the same except for the tongue position ( tt - lop ) , which was higher in class ii division i.conclusion:in general , there was no difference either in the pharyngeal airway anterioposterior dimension or in the position and relationship of the hyoid bone and tongue , between class i and class ii division i patients . these findings are consistent with the findings in studies . anterioposterior dimension of the upper airway is usually maintained by adaptation of both the tongue and the hyoid bone . the result should be viewed in the light of the fact that only anterioposterior dimensions were taken into consideration ; the vertical and transverse dimensions of these complex anatomical structures need to have newer three - dimensional ( 3-d ) imaging technique to find if a correlation existed between them , making future studies more comprehensive .

I A M Tongue study Hyoid bone study Upper airway study Angular measurement Area measurement R Pharynx-linear measurements Tongue Hyoid bone Area Total (Females and Males) D C

gobeille and bowman , cuozzo and bowman reported variations in the position of the hyoid bone relative to the mandible within a skeletal class i context , which led to the speculation that variability in the hyoid position is partly determined by mandibular posture . according to brodie ( 1950 ) , the position of the hyoid bone relative to the cranial base and the mandible has been of interest specifically as an indicator of tongue posture and function , and has an important role in maintaining the airway and upright natural head position . clinicians generally agree on a morphogenetic role of the tongue ; thus , any alteration or change in the spatial relationships of the hyoid bone , tongue , and airway have wide functional significance and reciprocal consequences . as of yet , there are only few studies relating the position of the pharyngeal airway , the hyoid bone and tongue to different skeletal patterns using the anb angle . the present study aims to evaluate the pharyngeal airway dimension , tongue and hyoid position by the use of lateral cephalograms in subjects having anb > 4 and anb < 4 . this study was undertaken to evaluate the pharyngeal airway dimension , tongue and hyoid position by the use of lateral cephalograms in subjects with normal nasorespiratory functions having anb > 4 and anb < 4. a total of 61 subjects ( class i , n = 29 ; class ii , n = 32 ) in an age range of 11 - 19 years ( mean ) were evaluated . the sagittal skeletal differentiation of subjects with angle 's class i and class ii division i molar relation was achieved with measurement of the anb angle . a lead foil was placed on the tip of the tongue with glass ionomer cement ( gic ) mixture and a barium paste coating was done on the tonguethe radiographs obtained were traced using lead acetate paper and 4h pencil , and various landmarks for evaluation of the tongue , hyoid bone , and pharynx were identified and marked [ figure 1 ] anb analysis was done for the sagittal apical base relationship on the study samples . a lead foil was placed on the tip of the tongue with glass ionomer cement ( gic ) mixture and a barium paste coating was done on the tongue the radiographs obtained were traced using lead acetate paper and 4h pencil , and various landmarks for evaluation of the tongue , hyoid bone , and pharynx were identified and marked [ figure 1 ] anb analysis was done for the sagittal apical base relationship on the study samples . the frankfort horizontal plane and a line perpendicular to it passing through sella the horizontal measurements made were : yl ; at - aph : distance of the most anterior point of the hyoid bone ( aph ) from the most anterior point of the atlas ( at ) , y2 ; s - aph : distance of aph from the center of sella turcica ( s ) , y3 ; pog - aph : distance of the anterior point of hyoid bone ( aph ) to pogonion ( pog ) ( measured indirectly through projections of both points on the line perpendicular to fh from s ) , y4 ; pog - aph : distance measured directly from aph to pog , y5 ; a - aph : distance of aph to point a ( measured through projections of both points on the line perpendicular to fh from s ) , y6 ; n - aph : distance of aph to nasion ( n ) ( measured through projections of both points on the line perpendicular to fh from s ) . the present cephalometric study determines and compares linear and angular parameters of the pharyngeal airway , the tongue and hyoid bone in angle 's class ii division i malocclusion subject with anb > 4 with those with anb < 4 . the comparative linear and angular parameters of the pharyngeal airway , tongue , and hyoid bone in subjects with the anb angle more than 4 are summarized below respectively . the mean pharynx - linear parameters of males of the two groups and the t - test revealed significantly different ( p < 0.05 ) and lower aa - h4 and ad2-ptm while significantly ( p < 0.05 ) higher e - p in class ii division i as compared to class i ; however , other parameters did not differ ( p > 0.05 ) between the two groups , i.e. pharynx linear and angular parameter ( meansd ) of class i and class ii division i males the mean pharynx - linear parameters of the two groups ( females and males ) and the t - test revealed significantly ( p < 0.05 or p < 0.01 ) different and lower mean s - n , s - pns , ho - pns , aa - hy , hy - rgn , up , lp , and ad2-ptm in class ii division i subjects as compared to class i ; however , it showed no significant difference between the two groups [ table 3 ] . pharynx linear and angular parameters ( meansd ) of two groups linear parameters of the tongue in the females of the two groups showed no significant difference and the t - test revealed significantly lower ( p < 0.05 ) tgl while higher ( p < 0.01 ) tt / lop was significantly higher ( p < 0.01 ) in class ii division i as compared to class i ; however , other parameters between the two groups were found to be statistically the same [ table 4 ] . tongue linear parameters ( meansd ) of class i and class ii division i females the mean male tongue linear parameters of class ii and t - test revealed significantly different ( p < 0.05 ) and higher tt / lop in class ii division i as compared to class i ; however , the difference between the two groups was not statistically significant ( p < 0.05 ) [ table 5 ] . tongue linear parameters ( mean sd ) of class i and class ii division i males the overall ( male and female ) mean tongue linear parameters of the two groups revealed higher tt / lop in class ii division i as compared to class i ; however , the difference in other parameters between the two groups was not statistically significant [ table 6 ] . tongue linear parameter levels ( meansd ) of two groups females : the hyoid bone linear and angular parameters of class i and class ii division i females are summarized in table 7 . comparing the mean hyoid bone linear parameters of females of two groups showed significantly different ( p < 0.05 ) and lower mean pog - aph in class ii division i as compared to class i ; however , other parameters did not differ significantly between the two groups [ table 7 ] . hyoid bone linear and angular parameters ( meansd ) of class i and class ii division i females the mean hyoid bone linear parameters of males of two groups and the t - test revealed significantly lower pog - aph ( p < 0.01 ) while significantly higher ( p < 0.05 ) a - aph and n - aph in class ii division i as compared to class i. other parameters between the two groups were found to be statistically similar [ table 8 ] . i and class ii division i males comparing the overall mean hyoid bone linear parameters of the two groups and the t - test revealed significantly lower aa - aph , s - aph , pog - aph , pog - aph , and pph - fh while significantly higher ( p < 0.05 or p < 0.01 ) a - aph , n - aph , and gop - pph in class ii division i as compared to class i ( p < 0.05 ) ; however , aph - fh and gop - pph did not differ between the two groups ( p > 0.05 ) [ table 9 ] . hyoid bone linear and angular parameter levels ( mean sd ) of two groups area of different parameters ( meansd ) of class i and class ii division i females the mean area of different parameters of females of the two groups and the t - test revealed that there was no statistically significant difference between the two groups . the mean area of different parameters of males of the two groups and the t - test revealed statistically significant difference ( p < 0.05 ) and higher cervical pharynx of class ii division i as compared to class i ; however , the difference in area of other parameters was not statistically significant ( p > 0.05 ) [ table 11 ] . area of different parameters ( meansd ) of class i and class ii division i malocclusion males the overall ( females and males ) area of different parameters of the two groups ( class i and class ii division i ) are summarized in table 12 . comparing the overall mean area of different parameters of the two groups and the t - test revealed statistically significant ( p < 0.05 ) difference and lower upper pharynx in class ii division i as compared to class i ; however , area of the other parameters did not differ ( p > 0.05 ) and were found to be statistically similar [ table 12 ] . linear and angular parameters ( meansd ) of class i and class ii division i females similarly , in the two groups the difference in the pharynx angular parameters of females was statistically significant [ table 1 ] . the mean pharynx - linear parameters of males of the two groups and the t - test revealed significantly different ( p < 0.05 ) and lower aa - h4 and ad2-ptm while significantly ( p < 0.05 ) higher e - p in class ii division i as compared to class i ; however , other parameters did not differ ( p > 0.05 ) between the two groups , i.e. pharynx linear and angular parameter ( meansd ) of class i and class ii division i males the mean pharynx - linear parameters of the two groups ( females and males ) and the t - test revealed significantly ( p < 0.05 or p < 0.01 ) different and lower mean s - n , s - pns , ho - pns , aa - hy , hy - rgn , up , lp , and ad2-ptm in class ii division i subjects as compared to class i ; however , it showed no significant difference between the two groups [ table 3 ] . linear parameters of the tongue in the females of the two groups showed no significant difference and the t - test revealed significantly lower ( p < 0.05 ) tgl while higher ( p < 0.01 ) tt / lop was significantly higher ( p < 0.01 ) in class ii division i as compared to class i ; however , other parameters between the two groups were found to be statistically the same [ table 4 ] . tongue linear parameters ( meansd ) of class i and class ii division i females the mean male tongue linear parameters of class ii and t - test revealed significantly different ( p < 0.05 ) and higher tt / lop in class ii division i as compared to class i ; however , the difference between the two groups was not statistically significant ( p < 0.05 ) [ table 5 ] . tongue linear parameters ( mean sd ) of class i and class ii division i males the overall ( male and female ) mean tongue linear parameters of the two groups revealed higher tt / lop in class ii division i as compared to class i ; however , the difference in other parameters between the two groups was not statistically significant [ table 6 ] . tongue linear parameter levels ( meansd ) of two groups females : the hyoid bone linear and angular parameters of class i and class ii division i females are summarized in table 7 . comparing the mean hyoid bone linear parameters of females of two groups showed significantly different ( p < 0.05 ) and lower mean pog - aph in class ii division i as compared to class i ; however , other parameters did not differ significantly between the two groups [ table 7 ] . hyoid bone linear and angular parameters ( meansd ) of class i and class ii division i females the mean hyoid bone linear parameters of males of two groups and the t - test revealed significantly lower pog - aph ( p < 0.01 ) while significantly higher ( p < 0.05 ) a - aph and n - aph in class ii division i as compared to class i. other parameters between the two groups were found to be statistically similar [ table 8 ] . hyoid bone linear and angular parameters ( meansd ) of class i and class ii division i males comparing the overall mean hyoid bone linear parameters of the two groups and the t - test revealed significantly lower aa - aph , s - aph , pog - aph , pog - aph , and pph - fh while significantly higher ( p < 0.05 or p < 0.01 ) a - aph , n - aph , and gop - pph in class ii division i as compared to class i ( p < 0.05 ) ; however , aph - fh and gop - pph did not differ between the two groups ( p > 0.05 ) [ table 9 ] . hyoid bone linear and angular parameter levels ( mean sd ) of two groups area of different parameters ( meansd ) of class i and class ii division i females the mean area of different parameters of females of the two groups and the t - test revealed that there was no statistically significant difference between the two groups . the mean area of different parameters of males of the two groups and the t - test revealed statistically significant difference ( p < 0.05 ) and higher cervical pharynx of class ii division i as compared to class i ; however , the difference in area of other parameters was not statistically significant ( p > 0.05 ) [ table 11 ] . area of different parameters ( meansd ) of class i and class ii division i malocclusion males the overall ( females and males ) area of different parameters of the two groups ( class i and class ii division i ) are summarized in table 12 . comparing the overall mean area of different parameters of the two groups and the t - test revealed statistically significant ( p < 0.05 ) difference and lower upper pharynx in class ii division i as compared to class i ; however , area of the other parameters did not differ ( p > 0.05 ) and were found to be statistically similar [ table 12 ] . area of different parameters ( meansd ) of two groups the present study compares linear and angular parameters of the pharyngeal airway , tongue , and hyoid bone between class i and class ii division i subjects based on the anb angle . roc analysis for the anb angle revealed it to be a significant predictor for skeletal sagittal differentiation into class i and class ii division i , as a criterion value of 4 in both females ( sensitivity : 100% , 95% class i = 81.3 - 100 ; specificity : 85.71% , 95% ci = 42.2 - 97.6 ) and males ( sensitivity : 100% , 95% ci = 76.7 - 100 ; specificity : 81.82% , 95% ci = 59.7 - 94 ) . these results conform to predictive value of the anb angle at 4 for differentiating into class i and class ii division i skeletal types for the lucknow population . in this cephalometric study , comparison between class i and class ii division i females revealed similar angular pharyngeal parameters ( p > 0.05 ) [ table 1 ] and significantly different and lower ( p < 0.05 or p < 0.001 ) pharyngeal linear parameters of ho - pns and up in class ii division i as compared to class i [ table 1 ] . a reduced vertical ( ho - pns ) and sagittal dimension ( up ) of the nasopharynx in class ii division i females as compared to class i females is contrary to the findings of some of the previous studies and needs to be interpreted in the light of the studies that have reported no sexual dimorphism of pharyngeal dimensions in relation to dentoskeletal patterns . although cephalometrics have been the preferred research technique , slight variation in the head position in the cephalostat , postural position of the spine , and the state of function have significant effect on the hyoid bone position . reported that the position of the tongue and the hyoid bone could be ascertained in a cephalogram taken with the head within the natural position between -5 and + 5 . the mean angular measures of the hyoid bone in both males and females did not differ statistically ( p > 0.05 ) between class i and class ii division i groups [ tables 7 , 8 ] . the linear parameters to differ in the two classes of males that were significantly lower ( p < 0.01 ) pog - aph and higher ( p < 0.05 ) a - aph and n - aph statistically significant values in class ii division i males as compared to class i males [ table 8 ] . the overall mean angular values also were not statistically different in both class i and class ii division i subjects [ table 9 ] . the linear values in the total subjects that were statistically lower ( p < 0.05 or p < 0.01 ) include s - aph , aa - aph , pog- aph and pph - fh and parameters that were statistically higher include a - aph , n - nph , and gop - pph in class ii division i as compared to class i subjects . the overall tongue linear measurements did not differ statistically except for the linear measurement of tt / lop , which was significantly positively higher ( p < 0.01 ) in the class ii division i group as compared to class i group . the inference for the tongue position and relation from these statistics are indicative of an inferiorly positioned tongue tip in relation to the lower occlusal plane in class ii division i subjects compared to class i subjects . in the pharynx , comparing the mean area of different parameters in males as well as females and overall between the two groups of class i and class ii division i subjects and the t - test revealed a similar ( p < 0.05 ) area of all parameters , i.e. they did not differ statistically except for significantly different ( p < 0.05 ) and higher hypopharyngeal area in class ii division i males as compared to class i males [ table 11 ] and significantly different ( p < 0.05 ) and lower nasopharyngeal area in total class ii division i subjects as compared to class i subjects . the higher hypopharyngeal area and the earlier finding of increased e - p linear distance in male class ii division i subjects is contrary to the findings of other studies that have reported a decrease in the dimensions of hypopharyngeal area in class ii division i subjects . the result should be viewed in the light of the fact that only anterioposterior dimensions were taken into consideration . the importance of understanding the spatial interactions between the pharyngeal airway and the surrounding structures for diagnosing and treating patients with class ii division i dentofacial patterns is emphasized in this study . generally speaking , there was no difference in the pharyngeal airway anterioposterior dimension and also in the position and relationship of the hyoid bone and tongue , between class i and class ii division i patients . these findings are consistent with those studies in which the anterioposterior dimension of the upper airway is usually maintained by adaptation of both the tongue and hyoid bone . the vertical and transverse dimensions of the complex three - dimensional ( 3-d ) anatomical structures need to be evaluated and correlated to arrive at a definitive conclusion .

mental health is an important area and influences health - related quality of life ( qol ) . depression , one of the most common mental diseases , is associated with worse qol and increased mortality . recently , depression has been found to be one of determinants for cardiovascular disease through alteration of the autonomic nervous system or inflammatory cytokines , which has been reported to have an association with glaucoma . patients with visual impairment have higher probability of being depressed than subjects without eye diseases . visual disability can aggravate qol through , for example , driving limitations and fear of falling , and it can be related to depression . glaucoma is a common , chronic , progressive optic nerve disease that can cause irreversible visual impairment . a 10% to 20% prevalence of depressive symptoms has been reported in subjects with glaucoma . despite the reported association between glaucoma and depression , no objective evaluation of vision , such as visual acuity ( va ) or visual field ( vf ) , the perception of disease itself could affect the sense of well - being in glaucoma patients through the fear of future vision loss , and the potential side effects of treatment might lead to mental health symptoms . evaluating mental health status or qol in undiagnosed glaucoma patients would remove the bias caused by acknowledgement of the disease itself , but no study has evaluated both psychological health and qol in undiagnosed glaucoma patients . the korea national health and nutrition examination survey ( knhanes ) is an ongoing , nationwide , population - based study that provides data on ophthalmologic examination , psychological health , and sociodemographic factors . therefore , we used data from the knhanes to investigate the association between mental health status and qol in undiagnosed glaucoma , along with the effects of va or vf damage . this cross - sectional study was based on data from the knhanes v ( 20102012 ) , a nationwide epidemiological survey conducted by the division of chronic disease and prevention , ministry of health and welfare , with the approval of the institutional review board . the survey follows the tenets of the declaration of helsinki for biomedical research involving humans . the knhanes uses a stratified , multistage , probability sampling method based on geographical area , age , and sex . this nationally representative study is composed of a health interview survey , health examination study , and a nutrition survey . among participants in the knhanes 20102012 v , subjects 40 years or older who underwent ophthalmological examination were included . participants who had previous retinal surgery , along with those whose fundus photograph displayed geographic atrophy or signs of wet age - related macular degeneration , such as retinal pigment epithelial ( rpe ) detachment , serous detachment of the sensory retina , subretinal or sub - rpe hemorrhage , or subretinal fibrous scars , were excluded . we excluded subjects with visually significant cataract ( best corrected visual acuity [ bcva ] of better eye worse than 0.5 ) and corneal opacity . cataract was defined as lens opacities ( locs iii score is 2 ) including nuclear , cortical , posterior subcapsular , anterior capsular , or combined type compared with standard photographs , similar to previous studies . individuals diagnosed with glaucoma by their doctor and those undergoing glaucoma treatment were also excluded . the knhanes includes questionnaires to assess psychological health , including depression , sleep duration , psychological stress , and suicidal ideations . to evaluate depression , has your life been disrupted by feelings of hopelessness or sadness for more than two weeks within the past year ? participants who responded psychological stress was graded from responses , which reflect cognitive complaints on a 4-point likert scale : 1 ( low ) , 2 ( moderate ) , 3 ( severe ) , and 4 ( very severe ) . self - reported sleep duration was divided into 5 categories : 5 , 6 , 7 , 8 , and 9 hours / day . if participants replied yes to the question , during the past year , have you ever had a suicidal ideation ? the knhanes includes the euroqol , which is composed of a health - status descriptive system ( euroqol 5-dimension , eq-5d ) and the eq visual analog scale ( eq - vas ) . the eq-5d assesses the level of self - reported problems according to the 5 dimensions of mobility ( eq-1 ) , self - care ( eq-2 ) , usual activities ( eq-3 ) , pain / discomfort ( eq-4 ) , and anxiety / depression ( eq-5 ) . three answers for each dimension were possible ( no problems , some problems , severe problems ) . the subjects who answered some or severe problem were defined as having a problem in the associated eq 5-d dimension . using a combination of the 5 dimensions , a summary index ( eq-5d index ; range , 0.171 to 1 ) was calculated using the korea valuation set developed by the korean centers for disease control and prevention . the maximum score of 1 on the eq5-d index represents the best health state with no self - reported problem in any of the 5 dimensions . participants also expressed their subjective health status on a vas ranging from 0 ( the worst imaginable health state ) to 100 ( the best imaginable health state ) . the knhanes also collects data on anthropometrics and demographic characteristics , including socioeconomic status , education level , and physical activity . data on age , sex , educational level , and medical comorbidities such as diabetes mellitus and hypertension were also collected as potential confounding variables . diabetes was defined as a fasting blood glucose level > 126 mg / dl or current use of systemic antidiabetic treatment . systemic hypertension was defined as a systolic blood pressure > 160 mmhg , diastolic blood pressure > 90 mmhg , or current use of systemic antihypertensive medication . participants were divided into nondrinkers , light - to - moderate drinkers ( 130 g alcohol / day ) , and heavy drinkers ( > 30.0 g alcohol / day ) . moderate physical activity involved performance of moderate - intensity physical activity for > 20 minutes 3 or 4 times / week , with moderate intensity exercise defined as that inducing a mild elevation in breathing or heart rate for at least 10 minutes . ophthalmologists measured intraocular pressure ( iop ) using a goldmann applanation tonometer ( haag - streit , inc , bern , switzerland ) and used a topcon kr8800 autorefractor to obtain refractive error . participant va was measured at a distance of 4 m using an international standard vision chart ( jin vision chart based on the logmar scale ) based on decimal fractions . the bcva was obtained with full subjective refraction using an autorefractometer , with the pinhole applied if the bcva with autorefractometry did not improve to 0.8 . the bcva was classified into 4 categories : 0.8 to 1.0 , 0.5 to 0.63 , 0.25 to 0.4 , and no light perception to 0.2 . participants with elevated iop ( 22 mmhg ) or a glaucomatous optic disc were examined using frequency - doubling technology ( fdt , humphrey matrix ; carl zeiss meditec , inc , dublin , ca ) with the n-301 program . a glaucomatous optic disc refers to any of the following : a horizontal or vertical cup - to - disc ratio ( cdr ) 0.5 , presence of optic disc hemorrhage , presence of a retinal nerve fiber layer ( rnfl ) defect , or a violation of the is nt rule ( neuroretinal rim thickness in the order of inferior > superior > nasal > temporal ) . a glaucoma diagnosis was made based on the isgeo criteria : category 1 requires a glaucomatous vf defect with a cdr 0.7 , asymmetry of a vertical cdr 0.2 , or presence of an rnfl defect . when the vf was unavailable or unsatisfactory , subjects were included in category 2 if their vertical cdr was 0.9 , vertical cdr asymmetry was 0.3 , or if they had an rnfl defect with violation of the is nt rule . if examination of the optic disc and a vf test were not possible , category 3 required a va<3/60 and iop exceeding 21 mmhg . vf score was measured as follows : one point for vf defects of p < 0.01 , 3 points for p < 0.005 , and 5 points for vf defects indicated as not seen at maximum in 18 locations of 1 eye . abnormality score of vf was calculated providing greater weight on the location in the paracenteral vf , as brusini et al suggested . the sum of points in both eyes was used to determine glaucoma stage ( mild versus advanced ) based on the median value of the summed points . 9.2 ; sas institute , inc . ) was used for statistical analyses in order to reflect sampling weights and offer nationally representative prevalence estimates . demographic factors between subjects with and without glaucoma were compared using student t test for continuous parameters and the test for categorical variables . continuous and categorical parameters are described as mean standard error and percentage , respectively . factors that showed a difference between the 2 groups with p < 0.05 were entered into a multivariate analysis , and odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated . a p value < 0.05 was considered to indicate statistical significance . this cross - sectional study was based on data from the knhanes v ( 20102012 ) , a nationwide epidemiological survey conducted by the division of chronic disease and prevention , ministry of health and welfare , with the approval of the institutional review board . the survey follows the tenets of the declaration of helsinki for biomedical research involving humans . the knhanes uses a stratified , multistage , probability sampling method based on geographical area , age , and sex . this nationally representative study is composed of a health interview survey , health examination study , and a nutrition survey . among participants in the knhanes 20102012 v , subjects 40 years or older who underwent ophthalmological examination were included . participants who had previous retinal surgery , along with those whose fundus photograph displayed geographic atrophy or signs of wet age - related macular degeneration , such as retinal pigment epithelial ( rpe ) detachment , serous detachment of the sensory retina , subretinal or sub - rpe hemorrhage , or subretinal fibrous scars , were excluded . we excluded subjects with visually significant cataract ( best corrected visual acuity [ bcva ] of better eye worse than 0.5 ) and corneal opacity . cataract was defined as lens opacities ( locs iii score is 2 ) including nuclear , cortical , posterior subcapsular , anterior capsular , or combined type compared with standard photographs , similar to previous studies . individuals diagnosed with glaucoma by their doctor and those undergoing glaucoma treatment were also excluded . the knhanes includes questionnaires to assess psychological health , including depression , sleep duration , psychological stress , and suicidal ideations . to evaluate depression , has your life been disrupted by feelings of hopelessness or sadness for more than two weeks within the past year ? participants who responded psychological stress was graded from responses , which reflect cognitive complaints on a 4-point likert scale : 1 ( low ) , 2 ( moderate ) , 3 ( severe ) , and 4 ( very severe ) . self - reported sleep duration was divided into 5 categories : 5 , 6 , 7 , 8 , and 9 hours / day . if participants replied yes to the question , during the past year , have you ever had a suicidal ideation ? the knhanes includes the euroqol , which is composed of a health - status descriptive system ( euroqol 5-dimension , eq-5d ) and the eq visual analog scale ( eq - vas ) . the eq-5d assesses the level of self - reported problems according to the 5 dimensions of mobility ( eq-1 ) , self - care ( eq-2 ) , usual activities ( eq-3 ) , pain / discomfort ( eq-4 ) , and anxiety / depression ( eq-5 ) . three answers for each dimension were possible ( no problems , some problems , severe problems ) . the subjects who answered some or severe problem were defined as having a problem in the associated eq 5-d dimension . using a combination of the 5 dimensions , a summary index ( eq-5d index ; range , 0.171 to 1 ) was calculated using the korea valuation set developed by the korean centers for disease control and prevention . the maximum score of 1 on the eq5-d index represents the best health state with no self - reported problem in any of the 5 dimensions . participants also expressed their subjective health status on a vas ranging from 0 ( the worst imaginable health state ) to 100 ( the best imaginable health state ) . the knhanes also collects data on anthropometrics and demographic characteristics , including socioeconomic status , education level , and physical activity . data on age , sex , educational level , and medical comorbidities such as diabetes mellitus and hypertension were also collected as potential confounding variables . diabetes was defined as a fasting blood glucose level > 126 mg / dl or current use of systemic antidiabetic treatment . systemic hypertension was defined as a systolic blood pressure > 160 mmhg , diastolic blood pressure > 90 mmhg , or current use of systemic antihypertensive medication . participants were divided into nondrinkers , light - to - moderate drinkers ( 130 g alcohol / day ) , and heavy drinkers ( > 30.0 g alcohol / day ) . moderate physical activity involved performance of moderate - intensity physical activity for > 20 minutes 3 or 4 times / week , with moderate intensity exercise defined as that inducing a mild elevation in breathing or heart rate for at least 10 minutes . ophthalmologists measured intraocular pressure ( iop ) using a goldmann applanation tonometer ( haag - streit , inc , bern , switzerland ) and used a topcon kr8800 autorefractor to obtain refractive error . participant va was measured at a distance of 4 m using an international standard vision chart ( jin vision chart based on the logmar scale ) based on decimal fractions . the bcva was obtained with full subjective refraction using an autorefractometer , with the pinhole applied if the bcva with autorefractometry did not improve to 0.8 . the bcva was classified into 4 categories : 0.8 to 1.0 , 0.5 to 0.63 , 0.25 to 0.4 , and no light perception to 0.2 . participants with elevated iop ( 22 mmhg ) or a glaucomatous optic disc were examined using frequency - doubling technology ( fdt , humphrey matrix ; carl zeiss meditec , inc , dublin , ca ) with the n-301 program . a glaucomatous optic disc refers to any of the following : a horizontal or vertical cup - to - disc ratio ( cdr ) 0.5 , presence of optic disc hemorrhage , presence of a retinal nerve fiber layer ( rnfl ) defect , or a violation of the is nt rule ( neuroretinal rim thickness in the order of inferior > superior > nasal > temporal ) . a glaucoma diagnosis was made based on the isgeo criteria : category 1 requires a glaucomatous vf defect with a cdr 0.7 , asymmetry of a vertical cdr 0.2 , or presence of an rnfl defect . when the vf was unavailable or unsatisfactory , subjects were included in category 2 if their vertical cdr was 0.9 , vertical cdr asymmetry was 0.3 , or if they had an rnfl defect with violation of the is nt rule . if examination of the optic disc and a vf test were not possible , category 3 required a va<3/60 and iop exceeding 21 mmhg . vf score was measured as follows : one point for vf defects of p < 0.01 , 3 points for p < 0.005 , and 5 points for vf defects indicated as not seen at maximum in 18 locations of 1 eye . abnormality score of vf was calculated providing greater weight on the location in the paracenteral vf , as brusini et al suggested . the sum of points in both eyes was used to determine glaucoma stage ( mild versus advanced ) based on the median value of the summed points . sas software ( ver . 9.2 ; sas institute , inc . ) was used for statistical analyses in order to reflect sampling weights and offer nationally representative prevalence estimates . demographic factors between subjects with and without glaucoma were compared using student t test for continuous parameters and the test for categorical variables . continuous and categorical parameters are described as mean standard error and percentage , respectively . factors that showed a difference between the 2 groups with p < 0.05 were entered into a multivariate analysis , and odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated . among the 25,534 participants in the knhanes 20102012 v , 12,976 subjects 40 years or older were included in the present study ( figure 1 ) . among them , 12,881 individuals who underwent ophthalmological examination were selected . of these , 155 participants who had previous retinal surgery , 89 subjects with a fundus photograph showing signs of wet age - related macular degeneration or geographic atrophy , and 18 individuals diagnosed with glaucoma or undergoing glaucoma treatment were excluded . in addition , 326 subjects were excluded because of incomplete medical data . ultimately , 12,079 participants ( 5235 males , 6844 females ) were included in this study . the prevalence of undiagnosed glaucoma was 4.27% 0.25% in the study population . the mean age standard error of subjects with glaucoma was 61.47 0.70 years ( table 1 ) . participants with glaucoma were older and less educated , less likely to live with a spouse , and exercised less regularly than those without glaucoma . according to univariate analysis , male sex , lower body mass index , presence of chronic disease such as diabetes or hypertension , and lower income status baseline characteristics table 2 shows the univariate associations between glaucoma and psychological health and qol parameters . glaucoma is related to short ( 5 h ) or long ( 9 h ) sleep duration ( p = 0.041 ) . suicidal ideation was greater in subjects with glaucoma than in those without glaucoma ( p = 0.005 ) . depression diagnosed by a doctor did not differ significantly between individuals with and without glaucoma . however , glaucoma was associated with depressive mood ( 2 weeks in a row ) ( p = 0.037 ) the proportion of anxiety / depression symptoms reported on the eq-5 was higher in subjects with glaucoma than in those without glaucoma ( p < 0.001 ) . the glaucoma group also presented a higher proportion of those who reported some or severe problems in all 5 dimensions of the eq-5 in comparison with the non - glaucoma group . the eq-5d index and the subjective health status reported by the eq - vas were lower in the subjects with glaucoma than in those without glaucoma . association between glaucoma and psychological health , health - related quality of life table 3 presents the differences in psychological health and qol between the subjects with and without glaucoma after adjusting for age ( model 1 ) and age , sex , body mass index , diabetes , hypertension , income status , education level , marital status , and regular exercise ( model 2 : demographic factors with p value < 0.05 in univariate analyses ) . in both models 1 and 2 , glaucoma subjects were more likely than those without glaucoma to have some or severe problems with anxiety / depression ( model 1 : or , 1.71 ; 95% ci 1.232.39 , model 2 : or 1.77 ; 95% ci , 1.262.49 ) . the multivariate or for the lowest quartile of eq - vas in the glaucoma group versus the nonglaucoma group was 1.55 ( 95% ci 1.211.98 ) in model 1 and 1.52 ( 95% ci 1.191.96 ) in model 2 . adjusted odds on multivariate analysis of the association between glaucoma and psychological health , health - related quality of life table 4 shows the results of subgroup analysis in individuals with glaucoma with regard to psychological health and qol according to bcva . as bcva decreased , the eq-5d index score also decreased ( p = 0.021 ) . subjects with lower bcva were more likely to have some or severe problems with anxiety / depression in the subgroup with glaucoma , but the relationship between them was not statistically significant ( p = 0.154 ) . subjects with points 0 and those with > 0 did not differ significantly in eq - vas or eq-5d ( p = 0.357 , 0.802 , respectively ) . eq-5 ( anxiety / depression ) was not significantly correlated with abnormality score of vf defects ( p = 0.784 ) . relationship of visual acuity and visual field damage and health - related quality of life in subjects with undiagnosed glaucoma we present the first report regarding both psychological health and qol in people with undiagnosed glaucoma . subjects with glaucoma showed greater odds of having some or severe problems with anxiety / depression . in addition , glaucoma can lower qol in patients even when they are unaware they have the disease . one previous hospital - based study showed that the prevalence of depressive symptoms did not differ according to the presence of glaucoma . however , a growing body of evidence , including a population - based study , suggests that glaucoma patients are more likely to be depressed than subjects without glaucoma . the us national health and nutrition examination survey reported that glaucoma is significantly associated with depression after adjusting for demographic factors . no large , cross - sectional studies have investigated the association between glaucoma and depression in asian countries . in this study , we discovered no significant difference between subjects with and without glaucoma in depression diagnosed by a doctor . however , glaucoma subjects were more likely to have self - reported depressive mood for 2 weeks ( p = 0.037 ) . a higher proportion of subjects with glaucoma ( 20.4% ) than without glaucoma ( 11.7% , p < 0.001 ) reported symptoms of some or severe anxiety / depression on the eq-5 . in multivariate analysis , only eq-5d ( anxiety / depression ) remained a significant correlation with glaucoma . therefore , depressive mood was more common in subjects with glaucoma , but the degree or duration of depressive symptoms was not severe enough to diagnose a major depressive disorder . when previous studies analyzed the relationships between glaucoma and depressive symptoms , they used different criteria for depressive mood . wang et al found that the prevalence of depression diagnosed by the diagnostic and statistical manual of mental disorders ( dsm - iv ) was 10.9% in glaucoma patients and 6.9% in subjects without glaucoma ( p = 0.02 ) . they used self - reported glaucoma to investigate the relationship between glaucoma and depression , which could explain the discrepancy with the results of our study . in our study , subjects with glaucoma undiagnosed by a doctor may have been free of the perception of the disease and the fear of the potential side effects of treatment . therefore , they might have experienced depressive symptoms that were not serious enough to warrant a depression diagnosis . the depressive symptoms were simply categorized for the subjects who answered some or severe problem for anxiety / depression ( eq-5 ) in this study . self - report instruments for common mental disorders include the patient health questionnaire , the center for epidemiological studies depression ( ces - d ) scale , beck depression inventory , and state - trait anxiety inventory . detailed grading for anxiety / depression such as state - trait anxiety inventory could not be performed in this study . in addition , it was not clear whether anxiety / depression is a temporary condition to symptom or a general tendency in subjects with undiagnosed glaucoma . it could be one of limitations that quantitative or qualitative evaluation for depression was not possible in this study , even though a brief screening questionnaire needed a shorter time . in addition , the information on mental health and qol came from self - reported questionnaires , so reporting bias is possible . for glaucoma patients , fear of blindness and the potential adverse effects of treatment can lead to depression . previous reports on the association of glaucoma with mental health status have concentrated on glaucoma patients with preexisting disease . jampel et al found that depressive mood was related to patient perceptions of worsening visual function but not to poor va or worse vf in newly diagnosed glaucoma patients . that study eliminated the effects of treatment itself because patients were not informed about their treatment at the time of the qol interview ; however , it could not adjust for the effects of perception of disease . our study , which excludes the effects of fear about the illness , corresponded to their study : the va and vf scores did not correlate with depressive symptoms . even without knowing about the disease itself , subjectively perceived poorer vision seems to be more closely related to depressive mood than the objective evaluation of vision . glaucoma influences qol in a degree similar to other chronic diseases such as dementia and diabetes . patients with glaucoma in a cross - sectional study acknowledged that their qol was decreased compared with subjects without glaucoma . in our study , subjects with glaucoma reported more problems with anxiety / depression on the eq-5d . to evaluate qol in glaucoma patients , general health - related , vision - specific , and glaucoma - specific instruments can be used . as general health - related questionnaires , eq-5d or short - form health surveys ( sf-36 ) have been widely used : as vision - specific measurements , national eye institute visual functioning questionnaire ( nei - vfq ) or activities of daily vision scale ( advs ) , visual function index ( vf-14 ) : as glaucoma - specific instruments , glaucoma quality of life ( gql)-15 , or symptom impact glaucoma ( sig ) . disease - specific instruments can focus on the issues of particular concern such as vision with more responsiveness ( sensitive to change ) . general qol instruments lack the responsiveness of the disease - specific instruments and the information focused on visual function . among glaucoma - specific instruments , gql-15 revealed that the most problematic activities for glaucoma patients were those associated with dark adaptation . those ocular dependent factors were not evaluated in general qol instruments such as the euro qol . that the euro qol does not assess vision - specific function was one of limitations of this study . however , valid and generic questionnaires may work even better with multidimensional evaluation of qol . combination of generic and disease - specific instruments may be a good approach to evaluate health - related qol in further studies . those results correspond with a previous hospital - based study that found that the eq-5d index was correlated with va in glaucoma patients . several studies reported that the severity of vf defects was linked to impaired qol or opposite results . the different study findings may result from dissimilar strategy used for grading qol or the proportion of individuals with advanced glaucoma , or the type of vf tests . in our study , fdt was used to evaluate vf damage , whereas other reports employed standard automated perimetry . using standard automated perimetry , visual field index was correlated with gql-15 , one of qol assessment tools , not corresponding to our findings . the visual field index is primarily based on the pattern deviation maps in eyes with a mean deviation better than 20 db with weighting toward the central areas of the vf , leading to be less affected by media opacities . n-301 program with a scoring algorithm in frequency doubling technology is not ideal for evaluating glaucoma stage and may be one of limitations of our study , even though that grading system has been found to be highly specific for glaucoma detection . a growing body of evidence shows that depression increases the risk of coronary artery disease to at least 1.5 times that of healthy subjects . depression is associated with autonomic dysfunction with increased sympathetic tone or endothelial dysfunction and inflammation . the relationship between undiagnosed glaucoma and depressive symptoms may also suggest a possibility that depression may increase the risk of glaucoma development . however , we can not confirm a cause and effect relationship from the present study because our cross - sectional study revealed only the association between glaucoma and depression . the strength of this study is that a population - based study is representative of individuals with undiagnosed glaucoma in the general population . the knhanes includes information on socioeconomic status and demographic factors that contribute to psychological health , such as income status , education level , marital status , and daily sun exposure . therefore , we could adjust all those factors to assess the association between glaucoma and mental health . in conclusion , individuals who had undiagnosed glaucoma might be more depressed than those without glaucoma , even though the degree of depression is not sufficient to diagnose depression . therefore , when initially diagnosing glaucoma , acknowledging that glaucoma patients might already have depressive symptoms could be helpful in treatment and follow - up .

abstractglaucoma is a leading cause of irreversible vision loss . mental health and quality of life ( qol ) are important issues for patients with glaucoma because visual impairment can be related to those . analysis of mental health status or qol in undiagnosed glaucoma patients can be free of the bias caused by awareness of the disease itself . in this study , the association between mental health status or qol and undiagnosed glaucoma , along with the effects of visual acuity or visual field damage was investigated . among individuals in the korea national health and nutrition examination survey ( knhanes ) v ( 20102012 ) , subjects 40 years or older were included . knhanes is a cross - sectional study using a stratified , multistage , probability sampling survey . the knhanes includes questionnaires to assess psychological health , including depression , sleep duration , psychological stress , and suicidal ideations . to evaluate qol , the knhanes includes the euroqol , which is composed of a health - status descriptive system ( euroqol 5-dimension , eq-5d ) and the eq visual analog scale ( eq - vas ) . the proportion of people reporting symptoms of some or severe problems in all 5 dimensions of the euroqol-5 instrument , including anxiety / depression , was higher in the glaucoma group than in the nonglaucoma group . the subjective health status reported by the eq - vas was lower in the subjects with glaucoma than in those without glaucoma . suicidal ideation was greater in subjects with glaucoma than in those without glaucoma ( p = 0.005 ) . after adjustment for demographic factors , glaucoma subjects were more likely than those without glaucoma to have some or severe problems with anxiety / depression ( odds ratio 1.77 ; 95% confidence interval , 1.262.49 ) . worse best corrected visual acuity was associated with more problems with a lower eq-5d score in glaucoma subjects . individuals who had undiagnosed glaucoma were more depressed than those without glaucoma . qol can be affected by glaucoma , especially in patients with worse visual acuity .

INTRODUCTION METHODS Study Population Outcome Variables Ophthalmological Evaluation Statistical Analyses RESULTS DISCUSSION

mental health is an important area and influences health - related quality of life ( qol ) . depression , one of the most common mental diseases , is associated with worse qol and increased mortality . recently , depression has been found to be one of determinants for cardiovascular disease through alteration of the autonomic nervous system or inflammatory cytokines , which has been reported to have an association with glaucoma . patients with visual impairment have higher probability of being depressed than subjects without eye diseases . visual disability can aggravate qol through , for example , driving limitations and fear of falling , and it can be related to depression . glaucoma is a common , chronic , progressive optic nerve disease that can cause irreversible visual impairment . a 10% to 20% prevalence of depressive symptoms has been reported in subjects with glaucoma . despite the reported association between glaucoma and depression , no objective evaluation of vision , such as visual acuity ( va ) or visual field ( vf ) , the perception of disease itself could affect the sense of well - being in glaucoma patients through the fear of future vision loss , and the potential side effects of treatment might lead to mental health symptoms . evaluating mental health status or qol in undiagnosed glaucoma patients would remove the bias caused by acknowledgement of the disease itself , but no study has evaluated both psychological health and qol in undiagnosed glaucoma patients . the korea national health and nutrition examination survey ( knhanes ) is an ongoing , nationwide , population - based study that provides data on ophthalmologic examination , psychological health , and sociodemographic factors . therefore , we used data from the knhanes to investigate the association between mental health status and qol in undiagnosed glaucoma , along with the effects of va or vf damage . this cross - sectional study was based on data from the knhanes v ( 20102012 ) , a nationwide epidemiological survey conducted by the division of chronic disease and prevention , ministry of health and welfare , with the approval of the institutional review board . the knhanes uses a stratified , multistage , probability sampling method based on geographical area , age , and sex . this nationally representative study is composed of a health interview survey , health examination study , and a nutrition survey . among participants in the knhanes 20102012 v , subjects 40 years or older who underwent ophthalmological examination were included . participants who had previous retinal surgery , along with those whose fundus photograph displayed geographic atrophy or signs of wet age - related macular degeneration , such as retinal pigment epithelial ( rpe ) detachment , serous detachment of the sensory retina , subretinal or sub - rpe hemorrhage , or subretinal fibrous scars , were excluded . we excluded subjects with visually significant cataract ( best corrected visual acuity [ bcva ] of better eye worse than 0.5 ) and corneal opacity . the knhanes includes questionnaires to assess psychological health , including depression , sleep duration , psychological stress , and suicidal ideations . participants who responded psychological stress was graded from responses , which reflect cognitive complaints on a 4-point likert scale : 1 ( low ) , 2 ( moderate ) , 3 ( severe ) , and 4 ( very severe ) . the knhanes includes the euroqol , which is composed of a health - status descriptive system ( euroqol 5-dimension , eq-5d ) and the eq visual analog scale ( eq - vas ) . the eq-5d assesses the level of self - reported problems according to the 5 dimensions of mobility ( eq-1 ) , self - care ( eq-2 ) , usual activities ( eq-3 ) , pain / discomfort ( eq-4 ) , and anxiety / depression ( eq-5 ) . the subjects who answered some or severe problem were defined as having a problem in the associated eq 5-d dimension . using a combination of the 5 dimensions , a summary index ( eq-5d index ; range , 0.171 to 1 ) was calculated using the korea valuation set developed by the korean centers for disease control and prevention . the maximum score of 1 on the eq5-d index represents the best health state with no self - reported problem in any of the 5 dimensions . participants also expressed their subjective health status on a vas ranging from 0 ( the worst imaginable health state ) to 100 ( the best imaginable health state ) . the knhanes also collects data on anthropometrics and demographic characteristics , including socioeconomic status , education level , and physical activity . a glaucomatous optic disc refers to any of the following : a horizontal or vertical cup - to - disc ratio ( cdr ) 0.5 , presence of optic disc hemorrhage , presence of a retinal nerve fiber layer ( rnfl ) defect , or a violation of the is nt rule ( neuroretinal rim thickness in the order of inferior > superior > nasal > temporal ) . when the vf was unavailable or unsatisfactory , subjects were included in category 2 if their vertical cdr was 0.9 , vertical cdr asymmetry was 0.3 , or if they had an rnfl defect with violation of the is nt rule . demographic factors between subjects with and without glaucoma were compared using student t test for continuous parameters and the test for categorical variables . factors that showed a difference between the 2 groups with p < 0.05 were entered into a multivariate analysis , and odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated . this cross - sectional study was based on data from the knhanes v ( 20102012 ) , a nationwide epidemiological survey conducted by the division of chronic disease and prevention , ministry of health and welfare , with the approval of the institutional review board . the knhanes uses a stratified , multistage , probability sampling method based on geographical area , age , and sex . this nationally representative study is composed of a health interview survey , health examination study , and a nutrition survey . among participants in the knhanes 20102012 v , subjects 40 years or older who underwent ophthalmological examination were included . participants who had previous retinal surgery , along with those whose fundus photograph displayed geographic atrophy or signs of wet age - related macular degeneration , such as retinal pigment epithelial ( rpe ) detachment , serous detachment of the sensory retina , subretinal or sub - rpe hemorrhage , or subretinal fibrous scars , were excluded . we excluded subjects with visually significant cataract ( best corrected visual acuity [ bcva ] of better eye worse than 0.5 ) and corneal opacity . the knhanes includes questionnaires to assess psychological health , including depression , sleep duration , psychological stress , and suicidal ideations . participants who responded psychological stress was graded from responses , which reflect cognitive complaints on a 4-point likert scale : 1 ( low ) , 2 ( moderate ) , 3 ( severe ) , and 4 ( very severe ) . self - reported sleep duration was divided into 5 categories : 5 , 6 , 7 , 8 , and 9 hours / day . the knhanes includes the euroqol , which is composed of a health - status descriptive system ( euroqol 5-dimension , eq-5d ) and the eq visual analog scale ( eq - vas ) . the eq-5d assesses the level of self - reported problems according to the 5 dimensions of mobility ( eq-1 ) , self - care ( eq-2 ) , usual activities ( eq-3 ) , pain / discomfort ( eq-4 ) , and anxiety / depression ( eq-5 ) . the subjects who answered some or severe problem were defined as having a problem in the associated eq 5-d dimension . using a combination of the 5 dimensions , a summary index ( eq-5d index ; range , 0.171 to 1 ) was calculated using the korea valuation set developed by the korean centers for disease control and prevention . the knhanes also collects data on anthropometrics and demographic characteristics , including socioeconomic status , education level , and physical activity . a glaucomatous optic disc refers to any of the following : a horizontal or vertical cup - to - disc ratio ( cdr ) 0.5 , presence of optic disc hemorrhage , presence of a retinal nerve fiber layer ( rnfl ) defect , or a violation of the is nt rule ( neuroretinal rim thickness in the order of inferior > superior > nasal > temporal ) . when the vf was unavailable or unsatisfactory , subjects were included in category 2 if their vertical cdr was 0.9 , vertical cdr asymmetry was 0.3 , or if they had an rnfl defect with violation of the is nt rule . demographic factors between subjects with and without glaucoma were compared using student t test for continuous parameters and the test for categorical variables . factors that showed a difference between the 2 groups with p < 0.05 were entered into a multivariate analysis , and odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated . among the 25,534 participants in the knhanes 20102012 v , 12,976 subjects 40 years or older were included in the present study ( figure 1 ) . of these , 155 participants who had previous retinal surgery , 89 subjects with a fundus photograph showing signs of wet age - related macular degeneration or geographic atrophy , and 18 individuals diagnosed with glaucoma or undergoing glaucoma treatment were excluded . ultimately , 12,079 participants ( 5235 males , 6844 females ) were included in this study . the mean age standard error of subjects with glaucoma was 61.47 0.70 years ( table 1 ) . participants with glaucoma were older and less educated , less likely to live with a spouse , and exercised less regularly than those without glaucoma . according to univariate analysis , male sex , lower body mass index , presence of chronic disease such as diabetes or hypertension , and lower income status baseline characteristics table 2 shows the univariate associations between glaucoma and psychological health and qol parameters . glaucoma is related to short ( 5 h ) or long ( 9 h ) sleep duration ( p = 0.041 ) . suicidal ideation was greater in subjects with glaucoma than in those without glaucoma ( p = 0.005 ) . however , glaucoma was associated with depressive mood ( 2 weeks in a row ) ( p = 0.037 ) the proportion of anxiety / depression symptoms reported on the eq-5 was higher in subjects with glaucoma than in those without glaucoma ( p < 0.001 ) . the glaucoma group also presented a higher proportion of those who reported some or severe problems in all 5 dimensions of the eq-5 in comparison with the non - glaucoma group . the eq-5d index and the subjective health status reported by the eq - vas were lower in the subjects with glaucoma than in those without glaucoma . association between glaucoma and psychological health , health - related quality of life table 3 presents the differences in psychological health and qol between the subjects with and without glaucoma after adjusting for age ( model 1 ) and age , sex , body mass index , diabetes , hypertension , income status , education level , marital status , and regular exercise ( model 2 : demographic factors with p value < 0.05 in univariate analyses ) . in both models 1 and 2 , glaucoma subjects were more likely than those without glaucoma to have some or severe problems with anxiety / depression ( model 1 : or , 1.71 ; 95% ci 1.232.39 , model 2 : or 1.77 ; 95% ci , 1.262.49 ) . the multivariate or for the lowest quartile of eq - vas in the glaucoma group versus the nonglaucoma group was 1.55 ( 95% ci 1.211.98 ) in model 1 and 1.52 ( 95% ci 1.191.96 ) in model 2 . adjusted odds on multivariate analysis of the association between glaucoma and psychological health , health - related quality of life table 4 shows the results of subgroup analysis in individuals with glaucoma with regard to psychological health and qol according to bcva . as bcva decreased , the eq-5d index score also decreased ( p = 0.021 ) . subjects with lower bcva were more likely to have some or severe problems with anxiety / depression in the subgroup with glaucoma , but the relationship between them was not statistically significant ( p = 0.154 ) . subjects with points 0 and those with > 0 did not differ significantly in eq - vas or eq-5d ( p = 0.357 , 0.802 , respectively ) . eq-5 ( anxiety / depression ) was not significantly correlated with abnormality score of vf defects ( p = 0.784 ) . relationship of visual acuity and visual field damage and health - related quality of life in subjects with undiagnosed glaucoma we present the first report regarding both psychological health and qol in people with undiagnosed glaucoma . subjects with glaucoma showed greater odds of having some or severe problems with anxiety / depression . in addition , glaucoma can lower qol in patients even when they are unaware they have the disease . however , a growing body of evidence , including a population - based study , suggests that glaucoma patients are more likely to be depressed than subjects without glaucoma . the us national health and nutrition examination survey reported that glaucoma is significantly associated with depression after adjusting for demographic factors . no large , cross - sectional studies have investigated the association between glaucoma and depression in asian countries . in this study , we discovered no significant difference between subjects with and without glaucoma in depression diagnosed by a doctor . however , glaucoma subjects were more likely to have self - reported depressive mood for 2 weeks ( p = 0.037 ) . a higher proportion of subjects with glaucoma ( 20.4% ) than without glaucoma ( 11.7% , p < 0.001 ) reported symptoms of some or severe anxiety / depression on the eq-5 . in multivariate analysis , only eq-5d ( anxiety / depression ) remained a significant correlation with glaucoma . therefore , depressive mood was more common in subjects with glaucoma , but the degree or duration of depressive symptoms was not severe enough to diagnose a major depressive disorder . wang et al found that the prevalence of depression diagnosed by the diagnostic and statistical manual of mental disorders ( dsm - iv ) was 10.9% in glaucoma patients and 6.9% in subjects without glaucoma ( p = 0.02 ) . they used self - reported glaucoma to investigate the relationship between glaucoma and depression , which could explain the discrepancy with the results of our study . in our study , subjects with glaucoma undiagnosed by a doctor may have been free of the perception of the disease and the fear of the potential side effects of treatment . the depressive symptoms were simply categorized for the subjects who answered some or severe problem for anxiety / depression ( eq-5 ) in this study . self - report instruments for common mental disorders include the patient health questionnaire , the center for epidemiological studies depression ( ces - d ) scale , beck depression inventory , and state - trait anxiety inventory . detailed grading for anxiety / depression such as state - trait anxiety inventory could not be performed in this study . in addition , it was not clear whether anxiety / depression is a temporary condition to symptom or a general tendency in subjects with undiagnosed glaucoma . it could be one of limitations that quantitative or qualitative evaluation for depression was not possible in this study , even though a brief screening questionnaire needed a shorter time . in addition , the information on mental health and qol came from self - reported questionnaires , so reporting bias is possible . for glaucoma patients , fear of blindness and the potential adverse effects of treatment can lead to depression . previous reports on the association of glaucoma with mental health status have concentrated on glaucoma patients with preexisting disease . that study eliminated the effects of treatment itself because patients were not informed about their treatment at the time of the qol interview ; however , it could not adjust for the effects of perception of disease . our study , which excludes the effects of fear about the illness , corresponded to their study : the va and vf scores did not correlate with depressive symptoms . even without knowing about the disease itself , subjectively perceived poorer vision seems to be more closely related to depressive mood than the objective evaluation of vision . patients with glaucoma in a cross - sectional study acknowledged that their qol was decreased compared with subjects without glaucoma . in our study , subjects with glaucoma reported more problems with anxiety / depression on the eq-5d . to evaluate qol in glaucoma patients , general health - related , vision - specific , and glaucoma - specific instruments can be used . as general health - related questionnaires , eq-5d or short - form health surveys ( sf-36 ) have been widely used : as vision - specific measurements , national eye institute visual functioning questionnaire ( nei - vfq ) or activities of daily vision scale ( advs ) , visual function index ( vf-14 ) : as glaucoma - specific instruments , glaucoma quality of life ( gql)-15 , or symptom impact glaucoma ( sig ) . general qol instruments lack the responsiveness of the disease - specific instruments and the information focused on visual function . combination of generic and disease - specific instruments may be a good approach to evaluate health - related qol in further studies . those results correspond with a previous hospital - based study that found that the eq-5d index was correlated with va in glaucoma patients . the different study findings may result from dissimilar strategy used for grading qol or the proportion of individuals with advanced glaucoma , or the type of vf tests . the visual field index is primarily based on the pattern deviation maps in eyes with a mean deviation better than 20 db with weighting toward the central areas of the vf , leading to be less affected by media opacities . however , we can not confirm a cause and effect relationship from the present study because our cross - sectional study revealed only the association between glaucoma and depression . the strength of this study is that a population - based study is representative of individuals with undiagnosed glaucoma in the general population . the knhanes includes information on socioeconomic status and demographic factors that contribute to psychological health , such as income status , education level , marital status , and daily sun exposure . therefore , we could adjust all those factors to assess the association between glaucoma and mental health . in conclusion , individuals who had undiagnosed glaucoma might be more depressed than those without glaucoma , even though the degree of depression is not sufficient to diagnose depression .

it is curious , began a letter to the lancet published in 1985 , that , in a language as descriptively rich as english , no clear distinction is made between a spontaneous and an induced expulsion of the contents of the uterus in early pregnancy. the communication , printed under the heading miscarriage or abortion? , came from a group at st mary 's hospital london led by richard beard , then professor of obstetrics and gynaecology.1 it continued : regardless of whether it was a spontaneous or induced event , yet our patients always speak of ' miscarriages unless they have had a termination of pregnancy . it seems likely that the words have been interchangeable for many centuries ... beard et al described the offence caused to those women who miscarry by the use of the word abortion to describe their condition. they appealed to doctors and all health professionals to start using the word miscarriage rather than abortion for a spontaneous pregnancy loss before 28 weeks of pregnancy. seven years later psychologist beverly chalmers asserted that publications in the british journal of obstetrics and gynaecology have taken heed. she observed a change in terminology used before and after the editorial ( sic ) by beard et al.2 in a 1997 british medical journal ( bmj ) editorial emeritus professor of obstetrics geoffrey chamberlain also credited richard beard with moving clinicians towards a more empathetic practice.3 if the terms miscarriage and abortion had , among doctors , been interchangeable for many centuries as beard and his colleagues suggested , why did they call for change in 1985 ? what factors , other than beard 's lancet letter , might explain a change in the medical language of early pregnancy loss ? with these questions in mind , i hand - searched the lancet , bmj and british journal of obstetrics and gynaecology for indexed references to miscarriage , abortion , spontaneous abortion and early pregnancy loss between the years 1960 and 2010 . these references were reviewed and coded in an effort to chart the changing use of terminology and understand those changes in the context of changing medical knowledge and clinical practice . i also obtained and reviewed consecutive editions of the seminal ten teachers textbooks of obstetrics and of gynaecology ( first published in london respectively as midwifery in 1917 and as diseases of women in 1919 and in print ever since ) . the 10th ( 1961 ) to 14th ( 1985 ) editions of obstetrics ( nee midwifery ) , and the 9th ( 1953 ) to 16th ( 1995 ) editions of gynaecology ( nee diseases of women ) were used to gain insight into established textbook knowledge and attitudes available to students and clinicians over time . my interpretation of these sources has been influenced by my experiences of working as a general practitioner , as a doctor in an emergency gynaecology department , and as a researcher conducting a clinical study of early pregnancy loss as well as by my readings of feminist accounts of abortion law,4 5 ultrasound,6 the medicalisation of pregnancy7 8 and of pregnancy loss.9 10 in 1992 , beverly chalmers perceived the impact of beard 's miscarriage or abortion? lancet letter upon papers published in the british journal of obstetrics and gynaecology.2 she had reviewed the titles of articles on the subject of early pregnancy loss at the beginning and end of the 1980s and found that whereas almost all the papers at the start of the decade used the term abortion in their titles , none of those published later did so. this trend can be analysed in greater detail . the graph in figure 1 plots the annual incidence of articles published on the subject of early pregnancy loss in the british journal of obstetrics and gynaecology from 1975 to 1999 ; it differentiates between those using abortion and miscarriage in their titles . around 1986 , a definitive flip occurred in the titles of articles referring to early pregnancy loss : from the exclusive use of . annual incidence of article titles using abortion and miscarriage to refer to early pregnancy loss in the british journal of obstetrics and gynaecology between 1975 and 1999 . spontaneous abortion in article titles referring to early pregnancy loss declined slowly between 1985 and 1995 ( see figure 2 ) : thereafter miscarriage is always employed . annual incidence of article titles using abortion and miscarriage to refer to early pregnancy loss in the bmj between 1975 and 2010 . in the lancet a shift in the terminology of early pregnancy loss is less obvious , but a divergence between the incidences of abortion and miscarriage becomes apparent after 1985 ( see figure 3 ) . annual incidence of article titles using abortion and miscarriage to refer to early pregnancy loss in the lancet between 1975 and 2010 . in the bmj and lancet , the gradual change in published article titles suggests a gradual shift in terminology used by contributors , rather than deliberate editorial policy on the part of the journals . closer inspection of published articles supports this conclusion of variable and slow - changing use of terms among doctors . in 1987 , a paper on management of threatened miscarriage published in the bmj consistently uses the term miscarriage throughout;11 yet while some correspondents responding to the article used only miscarriage in their letters,12 others used the term exclusively.13 similarly , in 1991 the bmj published an editorial titled recurrent miscarriage14 to which two correspondents responded : one letter headed recurrent miscarriage;15 the other , recurrent abortion.16 medical journals in fact appear slow to have acknowledged the changing use of language by their readers : miscarriage appears for the first time in the index of the bmj in 1978 , and until 1999 readers looking under as late as 1995 , the ten teachers ( who by then numbered two women alongside eight male contributors ) , in the 16th edition of their gynaecology text , continued , as in earlier editions , to refer to early pregnancy loss as abortion. they began the chapter on this subject by asserting the terms abortion and miscarriage are synonymous. they continued to use the terms interchangeably in their text , but conceded generally miscarriage carries connotations of spontaneous loss and is best used when talking to women. the actual clinical language used in conversation between doctors and patients at this time is not recorded . however , in 1988 one doctor felt compelled to write to the bmj , advising that : abortion was discussed during a recent phone in programme on bbc radio derby . a number of women listeners expressed their objection to the term abortion when it referred to the spontaneous event . they were unanimous in their wish that the terms miscarriage should be used instead ... a number of women listeners expressed their objection to the term abortion when it referred to the spontaneous event . they were unanimous in their wish that the terms miscarriage should be used instead ... 17 evidently , the shift in medical terminology occurred slowly among doctors as represented by the authors of journal articles , correspondence and textbooks . the following sections consider some of the factors apparent in the medical literature of the time that may have prompted richard beard to call for change in 1985 and that would have enabled a change in medical terminology . before the 1967 abortion act eased legal impediments to ending an unwanted pregnancy , the distinction between spontaneous and induced abortion alluded to in medical journals and textbooks of the time remained academic . although many abortions were carried out illegally , women who developed problems afterwards would rarely disclose their procedure for fear of criminalising themselves . meanwhile , clinicians would not normally have been able to distinguish between spontaneous and induced abortions , despite their recognition that many cases of early pregnancy bleeding ( particularly instances of septic abortion , in which pregnancy loss was complicated by infection ) were accounted for by ill - managed illegal abortion attempts . in 1961 an article in the bmj observed that : it is notoriously difficult to obtain reliable histories from patients suffering from septic abortion , and it is often suspected that criminal interference , which the patient will not acknowledge , has taken place.18 it is notoriously difficult to obtain reliable histories from patients suffering from septic abortion , and it is often suspected that criminal interference , which the patient will not acknowledge , has taken place.18 a 1964 bmj article on the subject of bleeding in early pregnancy spoke of criminal interference that such interference is so often denied , even when it is obvious , that many doctors do not inquire about the possibility.19 textbooks of the era exhibit similar suspicions . in 1966 , the year before the abortion act was passed , the 11th edition of obstetrics by ten teachers advised doctors on how they might discern an induced abortion when , presumably , it was not admitted by the patient : the presence of abrasions or lacerations in the genital tract ... strongly suggests criminal interference. nonetheless , criminal abortion was listed alongside other presumptive , but apparently indeterminable , causes of abortion that were to a certain extent , theoretical and arbitrary. these included : reflex stimulation of the uterus by emotional disturbances and coitus during the first 3 months of pregnancy. before 1967 , statutes relating to pregnancy termination prevented doctors discerning abortions that were the clinical varieties of abortion had long been distinguished and systematised in medical textbooks . with the aid of illustrative line drawings and monochrome photographs of pathological specimens , consecutive editions of the ten teachers texts had elaborated an inventory of : threatened , inevitable , complete , incomplete , septic , missed and habitual abortions . since they were first described , the difficulty of distinguishing these clinical varieties in practice had remained unchanged . before ultrasound technologies permitted clinicians to look inside the wombs of women experiencing pain or bleeding in early pregnancy , it was impossible to discern the varieties of abortion and distinguish these from a potentially life threatening tubal ( ectopic ) pregnancy . often the only effective diagnostic method was hospital admission and a prolonged period of observation . the pathological specimens featured in textbooks of this era attested to an impasse : until the uterus expelled its content , or emergency surgery was carried out , or the mother died , it might be impossible to discern the cause of bleeding occurring in early pregnancy . ultimate diagnostic knowledge was as likely to be obtained by the hospital pathologist as by the clinician . the diagnosis of early pregnancy failure by sonar20 and demonstrated the benefits of this technique over the traditional methods of urinary hormone assays and clinical judgement.21 the early years of the 1980s saw elaboration of understanding of ultrasound 's value in the specific context of early pregnancy problems22 23 along with growing acknowledgement of its routine potential in early pregnancy.24 demonstrating the timely relevance of ultrasound to understanding of spontaneous abortion and the speed of developments around this time , in 1984 a canadian research group claimed the second report on the frequency of spontaneous abortions in ultrasound - assessed intact pregnancies25 just 6 weeks after a dutch group had claimed the first report of this subject.26 in 1980 , the 13th edition of gynaecology by ten teachers gave ultrasound only a limited place in the assessment of early pregnancy bleeding ( threatened abortion ) . after the 12 week the ultrasonarscope ( e.g. sonicaid ) may be used to determine whether the fetus is alive. five years later , in the 14th edition of that textbook , the single sentence on the use of ultrasound in threatened abortion had expanded to become a paragraph that sounded very much more enthused : as soon as the initial bleeding has stopped an ultrasonic scan is performed . this will reveal whether or not the pregnancy is intact ...... with a high resolution real time mechanical sector scanner cardiac activity can consistently be recognised at 8 weeks . this will reveal whether or not the pregnancy is intact ... ... with a high resolution real time mechanical sector scanner cardiac activity can consistently be recognised at 8 weeks . during the early 1980s , developments in relation to ultrasound technology and its application in the context of early pregnancy problems changed clinical practice . by the middle of the decade , a survey of general practitioners management of bleeding in early pregnancy found that the patient was sent to hospital for ultrasound examination as an outpatient by 1045 ( 81% ) respondents.27 in 1990 , the 15th edition of the ten teachers text reflected that : the management of patients with bleeding early pregnancy has been enormously simplified by ultrasound . prior to ultrasound , patients with a missed abortion often spent many days in hospital until it became clinically clear that the pregnancy had ended . the management of patients with bleeding early pregnancy has been enormously simplified by ultrasound . prior to ultrasound , patients with a missed abortion often spent many days in hospital until it became clinically clear that the pregnancy had ended . textbooks replaced pictures of pathological specimens from early pregnancy with reproductions of ultrasound images and similar images could now be reproduced by doctors in clinics equipped with their own ultrasound scanner . for the first time in the medical history of early pregnancy , hospital clinicians with appropriate hardware could render technical , textbook knowledge of systematised early pregnancy pathologies directly and in real time from the female bodies lying before them . richard beard 's 1985 miscarriage or abortion? lancet letter attests to the potential impact of ultrasound technology upon medical terminology , and to the application of technologically determined medical language to women 's experiences:intrauterine death at x weeks is really a more satisfactory way of accurately describing what has happened and is appropriate now that ultrasound can distinguish between the blighted ovum and a fetus that has developed but died . intrauterine death at x weeks is really a more satisfactory way of accurately describing what has happened and is appropriate now that ultrasound can distinguish between the blighted ovum and a fetus that has developed but died . accurate descriptions and conformity in language would become a feature of new technology - derived medical knowledge and physicians new diagnostic authority . by the 1980s , thanks to legal and technical developments , early pregnancy loss could increasingly be diagnosed by doctors . however , it remained ambiguously designated : abortion was often used in the medical literature without a clarifying prefix ( spontaneous or induced ) to describe spontaneous pregnancy loss,28 induced termination of pregnancy29 or ( deliberately exploiting the dual meaning of the term ) both.30 early pregnancy loss also remained ambiguously positioned : it had not yet been claimed by a specialist group . research on the subject frequently appeared in the general medical journals and was carried out by a wide range of contributors ( including paediatricians,31 medical geneticists,32 psychiatrists,33 epidemiologists,34 general physicians35 and haematologists36 ) . in 1980 , the chapter on abortion ( miscarriage ) appeared for the last time in the 13th edition of obstetrics by ten teachers and materialised for the first time in the 13th edition of their gynaecology text . early pregnancy loss did not gain a home in a specialist niche until the growth of perinatology . richard beard ( of the 1985 miscarriage or abortion? lancet letter ) published the 1st edition of a defining textbook on fetal physiology and medicine in 1976 . aptly , the textbook was subtitled the basis of perinatology. 1976 also saw the founding of the british paediatric perinatal group ( bppg ) . the journal of perinatal medicine was founded in 1973 ; clinics in perinatology in 1974 ; advances in perinatal medicine in 1981 ; and the journal of perinatology in 1984 . in 1979 , the lancet published a series on better perinatal health . in 1982 , the bppg created a training programme offering accreditation in perinatal paediatrics and in 1985 the bppg dropped the paediatric from its title to become the british association of perinatal medicine . when the 2nd edition of beard 's fetal medicine textbook appeared it had expanded by over 50% : from 542 pages in 1976 to 823 pages in 1984 . with regard to spontaneous abortion , the 1976 1st edition referred only to the possible causative role of immunological factors ; in 1984 , the 2nd edition text referred in seven separate entries to the potential significance of a variety of hormones , immunological and infective agents . the growing knowledge base of perinatology was addressing the problems of early pregnancy . as professor and head of the department of obstetrics and gynaecology in st mary 's hospital , london , between 1972 and 1996 , richard william beard was a forerunner in the process of perinatal medicine 's subspecialisation . as well as his textbook on the subject , he published dozens of papers relaying the findings of his research in fetal physiology and monitoring . beard was also a frequent letter - writer : the correspondence columns of the lancet carry one or more of his contributions almost every year from the mid-1970s to mid-1980s . his letters evidence a concern for the improvement of women 's experiences of health and healthcare37 as well as efforts to defend his professional interests.38 in the miscarriage or abortion? lancet letter published in 1985 , beard was defining appropriate medical terminology for pregnancy loss and defining the subject 's position within his subspeciality , and defining his own authority in that field . richard beard 's acknowledgement of women 's emotional reactions to miscarriage and of the distress caused by the medical terminology of spontaneous abortion suggests the influence of contemporaneous non - medical developments . although miscarriage and the upset that it could cause women might have posed difficulties to feminists on account of its resonance with antiabortionist argument , some notable advocates sought to encourage a more woman - centred approach among health professionals . the miscarriage association had been established in 1982 to offer information and support to women affected by pregnancy loss . richard beard acknowledged in his lancet letter that the miscarriage association found that 85% of women who have had a miscarriage felt strongly that the word abortion should be changed. in 1984 , sociologists ann oakley and helen roberts along with general practitioner ann mcpherson had published miscarriage : the first british book of its kind on the subject. writing for women having miscarriages and those providing care for them , they sought to address the surprising lack of information that there is , especially in the areas of women 's emotional reactions to miscarriage , and with respect to what is considered appropriate treatment by the professionals. early in the book the point about terminology ( miscarriage preferred to richard beard in his lancet letter makes no reference to oakley , mcpherson and roberts , but their analysis of the issue is echoed distinctly in his . the high - profile and widely - reported case of wendy savage in 1985 prompted consideration within the medical profession of the role of women as doctors and as pregnant patients . a comprehensive bmj write - up of the savage case in june 1985 described a battle between the high tech , interventionist , hospital based school of obstetric practice and the community based , woman centred approach of savage.39 it was a battle undoubtedly won by the woman - centred approach . richard beard would align with that approach a few months later when he acknowledged that our patients always speak of ' miscarriages and called for all health professionals , to start using the word miscarriage rather than abortion. the conscious distinction by doctors of miscarriage from abortion ( induced and spontaneous ) may be seen to have reflected certain legal , technical , professional and societal developments . the distinction in language may also be read as part of the process of assigning meaning to those women to whom the language was applied , a process by which women who experience miscarriage could be defined as distinct from women who experience an induced abortion . political implications of beard 's miscarriage or abortion? letter became apparent only a fortnight after its publication when the lancet printed a response from caroline woodroffe , then general secretary of the brook advisory centres . woodroffe highlighted a further benefit of beard 's suggested change in terminology : that confusion in parliament and public debate would also be reduced.40 considerable public and parliamentary debate followed shortly when david alton introduced the abortion ( amendment ) bill to parliament in autumn 1987 . correspondence in the medical press around this time testifies to the strength of feeling among practitioners and the moral judgements of some doctors relating to abortion , both as an act ( killing foetuses ) and as an implication about sexual behaviour.41 beard refers tangentially to the moral stigma of abortion in his lancet letter suggesting that : most women associate the word with what in the past was an illegal act and therefore socially unacceptable ... for doctors ( like beard ) dealing with women who had miscarried the distinction of abortion permitted the distinguishing of their patients ( and themselves ) from a subject that was heavily stigmatised and socially unacceptable. in his letter to the lancet , beard portrays women who miscarry using the following words and phrases : uncomplaining and at their lowest ebb , an image of suffering consolidated by his final appeal for change on humanitarian grounds. meanwhile , he uses no adjectives or emotional descriptors in referring to women who experience abortion ( potentially a very difficult life - event ) . nor does beard consider what significance the socially unacceptable term abortion might carry for women who seek a termination of pregnancy ( a decision that might be made for a great variety of reasons by women in many different circumstances ) . beard 's letter demonstrates a selective empathy and represents a construction of the sensitivities of women who experience miscarriage as much as a response to those sensitivities . the distinction in language of abortion at this moment in history facilitated the splitting of two groups of women who could potentially be very differently constructed . after beard in 1985 asked doctors , indeed all health professionals , to start using the word miscarriage rather than abortion for a spontaneous pregnancy loss , other medical authors subsequently wrote articles whose titles addressed terminology used in early pregnancy loss2 and advocated that terminology for early pregnancy loss must be changed.42 in each of these instances use of the term miscarriage ( as might be suggested ) implies an inherent accusation in its literal allusion to a pregnancy miscarried by the mother 's womb or that ( as might be speculated ) the phrase spontaneous abortion ciphers ideas of hysteria and a typically irrational womb impulsively ejecting its content , it is quite apparent that neither of these two terms overtly acknowledges a pregnancy , or a loss . pregnancy loss was repeatedly employed by medical authors writing on the subject for other doctors , but was apparently not considered suitable for use in front of , or by , women themselves , is notable . the reluctance of doctors to offer pregnancy loss to women as a possible descriptor might suggest a professional denial of the experience or persisting professional efforts to maintain some sort of control . following beard 's appeal to doctors and the shift in medical language , there appears to have been less of a shift in the depth of actual woman - centredness likely to be found within the medical profession or in the degree of real empathy likely to be extended toward women who miscarried . two clinical review papers on the subject of miscarriage published in the bmj since the turn of the century contained no allusion to potential distress and emotional upset.43 44 conversely , during the 1990s and 2000s , medical researchers advocating for improved care after miscarriage tended to emphasise the psychiatric morbidity , including anxiety and depression among women and highlight psychiatric cases:45 46 arguably , by these and other means , miscarriage has tended to be pathologised , perhaps perceived as a potential precursor of psychiatric illness , rather than being acknowledged as a troubling event in its own right , one that lies within the breadth of human experience and that warrants greater physician empathy . the clinical terminology applied to women 's health experiences in britain changed after the mid-1980s when doctors consciously began using the term i have sought to convey the meaning of this change in language : the factors behind it and its subsequent significance . until now , richard beard 's 1985 letter to the lancet has tended to be perceived as an isolated stimulus to the shift in medical language and as a spontaneous response to women 's feelings . however , evidence from medical journals and textbooks of the time reveals how beard 's letter and the change in medical language can be better understood in the context of certain historical developments that enabled and encouraged both the letter - writing and language shift . from the late 1960s onwards , the changes in britain 's legislation allowing women greater access to abortion services also enabled them to discuss these issues with doctors without fear of being criminalised . in turn , doctors could engage in conversations with women without concern that they were being deceived : specifically , they could be confident that women describing symptoms of spontaneous pregnancy loss were not concealing a deliberate ( and previously illegal ) termination . doctors terminology for early pregnancy loss could now refer to something actually knowable . by the 1980s , developments in ultrasound technology enabled the content of a woman 's uterus and any early pregnancy pathologies to be visualised in real time . for the first time in the medical history of early pregnancy loss , following the legal and technological developments described above , diagnostic language could be immediately coupled to a clinically knowable reality . for clinicians , this would have conferred greater significance upon the medical terminology and may have heightened consciousness of language in this context . meanwhile , the need to define accurate and unambiguous terminology for describing diagnoses of fetal loss would have been given impetus by rapidly expanding medical knowledge of the fetus and the establishment of a new fetal - focused medical speciality . consistent with this , the communication that sought to define the terminology came from a senior figure at the forefront of this burgeoning speciality . the emotional experiences of women who miscarry were the most proximal stimulus to richard beard 's writing and the justification that he refers to in his letter . most probably , given the temporality of other writing on this issue , these experiences were acknowledged after being channelled by women 's associations and feminist commentators . the fact that the emotional experiences of women formed the basis for beard 's appeal to doctors suggests that these experiences , in theory at least , had some importance for clinicians , reflecting a growing value placed upon empathy and the patient 's experience . despite the prominence of empathy implied in the call for doctors to use the term when referring to early pregnancy loss , it is doubtful whether the subsequent shift in language among clinicians was accompanied by any similar shift toward more genuinely empathic medical care for women who experienced miscarriage . conceivably , the emphasis upon empathic terminology made the real challenge ( an empathetic response to the loss not only of a pregnancy , but of an expected child , motherhood and apparent certainties ) appear amenable to a simple technical solution ( the substituting of one word for another ) . this may have aided the language shift among doctors , and brought some small improvement to the experiences of women who miscarried , but it did not necessarily betoken a more empathic or women - centred approach . in 1998 , abortion was really the medical profession 's most effective intervention in the context of pregnancy loss . it warned that changing medical language while laudable in its intentions , may not be enough to alleviate mothers dissatisfaction with the care that they receive . the risk is that mere use of correct terminology ... could lead to professional complacency.47 the importance of ensuring appropriate standards of care is underscored by the estimation that one in five pregnancies will miscarry , and most of these women will seek medical attention . research findings have demonstrated repeatedly that mothers dissatisfaction with medical care in the context of early pregnancy loss has not yet been alleviated in britain.48 49 in october 2011 , the internet based charity mumsnet felt compelled to launch a campaign for better miscarriage care.50 the first ever nhs guideline dealing specifically with the issue of miscarriage was published in december 2012 ; it placed considerable emphasis upon the need for support , information - giving and the offer of follow - up for women.51 this may represent a significant development , but the shift of medical language from abortion to miscarriage reminds us that it will take more than words to truly improve patients experiences .

clinical language applied to early pregnancy loss changed in late twentieth century britain when doctors consciously began using the term miscarriage instead of abortion to refer to this subject . medical professionals at the time and since have claimed this change as an intuitive empathic response to women 's experiences . however , a reading of medical journals and textbooks from the era reveals how the change in clinical language reflected legal , technological , professional and social developments . the shift in language is better understood in the context of these historical developments , rather than as the consequence of more empathic medical care for women who experience miscarriage .

Introduction: miscarriage or abortion? Terminology in medical journals and textbooks Abortion law Ultrasound technology The basis of perinatology A woman-centred approach Future meanings Discussion

it is curious , began a letter to the lancet published in 1985 , that , in a language as descriptively rich as english , no clear distinction is made between a spontaneous and an induced expulsion of the contents of the uterus in early pregnancy. it seems likely that the words have been interchangeable for many centuries ... beard et al described the offence caused to those women who miscarry by the use of the word abortion to describe their condition. they appealed to doctors and all health professionals to start using the word miscarriage rather than abortion for a spontaneous pregnancy loss before 28 weeks of pregnancy. seven years later psychologist beverly chalmers asserted that publications in the british journal of obstetrics and gynaecology have taken heed. she observed a change in terminology used before and after the editorial ( sic ) by beard et al.2 in a 1997 british medical journal ( bmj ) editorial emeritus professor of obstetrics geoffrey chamberlain also credited richard beard with moving clinicians towards a more empathetic practice.3 if the terms miscarriage and abortion had , among doctors , been interchangeable for many centuries as beard and his colleagues suggested , why did they call for change in 1985 ? what factors , other than beard 's lancet letter , might explain a change in the medical language of early pregnancy loss ? with these questions in mind , i hand - searched the lancet , bmj and british journal of obstetrics and gynaecology for indexed references to miscarriage , abortion , spontaneous abortion and early pregnancy loss between the years 1960 and 2010 . these references were reviewed and coded in an effort to chart the changing use of terminology and understand those changes in the context of changing medical knowledge and clinical practice . my interpretation of these sources has been influenced by my experiences of working as a general practitioner , as a doctor in an emergency gynaecology department , and as a researcher conducting a clinical study of early pregnancy loss as well as by my readings of feminist accounts of abortion law,4 5 ultrasound,6 the medicalisation of pregnancy7 8 and of pregnancy loss.9 10 in 1992 , beverly chalmers perceived the impact of beard 's miscarriage or abortion? lancet letter upon papers published in the british journal of obstetrics and gynaecology.2 she had reviewed the titles of articles on the subject of early pregnancy loss at the beginning and end of the 1980s and found that whereas almost all the papers at the start of the decade used the term abortion in their titles , none of those published later did so. the graph in figure 1 plots the annual incidence of articles published on the subject of early pregnancy loss in the british journal of obstetrics and gynaecology from 1975 to 1999 ; it differentiates between those using abortion and miscarriage in their titles . around 1986 , a definitive flip occurred in the titles of articles referring to early pregnancy loss : from the exclusive use of . annual incidence of article titles using abortion and miscarriage to refer to early pregnancy loss in the british journal of obstetrics and gynaecology between 1975 and 1999 . spontaneous abortion in article titles referring to early pregnancy loss declined slowly between 1985 and 1995 ( see figure 2 ) : thereafter miscarriage is always employed . annual incidence of article titles using abortion and miscarriage to refer to early pregnancy loss in the bmj between 1975 and 2010 . in the lancet a shift in the terminology of early pregnancy loss is less obvious , but a divergence between the incidences of abortion and miscarriage becomes apparent after 1985 ( see figure 3 ) . annual incidence of article titles using abortion and miscarriage to refer to early pregnancy loss in the lancet between 1975 and 2010 . in the bmj and lancet , the gradual change in published article titles suggests a gradual shift in terminology used by contributors , rather than deliberate editorial policy on the part of the journals . in 1987 , a paper on management of threatened miscarriage published in the bmj consistently uses the term miscarriage throughout;11 yet while some correspondents responding to the article used only miscarriage in their letters,12 others used the term exclusively.13 similarly , in 1991 the bmj published an editorial titled recurrent miscarriage14 to which two correspondents responded : one letter headed recurrent miscarriage;15 the other , recurrent abortion.16 medical journals in fact appear slow to have acknowledged the changing use of language by their readers : miscarriage appears for the first time in the index of the bmj in 1978 , and until 1999 readers looking under as late as 1995 , the ten teachers ( who by then numbered two women alongside eight male contributors ) , in the 16th edition of their gynaecology text , continued , as in earlier editions , to refer to early pregnancy loss as abortion. they continued to use the terms interchangeably in their text , but conceded generally miscarriage carries connotations of spontaneous loss and is best used when talking to women. the actual clinical language used in conversation between doctors and patients at this time is not recorded . however , in 1988 one doctor felt compelled to write to the bmj , advising that : abortion was discussed during a recent phone in programme on bbc radio derby . they were unanimous in their wish that the terms miscarriage should be used instead ... a number of women listeners expressed their objection to the term abortion when it referred to the spontaneous event . they were unanimous in their wish that the terms miscarriage should be used instead ... 17 evidently , the shift in medical terminology occurred slowly among doctors as represented by the authors of journal articles , correspondence and textbooks . the following sections consider some of the factors apparent in the medical literature of the time that may have prompted richard beard to call for change in 1985 and that would have enabled a change in medical terminology . before the 1967 abortion act eased legal impediments to ending an unwanted pregnancy , the distinction between spontaneous and induced abortion alluded to in medical journals and textbooks of the time remained academic . meanwhile , clinicians would not normally have been able to distinguish between spontaneous and induced abortions , despite their recognition that many cases of early pregnancy bleeding ( particularly instances of septic abortion , in which pregnancy loss was complicated by infection ) were accounted for by ill - managed illegal abortion attempts . in 1961 an article in the bmj observed that : it is notoriously difficult to obtain reliable histories from patients suffering from septic abortion , and it is often suspected that criminal interference , which the patient will not acknowledge , has taken place.18 it is notoriously difficult to obtain reliable histories from patients suffering from septic abortion , and it is often suspected that criminal interference , which the patient will not acknowledge , has taken place.18 a 1964 bmj article on the subject of bleeding in early pregnancy spoke of criminal interference that such interference is so often denied , even when it is obvious , that many doctors do not inquire about the possibility.19 textbooks of the era exhibit similar suspicions . in 1966 , the year before the abortion act was passed , the 11th edition of obstetrics by ten teachers advised doctors on how they might discern an induced abortion when , presumably , it was not admitted by the patient : the presence of abrasions or lacerations in the genital tract ... strongly suggests criminal interference. nonetheless , criminal abortion was listed alongside other presumptive , but apparently indeterminable , causes of abortion that were to a certain extent , theoretical and arbitrary. before 1967 , statutes relating to pregnancy termination prevented doctors discerning abortions that were the clinical varieties of abortion had long been distinguished and systematised in medical textbooks . before ultrasound technologies permitted clinicians to look inside the wombs of women experiencing pain or bleeding in early pregnancy , it was impossible to discern the varieties of abortion and distinguish these from a potentially life threatening tubal ( ectopic ) pregnancy . the diagnosis of early pregnancy failure by sonar20 and demonstrated the benefits of this technique over the traditional methods of urinary hormone assays and clinical judgement.21 the early years of the 1980s saw elaboration of understanding of ultrasound 's value in the specific context of early pregnancy problems22 23 along with growing acknowledgement of its routine potential in early pregnancy.24 demonstrating the timely relevance of ultrasound to understanding of spontaneous abortion and the speed of developments around this time , in 1984 a canadian research group claimed the second report on the frequency of spontaneous abortions in ultrasound - assessed intact pregnancies25 just 6 weeks after a dutch group had claimed the first report of this subject.26 in 1980 , the 13th edition of gynaecology by ten teachers gave ultrasound only a limited place in the assessment of early pregnancy bleeding ( threatened abortion ) . five years later , in the 14th edition of that textbook , the single sentence on the use of ultrasound in threatened abortion had expanded to become a paragraph that sounded very much more enthused : as soon as the initial bleeding has stopped an ultrasonic scan is performed . during the early 1980s , developments in relation to ultrasound technology and its application in the context of early pregnancy problems changed clinical practice . by the middle of the decade , a survey of general practitioners management of bleeding in early pregnancy found that the patient was sent to hospital for ultrasound examination as an outpatient by 1045 ( 81% ) respondents.27 in 1990 , the 15th edition of the ten teachers text reflected that : the management of patients with bleeding early pregnancy has been enormously simplified by ultrasound . for the first time in the medical history of early pregnancy , hospital clinicians with appropriate hardware could render technical , textbook knowledge of systematised early pregnancy pathologies directly and in real time from the female bodies lying before them . lancet letter attests to the potential impact of ultrasound technology upon medical terminology , and to the application of technologically determined medical language to women 's experiences:intrauterine death at x weeks is really a more satisfactory way of accurately describing what has happened and is appropriate now that ultrasound can distinguish between the blighted ovum and a fetus that has developed but died . by the 1980s , thanks to legal and technical developments , early pregnancy loss could increasingly be diagnosed by doctors . however , it remained ambiguously designated : abortion was often used in the medical literature without a clarifying prefix ( spontaneous or induced ) to describe spontaneous pregnancy loss,28 induced termination of pregnancy29 or ( deliberately exploiting the dual meaning of the term ) both.30 early pregnancy loss also remained ambiguously positioned : it had not yet been claimed by a specialist group . research on the subject frequently appeared in the general medical journals and was carried out by a wide range of contributors ( including paediatricians,31 medical geneticists,32 psychiatrists,33 epidemiologists,34 general physicians35 and haematologists36 ) . in 1980 , the chapter on abortion ( miscarriage ) appeared for the last time in the 13th edition of obstetrics by ten teachers and materialised for the first time in the 13th edition of their gynaecology text . early pregnancy loss did not gain a home in a specialist niche until the growth of perinatology . the growing knowledge base of perinatology was addressing the problems of early pregnancy . beard was also a frequent letter - writer : the correspondence columns of the lancet carry one or more of his contributions almost every year from the mid-1970s to mid-1980s . his letters evidence a concern for the improvement of women 's experiences of health and healthcare37 as well as efforts to defend his professional interests.38 in the miscarriage or abortion? lancet letter published in 1985 , beard was defining appropriate medical terminology for pregnancy loss and defining the subject 's position within his subspeciality , and defining his own authority in that field . the miscarriage association had been established in 1982 to offer information and support to women affected by pregnancy loss . richard beard acknowledged in his lancet letter that the miscarriage association found that 85% of women who have had a miscarriage felt strongly that the word abortion should be changed. writing for women having miscarriages and those providing care for them , they sought to address the surprising lack of information that there is , especially in the areas of women 's emotional reactions to miscarriage , and with respect to what is considered appropriate treatment by the professionals. early in the book the point about terminology ( miscarriage preferred to richard beard in his lancet letter makes no reference to oakley , mcpherson and roberts , but their analysis of the issue is echoed distinctly in his . richard beard would align with that approach a few months later when he acknowledged that our patients always speak of ' miscarriages and called for all health professionals , to start using the word miscarriage rather than abortion. the conscious distinction by doctors of miscarriage from abortion ( induced and spontaneous ) may be seen to have reflected certain legal , technical , professional and societal developments . the distinction in language may also be read as part of the process of assigning meaning to those women to whom the language was applied , a process by which women who experience miscarriage could be defined as distinct from women who experience an induced abortion . woodroffe highlighted a further benefit of beard 's suggested change in terminology : that confusion in parliament and public debate would also be reduced.40 considerable public and parliamentary debate followed shortly when david alton introduced the abortion ( amendment ) bill to parliament in autumn 1987 . correspondence in the medical press around this time testifies to the strength of feeling among practitioners and the moral judgements of some doctors relating to abortion , both as an act ( killing foetuses ) and as an implication about sexual behaviour.41 beard refers tangentially to the moral stigma of abortion in his lancet letter suggesting that : most women associate the word with what in the past was an illegal act and therefore socially unacceptable ... for doctors ( like beard ) dealing with women who had miscarried the distinction of abortion permitted the distinguishing of their patients ( and themselves ) from a subject that was heavily stigmatised and socially unacceptable. in his letter to the lancet , beard portrays women who miscarry using the following words and phrases : uncomplaining and at their lowest ebb , an image of suffering consolidated by his final appeal for change on humanitarian grounds. meanwhile , he uses no adjectives or emotional descriptors in referring to women who experience abortion ( potentially a very difficult life - event ) . nor does beard consider what significance the socially unacceptable term abortion might carry for women who seek a termination of pregnancy ( a decision that might be made for a great variety of reasons by women in many different circumstances ) . beard 's letter demonstrates a selective empathy and represents a construction of the sensitivities of women who experience miscarriage as much as a response to those sensitivities . the distinction in language of abortion at this moment in history facilitated the splitting of two groups of women who could potentially be very differently constructed . after beard in 1985 asked doctors , indeed all health professionals , to start using the word miscarriage rather than abortion for a spontaneous pregnancy loss , other medical authors subsequently wrote articles whose titles addressed terminology used in early pregnancy loss2 and advocated that terminology for early pregnancy loss must be changed.42 in each of these instances use of the term miscarriage ( as might be suggested ) implies an inherent accusation in its literal allusion to a pregnancy miscarried by the mother 's womb or that ( as might be speculated ) the phrase spontaneous abortion ciphers ideas of hysteria and a typically irrational womb impulsively ejecting its content , it is quite apparent that neither of these two terms overtly acknowledges a pregnancy , or a loss . pregnancy loss was repeatedly employed by medical authors writing on the subject for other doctors , but was apparently not considered suitable for use in front of , or by , women themselves , is notable . the reluctance of doctors to offer pregnancy loss to women as a possible descriptor might suggest a professional denial of the experience or persisting professional efforts to maintain some sort of control . following beard 's appeal to doctors and the shift in medical language , there appears to have been less of a shift in the depth of actual woman - centredness likely to be found within the medical profession or in the degree of real empathy likely to be extended toward women who miscarried . two clinical review papers on the subject of miscarriage published in the bmj since the turn of the century contained no allusion to potential distress and emotional upset.43 44 conversely , during the 1990s and 2000s , medical researchers advocating for improved care after miscarriage tended to emphasise the psychiatric morbidity , including anxiety and depression among women and highlight psychiatric cases:45 46 arguably , by these and other means , miscarriage has tended to be pathologised , perhaps perceived as a potential precursor of psychiatric illness , rather than being acknowledged as a troubling event in its own right , one that lies within the breadth of human experience and that warrants greater physician empathy . the clinical terminology applied to women 's health experiences in britain changed after the mid-1980s when doctors consciously began using the term i have sought to convey the meaning of this change in language : the factors behind it and its subsequent significance . until now , richard beard 's 1985 letter to the lancet has tended to be perceived as an isolated stimulus to the shift in medical language and as a spontaneous response to women 's feelings . however , evidence from medical journals and textbooks of the time reveals how beard 's letter and the change in medical language can be better understood in the context of certain historical developments that enabled and encouraged both the letter - writing and language shift . from the late 1960s onwards , the changes in britain 's legislation allowing women greater access to abortion services also enabled them to discuss these issues with doctors without fear of being criminalised . in turn , doctors could engage in conversations with women without concern that they were being deceived : specifically , they could be confident that women describing symptoms of spontaneous pregnancy loss were not concealing a deliberate ( and previously illegal ) termination . doctors terminology for early pregnancy loss could now refer to something actually knowable . for the first time in the medical history of early pregnancy loss , following the legal and technological developments described above , diagnostic language could be immediately coupled to a clinically knowable reality . most probably , given the temporality of other writing on this issue , these experiences were acknowledged after being channelled by women 's associations and feminist commentators . despite the prominence of empathy implied in the call for doctors to use the term when referring to early pregnancy loss , it is doubtful whether the subsequent shift in language among clinicians was accompanied by any similar shift toward more genuinely empathic medical care for women who experienced miscarriage . conceivably , the emphasis upon empathic terminology made the real challenge ( an empathetic response to the loss not only of a pregnancy , but of an expected child , motherhood and apparent certainties ) appear amenable to a simple technical solution ( the substituting of one word for another ) . this may have aided the language shift among doctors , and brought some small improvement to the experiences of women who miscarried , but it did not necessarily betoken a more empathic or women - centred approach . in 1998 , abortion was really the medical profession 's most effective intervention in the context of pregnancy loss . research findings have demonstrated repeatedly that mothers dissatisfaction with medical care in the context of early pregnancy loss has not yet been alleviated in britain.48 49 in october 2011 , the internet based charity mumsnet felt compelled to launch a campaign for better miscarriage care.50 the first ever nhs guideline dealing specifically with the issue of miscarriage was published in december 2012 ; it placed considerable emphasis upon the need for support , information - giving and the offer of follow - up for women.51 this may represent a significant development , but the shift of medical language from abortion to miscarriage reminds us that it will take more than words to truly improve patients experiences .

application of magnetic nanoparticles in magnetic resonance imaging ( mri ) and immunoassay is mainly dependent on the best performance of superparamagnetism below a critical size value.1 the superparamagnetic properties make magnetic nanoparticles monodisperse and exhibit high enzyme mimetic activity . among the various magnetic nanoparticles , magnetoferritin was first synthesized within horse spleen ferritin ( hosf ) cavity through removing native ferrihydrite core and replacing magnetite / maghemite core.24 recently , a novel magnetoferritin with a magnetite core , named as m - hfn , was successfully synthesized by using genetically engineered recombinant human h chain ferritin ( hfn ) as a biotemplate.57 the hfn shell is stable as a hydrophilic protein which is tolerant to heat and urea or guanidinium chloride due to the special structure with outer diameter of 12 nm and inner cavity diameter of 8 nm.8 several studies have demonstrated that without tumor - targeted ligand modification , the hfn shell of m - hfn nanoparticles can be specifically targeted to transferrin receptor 1 ( tfr1 ) overexpressed on various cancer cells,913 which allow efficiently distinguishing cancerous cells from normal cells both in vitro and in vivo . because of their high transverse relaxivity and good peroxidase - like activity,11,14 the m - hfn nanoparticles are found to be a very promising contrast agent for the in vivo detection of microscopic ( < 12 mm ) tumors by mri and in vitro detection of tumor tissues with a high sensitivity of 98% and specificity of 95%.10,11 in addition , tumor - targeted arginine aspartate peptide can be easily loaded on the hfn shell via genetic means , which make m - hfn nanoparticles specifically bind to v3 integrin implicated in tumor of a variety of cancer types.5,15 the nanoscale size plays a key role in avoiding from agglomeration , usage of mri , clinical diagnosis , and therapeutics.1620 compared with traditional magnetoferritin synthesized using hosf cavity as a template , the genetic engineering of hfn cavity provides a superior nanoplatform for synthesizing monodispersed , noninteracting , and stoichiometric magnetite nanoparticles with nearly the same spherical shape , narrow size distribution , and high crystallinity.57,14,21 thus , the m - hfn nanoparticles are ideal for studying the nanoscale size effects on magnetic properties , mri and immunoassay , and so forth . in this study , m - hfn nanoparticles with different sizes of magnetite cores were successfully synthesized through strictly controlling iron loading into hfn shells . the nanoscale size effects on application of m - hfn nanoparticles in mri and immunohistochemical staining of cancer cells were analyzed and discussed . the recombinant plasmid pet12b - hfn , which contains the sequence coding of the human hfn , was transformed into escherichia coli strain rosetta . bertani medium , with ampicillin and chloromycetin acting as selection markers , for 3 hours . isopropyl--d - thiogalactopyranoside ( 1 mm ) was used as an inductive agent to induce the expression of ferritin . was then acquired by centrifugation . to remove other proteins , the supernatant was heated at 75c for 15 minutes . finally , the hfn was purified through size exclusion chromatography ( sepharose 6b , ge healthcare ) , and the purity of hfn was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( page ) . the protein concentration was determined by bicinchoninic acid protein assay reagent ( pierce ) with bovine serum albumin as standard . the m - hfn nanoparticles were synthesized using our previous experimental procedure with minor modifications.6 fe ( ii ) was added in a rate of 50 fe / protein / minute . simultaneously , freshly prepared h2o2 was added as an oxidant in accordance with stoichiometric equivalents ( 1:3 , h2o2:fe ) . after adding theoretical 1,000 , 3,000 , 5,000 , and 7,000 iron atoms per protein cage to the reaction vessel , then , 200 l of 300 mm sodium citrate was added to chelate any free iron to each sample . these four sets of synthesized m - hfn nanoparticles are named here as m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 , respectively . the core structure and composition of m - hfn nanoparticles tem observations were performed on a fei - tecnai g f20 electron microscope with an accelerating voltage of 200 kev . the size of m - hfn core for each sample set was determined by measuring 800 particles . the crystalline structure of cores was examined by selected area electron diffraction ( saed ) patterns . the stability and integrity of m - hfn protein cages were examined by native page and circular dichroism ( cd ) spectra analysis . the gels were used to perform protein staining with coomassie brilliant blue r250 and iron staining with potassium ferrocyanide . the cd spectra were performed using a chirascan-plus cd spectrometer ( applied photophysics , leatherhead , uk ) at 190300 nm ( bandwidth : 1 nm ) with protein concentration of 0.15 mg ml . the magnetic measurements were performed on a quantum design mpms squid magnetometer ( model xp-5xl , with magnetic moment sensitivity of 5.010 am ) . low - field magnetization curves were measured between 5 k and 200 k in a field of 1.5 mt , after the sample was cooled to 5 k in zero field ( zero - field cooling ) and after cooling in a 1.5 mt field ( field cooling ) . to evaluate magnetostatic interactions of these m - hfn nanoparticles , the acquisition of isothermal remanent magnetization and dc field demagnetization curves were measured at 5 k in dc field up to 1,000 mt . for each field step , a constant magnetic field was applied for 120 seconds ; the remanent moment was measured after removal of the field and a delay of 120 seconds . cisowski test.22 the hysteresis loops at 5 k were also measured in fields of 3 t. the real iron content in the m - hfn nanoparticles was determined by inductively coupled plasma mass spectrometry ( icp - ms ) ( agilent , 7500a ) . nitration of all samples was performed with high - purity hydrogen nitrate at high temperature in a clean room . the solution volume of all samples was quantified to a constant value of 1 ml . the transverse relaxation times ( t2 ) in phosphate - buffered saline ( pbs ) ( iron concentration of 01 mm ) were measured using multi - slice multi - echo ( msme ) sequence with the following parameters : field of view ( fov ) = 5 cm 5 cm , matrix = 12896 , repetition time ( tr ) = 3,000 ms , echo time ( te ) = 8.585 ms , and ten echoes . to measure the longitudinal relaxation times ( t1 ) , msme sequence was used with the following parameters : fov = 5 cm 5 cm , matrix = 12896 , tr = 90 ms , 150 ms , 300 ms , 500 ms , 800 ms , 1,200 ms , 2,000 ms , and 3,000 ms , and te = 8.5 ms . the t1 for each sample was calculated in matlab ( the mathworks , r2012b ) by using the equation of f(x)=a+c(1exp(1x / b ) ) . in order to assess the size effect on cancer detection applications , the mda - mb-231 breast cancer cell line with high tfr1 expression was chosen based on our previous studies.10,11 cancer cells were cultured by using dulbecco s modified eagle s medium ( dmem , cat 12800 - 058 , invitrogen ) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin solution ( cat c0222 , beyotime ) at 37c in 5% co2 atmosphere . in the present study , cells were seeded into six - well polystyrene plate , and then , m - hfn nanoparticles were added until the cell attachment rate reaches about 80% , while the control group of cells was incubated in the absence of m - hfn nanoparticles . the final concentration of m - hfn nanoparticles in experimental groups was diluted to 0.3 mg ml ( protein concentration ) in dmem . after 24 hours of incubation , the medium was removed , and cells were washed three times with pbs . finally , cells were resuspended in pbs and counted after collecting by using trypsin solution , and then , the cell pellets were dissolved in hydrogen nitrate after being centrifuged and washed in distilled water several times . the amount of fe taken up by cells was measured by icp - ms ( agilent , 7700 ) . for mri analysis , the cancer cells were treated as described earlier in this paper ; uniform gel suspensions ( 1% agarose gel ) were prepared with cell concentration of 310 cells ml , and placed in a 96-well microtiter plate . sampling was performed at 7 t on a bruker biospec imager using msme sequence with the following parameters : fov = 3.5 cm 3.5 cm , matrix = 256256 , tr = 2,500 ms , te = 20320 ms , and 16 echoes . to evaluate the efficiency of m - hfn7000 , cells of different concentrations were prepared for mri . to visualize m - hfn nanoparticles uptaken by cancer cells , a 12-well polystyrene plate - sized glass slide was put in the well before cells seeding . the cells were fixed with 4% formaldehyde subsequently for 1 hour , and then stained with prussian blue and counterstained with eosin . the peroxidase activity was examined at room temperature . constant protein concentration of m - hfn ( 0.25 mg ml ) nanoparticles and 500 mm h2o2 were used in 0.2 m sodium acetate buffer ( ph 4.5 ) , and 0.2 mg ml of 3,3,5,5-tetramethylbenzidine ( tmb , sigma ) was used as the substrate . 0.05 m tris hcl ( ph 7.5 ) was used as the color reaction buffer , while 3,3-diaminobenzidine tetrahydrochloride ( dab , sigma ) was used as the substrate . to further characterize the peroxidase - like activity of m - hfn nanoparticles , quadruple amount of sodium acetate buffer and half amount of m - hfn , h2o2 , and tmb were used in the color reaction ; mda - mb-231 tumor implantation was performed according to previously used procedure.14 the tumor tissues were harvested and kept in the form of paraffin embedded . for immunohistochemical staining , the paraffin - embedded tissue sections were firstly heated at 55c for 60 minutes and then deparaffinized by washing twice in xylene for 10 minutes , using an ethanol gradient for sections hydrating . antigen retrieval was carried out in microwave oven where sections were boiled in 0.01 m citrate buffer ( ph 6.0 ) for 30 minutes . to quench endogenous peroxidase activity , tissue sections were incubated with 0.3% h2o2 in methanol for 20 minutes after cooling to room temperature and rinsing in pbs . five percent goat serum in pbs was used to block tissue sections for 1 hour at 37c , and after rinsing , the tissue sections were independently incubated with m - hfn nanoparticles of different core sizes ( 2 m ) overnight at 4c . freshly prepared dab was used for color developing , while hematoxylin was used for counterstaining . the analysis of stained sections was done under a microscope , and quantitative analysis was done using image pro plus software ( version 6.0 ) . the measurement parameter was density mean , calculated by integrated optical density divided by area sum , and the intensity was averaged from five fovs for ten tissue sections . the recombinant plasmid pet12b - hfn , which contains the sequence coding of the human hfn , was transformed into escherichia coli strain rosetta . bertani medium , with ampicillin and chloromycetin acting as selection markers , for 3 hours . isopropyl--d - thiogalactopyranoside ( 1 mm ) was used as an inductive agent to induce the expression of ferritin . was then acquired by centrifugation . to remove other proteins , the supernatant was heated at 75c for 15 minutes . finally , the hfn was purified through size exclusion chromatography ( sepharose 6b , ge healthcare ) , and the purity of hfn was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( page ) . the protein concentration was determined by bicinchoninic acid protein assay reagent ( pierce ) with bovine serum albumin as standard . the m - hfn nanoparticles were synthesized using our previous experimental procedure with minor modifications.6 fe ( ii ) was added in a rate of 50 fe / protein / minute . simultaneously , freshly prepared h2o2 was added as an oxidant in accordance with stoichiometric equivalents ( 1:3 , h2o2:fe ) . after adding theoretical 1,000 , 3,000 , 5,000 , and 7,000 iron atoms per protein cage to the reaction vessel , then , 200 l of 300 mm sodium citrate was added to chelate any free iron to each sample . these four sets of synthesized m - hfn nanoparticles are named here as m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 , respectively . the core structure and composition of m - hfn nanoparticles were analyzed by transmission electron microscope ( tem ) . tem observations were performed on a fei - tecnai g f20 electron microscope with an accelerating voltage of 200 kev . the size of m - hfn core for each sample set was determined by measuring 800 particles . the crystalline structure of cores was examined by selected area electron diffraction ( saed ) patterns . the stability and integrity of m - hfn protein cages were examined by native page and circular dichroism ( cd ) spectra analysis . the gels were used to perform protein staining with coomassie brilliant blue r250 and iron staining with potassium ferrocyanide . the cd spectra were performed using a chirascan-plus cd spectrometer ( applied photophysics , leatherhead , uk ) at 190300 nm ( bandwidth : 1 nm ) with protein concentration of 0.15 mg ml . the magnetic measurements were performed on a quantum design mpms squid magnetometer ( model xp-5xl , with magnetic moment sensitivity of 5.010 am ) . low - field magnetization curves were measured between 5 k and 200 k in a field of 1.5 mt , after the sample was cooled to 5 k in zero field ( zero - field cooling ) and after cooling in a 1.5 mt field ( field cooling ) . to evaluate magnetostatic interactions of these m - hfn nanoparticles , the acquisition of isothermal remanent magnetization and dc field demagnetization curves were measured at 5 k in dc field up to 1,000 mt . for each field step , a constant magnetic field was applied for 120 seconds ; the remanent moment was measured after removal of the field and a delay of 120 seconds . cisowski test.22 the hysteresis loops at 5 k were also measured in fields of 3 t. the real iron content in the m - hfn nanoparticles was determined by inductively coupled plasma mass spectrometry ( icp - ms ) ( agilent , 7500a ) . nitration of all samples was performed with high - purity hydrogen nitrate at high temperature in a clean room . the solution volume of all samples was quantified to a constant value of 1 ml . the transverse relaxation times ( t2 ) in phosphate - buffered saline ( pbs ) ( iron concentration of 01 mm ) were measured using multi - slice multi - echo ( msme ) sequence with the following parameters : field of view ( fov ) = 5 cm 5 cm , matrix = 12896 , repetition time ( tr ) = 3,000 ms , echo time ( te ) = 8.585 ms , and ten echoes . to measure the longitudinal relaxation times ( t1 ) , msme sequence was used with the following parameters : fov = 5 cm 5 cm , matrix = 12896 , tr = 90 ms , 150 ms , 300 ms , 500 ms , 800 ms , 1,200 ms , 2,000 ms , and 3,000 ms , and te = 8.5 ms . the t1 for each sample was calculated in matlab ( the mathworks , r2012b ) by using the equation of f(x)=a+c(1exp(1x / b ) ) . in order to assess the size effect on cancer detection applications , the mda - mb-231 breast cancer cell line with high tfr1 expression was chosen based on our previous studies.10,11 cancer cells were cultured by using dulbecco s modified eagle s medium ( dmem , cat 12800 - 058 , invitrogen ) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin solution ( cat c0222 , beyotime ) at 37c in 5% co2 atmosphere . in the present study , cells were seeded into six - well polystyrene plate , and then , m - hfn nanoparticles were added until the cell attachment rate reaches about 80% , while the control group of cells was incubated in the absence of m - hfn nanoparticles . the final concentration of m - hfn nanoparticles in experimental groups was diluted to 0.3 mg ml ( protein concentration ) in dmem . after 24 hours of incubation , the medium was removed , and cells were washed three times with pbs . finally , cells were resuspended in pbs and counted after collecting by using trypsin solution , and then , the cell pellets were dissolved in hydrogen nitrate after being centrifuged and washed in distilled water several times . the amount of fe taken up by cells was measured by icp - ms ( agilent , 7700 ) . for mri analysis , the cancer cells were treated as described earlier in this paper ; uniform gel suspensions ( 1% agarose gel ) were prepared with cell concentration of 310 cells ml , and placed in a 96-well microtiter plate . sampling was performed at 7 t on a bruker biospec imager using msme sequence with the following parameters : fov = 3.5 cm 3.5 cm , matrix = 256256 , tr = 2,500 ms , te = 20320 ms , and 16 echoes . to evaluate the efficiency of m - hfn7000 , cells of different concentrations were prepared for mri . to visualize m - hfn nanoparticles uptaken by cancer cells , a 12-well polystyrene plate - sized glass slide was put in the well before cells seeding . the cells were fixed with 4% formaldehyde subsequently for 1 hour , and then stained with prussian blue and counterstained with eosin . the peroxidase activity was examined at room temperature . constant protein concentration of m - hfn ( 0.25 mg ml ) nanoparticles and 500 mm h2o2 were used in 0.2 m sodium acetate buffer ( ph 4.5 ) , and 0.2 mg ml of 3,3,5,5-tetramethylbenzidine ( tmb , sigma ) was used as the substrate . 0.05 m tris hcl ( ph 7.5 ) was used as the color reaction buffer , while 3,3-diaminobenzidine tetrahydrochloride ( dab , sigma ) was used as the substrate . to further characterize the peroxidase - like activity of m - hfn nanoparticles , quadruple amount of sodium acetate buffer and half amount of m - hfn , h2o2 , and tmb were used in the color reaction ; mda - mb-231 tumor implantation was performed according to previously used procedure.14 the tumor tissues were harvested and kept in the form of paraffin embedded . for immunohistochemical staining , the paraffin - embedded tissue sections were firstly heated at 55c for 60 minutes and then deparaffinized by washing twice in xylene for 10 minutes , using an ethanol gradient for sections hydrating . antigen retrieval was carried out in microwave oven where sections were boiled in 0.01 m citrate buffer ( ph 6.0 ) for 30 minutes . to quench endogenous peroxidase activity , tissue sections were incubated with 0.3% h2o2 in methanol for 20 minutes after cooling to room temperature and rinsing in pbs . five percent goat serum in pbs was used to block tissue sections for 1 hour at 37c , and after rinsing , the tissue sections were independently incubated with m - hfn nanoparticles of different core sizes ( 2 m ) overnight at 4c . freshly prepared dab was used for color developing , while hematoxylin was used for counterstaining . the analysis of stained sections was done under a microscope , and quantitative analysis was done using image pro plus software ( version 6.0 ) . the measurement parameter was density mean , calculated by integrated optical density divided by area sum , and the intensity was averaged from five fovs for ten tissue sections . by loading 1,000 , 3,000 , 5,000 , and 7,000 iron atoms , the synthesized m - hfn nanoparticle samples m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 , respectively , in the present study are monodispersed ( figure 1a ) , having well - crystalline cores with clear lattice fringes ( figure 1b ) . the saed patterns consist of five distinct rings ( figure 1c ) , indexed to be the ( 220 ) , ( 311 ) , ( 400 ) , ( 511 ) , and ( 440 ) lattice planes of magnetite . the mean size of the magnetite cores in the m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 nanoparticles is 2.70.6 nm , 3.30.8 nm , 4.41.2 nm , and 5.31.3 nm , respectively ( figure 1d and table 1 ) , with major_axis / minor_axis ratios of 1.15 , 1.13 , 1.12 , and 1.11 , respectively . iron content of the synthesized m - hfn nanoparticles determined by using icp - ms ( agilent , 7500a ) with external and internal standard iron calibration showed that the average iron content per protein of the synthesized m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 was 786 , 1,707 , 2,456 , and 4,709 fe atoms , respectively . the loss of iron might be due to the formation of iron oxide or remaining as free iron outside of protein shells during the reaction period used in this study , which were removed . native page and cd spectra of the synthesized m - hfn nanoparticles show that the synthesized m - hfn nanoparticles have intact protein cages ( figure 2 ) . the gradual darkening of blue color from lane 2 to lane 5 ( figure 2a ) indicates the increase in iron contents in m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 . comparable protein - staining bands from lane 2 to lane 5 with native hfn protein cage in lane 1 ( figure 2b ) and the cd spectra ( figure 2c ) indicate that the protein structure of hfn cages / shells in the synthesized m - hfn nanoparticles is likely intact after the mineralization process . the r - value of the wohlfarth cisowski test of m - hfn1000 is 0.5 , suggesting no magnetostatic interactions between the m - hfn cores . however , the r - value of m - hfn3000 , m - hfn5000 , and m - hfn7000 decreases slightly to 0.48 , 0.44 , and 0.40 , respectively , suggesting weak magnetic interaction in these samples ( table 1 and figure s1 ) . with the increase in core sizes , the saturation magnetization ( ms ) , coercivity ( hc ) , and blocking temperature ( tb ) of those samples measured at 5 k are increasing . specifically , the m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 have ms values of 5.9 am kg , 15.2 am kg , 27.6 am kg , and 37.3 am kg , hc values of 4.62 mt , 8.28 mt , 19.87 mt , and 24.41 mt , and tb values of 10 k , 13 k , 44 k , and 170 k , respectively ( figures 3 and s2 and table 1 ) . the determined transverse relaxivity ( r2 ) and longitudinal relaxivity ( r1 ) were acquired by linear mapping of iron concentration , and 1/t2 ( r2 ) and 1/t1 ( r1 ) ( figures s3 and s4 ) . the r2 values of m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 are 22.5 mm s , 63.2 mm s , 99.4 mm s , and 320.9 mm s , respectively . the r1 values of these m - hfn nanoparticles are 10.4 mm s , 14.5 mm s , 17.4 mm s , and 22.0 mm s. this strongly indicates that the transverse and longitudinal relaxivity increase with the size of m - hfn cores . the values of r2/r1 are 2.2 , 4.4 , 5.7 , and 14.6 for m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 , respectively ( table 1 ) . peroxidase - like activity of m - hfn nanoparticles tested at constant concentration of hfn nicely increased with size of m - hfn cores ( figure 4 ) . m - hfn nanoparticles catalyze the oxidation of peroxidase substrates tmb and dab in the presence of h2o2 to give a blue color ( figure 4a ) and a brown color product ( figure 4b ) , respectively . it is clear that the m - hfn nanoparticles with larger size of cores have higher peroxidase - like activity and absorbance . the m - hfn7000 nanoparticles have nearly five times absorbance compared to m - hfn1000 at 10 minutes ( figure 4c ) . in vitro mri of mda - mb-231 tumor cells after incubation with m - hfn nanoparticles shows obvious dark images compared to cells - only control ( figure 5a ) . the mda - mb-231 cancer cells incubated with m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 have t2 values equal to 110.9 ms , 99.1 ms , 90.0 ms , and 81.2 ms , respectively , while the cells - only control ( incubated without m - hfn nanoparticles ) has a t2 value of 122.5 ms . as shown in figure 5b , the t2 values of mda - mb-231 tumor cells with cell densities of 110 cells ml , 110 cells ml , 210 cells ml , and 310 cells ml incubated with m - hfn7000 reduce to 73.5% , 66.3% , 63.2% , and 57.8% , respectively , compared with the t2 value of control ( 0 cells ml ) . to further identify iron uptaken by cancer cells , we performed prussian blue staining for cancer cells after incubation with the m - hfn nanoparticles . it can be seen that there is more blue deposition in cancer cells incubated with m - hfn nanoparticles with larger core size . we also assessed the iron content in cancer cells by icp - ms and found that cancer cells accumulated 0.16 pg cell , 0.29 pg cell , 0.80 pg cell , and 1.23 pg cell of fe after being incubated with equal amount of m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 for 24 hours , respectively ( figure 6f ) . figure 7 shows the m - hfn - based peroxidase staining performed on xenografted mda - mb-231 tumor tissues . tumor tissues incubated with m - hfn nanoparticles showed brown color , which is in contrast to purple color of the normal tissues counterstained by hematoxylin . quantitative analysis of effect on staining with m - hfn nanoparticles was performed using image pro plus software , which shows that m - hfn nanoparticles with large core size generate high value of density mean . by loading 1,000 , 3,000 , 5,000 , and 7,000 iron atoms , the synthesized m - hfn nanoparticle samples m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 , respectively , in the present study are monodispersed ( figure 1a ) , having well - crystalline cores with clear lattice fringes ( figure 1b ) . the saed patterns consist of five distinct rings ( figure 1c ) , indexed to be the ( 220 ) , ( 311 ) , ( 400 ) , ( 511 ) , and ( 440 ) lattice planes of magnetite . the mean size of the magnetite cores in the m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 nanoparticles is 2.70.6 nm , 3.30.8 nm , 4.41.2 nm , and 5.31.3 nm , respectively ( figure 1d and table 1 ) , with major_axis / minor_axis ratios of 1.15 , 1.13 , 1.12 , and 1.11 , respectively . iron content of the synthesized m - hfn nanoparticles determined by using icp - ms ( agilent , 7500a ) with external and internal standard iron calibration showed that the average iron content per protein of the synthesized m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 was 786 , 1,707 , 2,456 , and 4,709 fe atoms , respectively . the loss of iron might be due to the formation of iron oxide or remaining as free iron outside of protein shells during the reaction period used in this study , which were removed . native page and cd spectra of the synthesized m - hfn nanoparticles show that the synthesized m - hfn nanoparticles have intact protein cages ( figure 2 ) . the gradual darkening of blue color from lane 2 to lane 5 ( figure 2a ) indicates the increase in iron contents in m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 . comparable protein - staining bands from lane 2 to lane 5 with native hfn protein cage in lane 1 ( figure 2b ) and the cd spectra ( figure 2c ) indicate that the protein structure of hfn cages / shells in the synthesized m - hfn nanoparticles is likely intact after the mineralization process . cisowski test of m - hfn1000 is 0.5 , suggesting no magnetostatic interactions between the m - hfn cores . however , the r - value of m - hfn3000 , m - hfn5000 , and m - hfn7000 decreases slightly to 0.48 , 0.44 , and 0.40 , respectively , suggesting weak magnetic interaction in these samples ( table 1 and figure s1 ) . with the increase in core sizes , the saturation magnetization ( ms ) , coercivity ( hc ) , and blocking temperature ( tb ) of those samples measured at 5 k are increasing . specifically , the m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 have ms values of 5.9 am kg , 15.2 am kg , 27.6 am kg , and 37.3 am kg , hc values of 4.62 mt , 8.28 mt , 19.87 mt , and 24.41 mt , and tb values of 10 k , 13 k , 44 k , and 170 k , respectively ( figures 3 and s2 and table 1 ) . the determined transverse relaxivity ( r2 ) and longitudinal relaxivity ( r1 ) were acquired by linear mapping of iron concentration , and 1/t2 ( r2 ) and 1/t1 ( r1 ) ( figures s3 and s4 ) . the r2 values of m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 are 22.5 mm s , 63.2 mm s , 99.4 mm s , and 320.9 mm s , respectively . the r1 values of these m - hfn nanoparticles are 10.4 mm s , 14.5 mm s , 17.4 mm s , and 22.0 mm s. this strongly indicates that the transverse and longitudinal relaxivity increase with the size of m - hfn cores . the values of r2/r1 are 2.2 , 4.4 , 5.7 , and 14.6 for m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 , respectively ( table 1 ) . peroxidase - like activity of m - hfn nanoparticles tested at constant concentration of hfn nicely increased with size of m - hfn cores ( figure 4 ) . m - hfn nanoparticles catalyze the oxidation of peroxidase substrates tmb and dab in the presence of h2o2 to give a blue color ( figure 4a ) and a brown color product ( figure 4b ) , respectively . it is clear that the m - hfn nanoparticles with larger size of cores have higher peroxidase - like activity and absorbance . the m - hfn7000 nanoparticles have nearly five times absorbance compared to m - hfn1000 at 10 minutes ( figure 4c ) . in vitro mri of mda - mb-231 tumor cells after incubation with m - hfn nanoparticles shows obvious dark images compared to cells - only control ( figure 5a ) . the mda - mb-231 cancer cells incubated with m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 have t2 values equal to 110.9 ms , 99.1 ms , 90.0 ms , and 81.2 ms , respectively , while the cells - only control ( incubated without m - hfn nanoparticles ) has a t2 value of 122.5 ms . as shown in figure 5b , the t2 values of mda - mb-231 tumor cells with cell densities of 110 cells ml , 110 cells ml , 210 cells ml , and 310 cells ml incubated with m - hfn7000 reduce to 73.5% , 66.3% , 63.2% , and 57.8% , respectively , compared with the t2 value of control ( 0 cells ml ) . to further identify iron uptaken by cancer cells , we performed prussian blue staining for cancer cells after incubation with the m - hfn nanoparticles . it can be seen that there is more blue deposition in cancer cells incubated with m - hfn nanoparticles with larger core size . we also assessed the iron content in cancer cells by icp - ms and found that cancer cells accumulated 0.16 pg cell , 0.29 pg cell , 0.80 pg cell , and 1.23 pg cell of fe after being incubated with equal amount of m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 for 24 hours , respectively ( figure 6f ) . figure 7 shows the m - hfn - based peroxidase staining performed on xenografted mda - mb-231 tumor tissues . tumor tissues incubated with m - hfn nanoparticles showed brown color , which is in contrast to purple color of the normal tissues counterstained by hematoxylin . quantitative analysis of effect on staining with m - hfn nanoparticles was performed using image pro plus software , which shows that m - hfn nanoparticles with large core size generate high value of density mean . the m - hfn nanoparticles are different from previously synthesized m - hosf nanoparticles , which were reconstituted in hosf . the m - hfn nanoparticles are directly synthesized in recombinant human hfn cavity under well - controlled condition in anaerobic box with stat ph , temperature , and addition rate of fe ( ii ) and oxidant , while the m - hosf needs to remove native ferrihydrite cores before reconstitution of magnetite cores . the removal process and the difference of ferritins may affect the mineralization of the core . the uniform size and nearly spherical shape of m - hfn nanoparticles imply that the mineralization of iron oxide core ( magnetite ) is highly controlled by the hfn protein shells , in spite of non - physiological condition ( 65c , ph 8.5 ) . it has been estimated that ferritin cavity can be capable of sorting up to 4,500 fe atoms as a hydrated ferrihydrite and 8,400 fe atoms as a magnetite.21,23 in this study , out of theoretically added 7,000 iron atoms , the m - hfn nanoparticle has taken 4,709 fe atoms per hfn ( determined by icp - ms ) , forming magnetite core of 5.3 nm . figure 2 indicates that the protein structure of hfn cages / shells in the synthesized m - hfn nanoparticles is nearly intact after the mineralization process . owing to the nano - size effect , smaller nanoparticles may have longer circulation half - life in the body and exhibit better contrast.24,25 the smaller size of nanoparticles also influences the relaxivity and contrast enhancement.2628 it is noted in this study that the m - hfn7000 with the largest core size ( 5.3 nm ) exhibits the highest value of r2 ( 320.9 mm s ) , which is ten times higher than that of the m - hfn1000 with the smallest magnetite cores ( 2.7 nm ) . the increase in transverse relaxivity with increased size of m - hfn cores is well consistent with typical behavior of relatively small particles in motional averaging regime.19 this is mainly because the transverse relaxivity is proportional to the magnetic moment;29 that is , the larger the magnetization particles are , the higher the r2 the particles exhibit.30 the saturation magnetization of m - hfn nanoparticles synthesized here increased from 5.9 am kg to 37.3 am kg with the increasing size from 2.7 nm to 5.3 nm . the increasing nanoparticle size may enhance the water protons chemically exchanging with surface paramagnetic centers of particles ; consequently , t1 relaxation enhanced.31 the values of r1 and ratio of r2/r1 are important parameters for evaluating magnetic nanoparticles as mr - positive contrast agents . in general , t1 agents have r2/r1 ratios of 12.32 for m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 nanoparticles , the r2/r1 ratios are 2.2 , 4.4 , 5.7 , and 14.6 , respectively ( table 1 ) . previous study showed that the ultrasmall magnetic iron oxide nanoparticles of 3.3 nm have r2/r1 ratio of 4.2 and they could be used as both positive contrast agents and negative contrast agents.33 the relatively low r2/r1 ratios of m - hfn1000 and m - hfn3000 suggest that they may be potential dual - contrast agents . as a negative contrast agent , magnetic nanoparticles primarily act to alter transverse relaxation time ( t2 ) values of the water proton surrounding the particles.34 when these nanoparticles present in cells ( or tissues ) and are subject to an external magnetic field , the magnetic moments of the magnetite crystals align to create large heterogeneous field gradients and dipolar coupling between the magnetic moments of water protons and the magnetic moments of particles , and cause a decrease in signal intensity in t2- and t2 * -weighted mri . magnetic nanoparticles with higher magnetic moments and transverse relaxivity can significantly shorten the relaxation time of protons . as seen in figure 5 , t2 maps of mda - mb-231 cancer cells incubated with larger m - hfn nanoparticles show darker images . for mri detection , enhancing the intracellular cell - labeling efficiency of iron oxide nanoparticles is desired.35 in this study , it shows that the largest m - hfn ( m - hfn7000 ) nanoparticles can sensitively detect cancer cells with very low cell density of 110 cells ml of mda - mb-231 cancer cells , suggesting a promising application in cancer detection . previous studies have shown that circulation half - life of magnetic nanoparticles can be reduced drastically if their size is below 10 nm,36,37 while the magnetic nanoparticles will easily be cleared by phagocyte systems , if the size is too large.38,39 for in vivo tumor imaging , m - hfn nanoparticles can be accumulated in tumor tissues via receptor - mediated pathway and not be cleared quickly.11 it was also found by acute toxic study on mice that there was no lethal effect with injection of the m - hfn nanoparticles at proposed high dose over 14 days.11 figure 6 shows that the quantitative analysis of fe uptaken is similar to the prussian blue staining . it can be seen that there is more blue deposition in cancer cells incubated with m - hfn nanoparticles with larger core size , indicating that more iron of m - hfn with larger size was endocytosed by cancer cells . this suggests that more fe atoms could be loaded into ferritin while keeping the ferritin shell unchanged ; the synthesized m - hfn nanoparticles would be more efficient for t2-weighted mri . possessing intrinsic peroxidase - like activity is one of the pronounced features of magnetic nanoparticles.4042 figures 4 and 7 show that m - hfn nanoparticles with large size of cores have better performance of the peroxidase activity . this can be attributed to the larger magnetite core having more fe sites on surface than the smaller size crystals . during the color reaction process of m - hfn nanoparticles , in the presence of h2o2 and oxidant substrate , a key issue is the generation of oh when h2o2 enters the ferritin cavity and interacts with the iron oxide core of m - hfn nanoparticles . this reaction is strongly influenced by the surface fe.43 the peroxidase - like activity of nanoparticles could be used in various applications such as glucose detection and immunohistochemical detection.44,45 the results in this study also clearly demonstrate that the m - hfn nanoparticles with larger size are capable of being more efficient for immunohistochemical staining of tumor tissues . in other words , large cores can enhance the performance of immunohistochemical staining by taking advantage of peroxidase - like activity of m - hfn nanoparticles . many efforts have been made on improving the specificity of magnetic nanoparticles to target molecules by surface - coating modification as well as improving the magnetic properties of the core . for example , ferritin shell was reengineered by fusing tumor cell - specific peptide arginine aspartate and green fluorescent protein , forming a new nanostructure for multifunctional imaging of tumor cells.15 on the other hand , gold clusters were successfully synthesized within ferritin shells , and these nanoparticles were used for kidney and liver imaging in live animals.46 the present study demonstrated that , by simply controlling the size of magnetite core , the contrast ability of m - hfn nanoparticles can be finely tuned . finally , the peroxidase - like activity of magnetic nanoparticles can be utilized to efficiently catalyze the oxidation of phenols.47 possibly , m - hfn nanoparticles with higher peroxidase - like activity could also be applied in environmental studies . the m - hfn nanoparticles with different magnetite core sizes can be synthesized by controlling the quantity of iron loading . the synthesized m - hfn nanoparticles have well - crystalline , near spherical - shaped , and ferrimagnetic magnetite cores with intact ferritin shells . lines of evidences have demonstrated that the magnetic properties , relaxivity , and peroxidase - like activity of m - hfn nanoparticles are clearly size dependent . the m - hfn nanoparticles with larger magnetite cores have superior performance in mri and immunoassay . cisowski test curves of ( a ) m - hfn1000 , ( b ) m - hfn3000 , ( c ) m - hfn5000 , and ( d ) m - hfn7000 at 5 k. r values are 0.5 , 0.48 , 0.44 , and 0.40 for m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 , respectively . abbreviations : m - hfn , ferrimagnetic h - ferritin ; irm , isothermal remanent magnetization ; h , applied magnetic field ; dcd , direct current field demagnetization ; r , magnetostatic interaction parameter . notes : hysteresis loops of m - hfn nanoparticles measured at ( a ) 300 k and ( b ) 5 k. ( c ) low - field ( 1.5 mt ) magnetization curves as a function of temperature measured after zfc and fc treatments of the m - hfn nanoparticles . abbreviations : m - hfn , ferrimagnetic h - ferritin ; zfc , zero - field cooling ; fc , field cooling ; m , magnetization ; h , magnetic field ; tb , blocking temperature . analysis of transverse relaxivity of m - hfn nanoparticles by linear mapping iron concentration and 1/t2 . notes : transverse relaxivity ( r2 ) analysis of ( a ) m - hfn1000 , ( b ) m - hfn3000 , ( c ) m - hfn5000 , and ( d ) m - hfn7000 . abbreviations : m - hfn , ferrimagnetic h - ferritin ; t2 , transverse relaxation times . analysis of longitudinal relaxivity of m - hfn nanoparticles by linear mapping iron concentration and 1/t1 . notes : longitudinal relaxivity ( r1 ) analysis of ( a ) m - hfn1000 , ( b ) m - hfn3000 , ( c ) m - hfn5000 , and ( d ) m - hfn7000 . abbreviations : m - hfn , ferrimagnetic h - ferritin ; t1 , longitudinal relaxation times .

purposethis study is to demonstrate the nanoscale size effect of ferrimagnetic h - ferritin ( m - hfn ) nanoparticles on magnetic properties , relaxivity , enzyme mimetic activities , and application in magnetic resonance imaging ( mri ) and immunohistochemical staining of cancer cells.materials and methodsm - hfn nanoparticles with different sizes of magnetite cores in the range of 2.75.3 nm were synthesized through loading different amounts of iron into recombinant human h chain ferritin ( hfn ) shells . core size , crystallinity , and magnetic properties of those m - hfn nanoparticles were analyzed by transmission electron microscope and low - temperature magnetic measurements . the mda - mb-231 cancer cells were incubated with synthesized m - hfn nanoparticles for 24 hours in dulbecco s modified eagle s medium . in vitro mri of cell pellets after m - hfn labeling was performed at 7 t. iron uptake of cells was analyzed by prussian blue staining and inductively coupled plasma mass spectrometry . immunohistochemical staining by using the peroxidase - like activity of m - hfn nanoparticles was carried out on mda - mb-231 tumor tissue paraffin sections.resultsthe saturation magnetization ( ms ) , relaxivity , and peroxidase - like activity of synthesized m - hfn nanoparticles were monotonously increased with the size of ferrimagnetic cores . the m - hfn nanoparticles with the largest core size of 5.3 nm exhibit the strongest saturation magnetization , the highest peroxidase activity in immunohistochemical staining , and the highest r2 of 321 mm1 s1 , allowing to detect mda - mb-231 breast cancer cells as low as 104 cells ml1.conclusionthe magnetic properties , relaxivity , and peroxidase - like activity of m - hfn nanoparticles are size dependent , which indicates that m - hfn nanoparticles with larger magnetite core can significantly enhance performance in mri and staining of cancer cells .

Introduction Materials and methods Preparation of recombinant ferritin cage Synthesis of M-HFn nanoparticles Characterization of synthesized M-HFn nanoparticles Uptake assay in cancer cells and MRI Peroxidase activity assay and staining of tumors Results Structure of M-HFn nanoparticles Magnetism of M-HFn nanoparticles Relaxivity and peroxidase activity of M-HFn nanoparticles Uptaken assay and MRI of cancer cells Immunohistochemical-like staining of xenografted tumor tissues Discussion Conclusion Supplementary materials

application of magnetic nanoparticles in magnetic resonance imaging ( mri ) and immunoassay is mainly dependent on the best performance of superparamagnetism below a critical size value.1 the superparamagnetic properties make magnetic nanoparticles monodisperse and exhibit high enzyme mimetic activity . among the various magnetic nanoparticles , magnetoferritin was first synthesized within horse spleen ferritin ( hosf ) cavity through removing native ferrihydrite core and replacing magnetite / maghemite core.24 recently , a novel magnetoferritin with a magnetite core , named as m - hfn , was successfully synthesized by using genetically engineered recombinant human h chain ferritin ( hfn ) as a biotemplate.57 the hfn shell is stable as a hydrophilic protein which is tolerant to heat and urea or guanidinium chloride due to the special structure with outer diameter of 12 nm and inner cavity diameter of 8 nm.8 several studies have demonstrated that without tumor - targeted ligand modification , the hfn shell of m - hfn nanoparticles can be specifically targeted to transferrin receptor 1 ( tfr1 ) overexpressed on various cancer cells,913 which allow efficiently distinguishing cancerous cells from normal cells both in vitro and in vivo . because of their high transverse relaxivity and good peroxidase - like activity,11,14 the m - hfn nanoparticles are found to be a very promising contrast agent for the in vivo detection of microscopic ( < 12 mm ) tumors by mri and in vitro detection of tumor tissues with a high sensitivity of 98% and specificity of 95%.10,11 in addition , tumor - targeted arginine aspartate peptide can be easily loaded on the hfn shell via genetic means , which make m - hfn nanoparticles specifically bind to v3 integrin implicated in tumor of a variety of cancer types.5,15 the nanoscale size plays a key role in avoiding from agglomeration , usage of mri , clinical diagnosis , and therapeutics.1620 compared with traditional magnetoferritin synthesized using hosf cavity as a template , the genetic engineering of hfn cavity provides a superior nanoplatform for synthesizing monodispersed , noninteracting , and stoichiometric magnetite nanoparticles with nearly the same spherical shape , narrow size distribution , and high crystallinity.57,14,21 thus , the m - hfn nanoparticles are ideal for studying the nanoscale size effects on magnetic properties , mri and immunoassay , and so forth . in this study , m - hfn nanoparticles with different sizes of magnetite cores were successfully synthesized through strictly controlling iron loading into hfn shells . the nanoscale size effects on application of m - hfn nanoparticles in mri and immunohistochemical staining of cancer cells were analyzed and discussed . these four sets of synthesized m - hfn nanoparticles are named here as m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 , respectively . cisowski test.22 the hysteresis loops at 5 k were also measured in fields of 3 t. the real iron content in the m - hfn nanoparticles was determined by inductively coupled plasma mass spectrometry ( icp - ms ) ( agilent , 7500a ) . in order to assess the size effect on cancer detection applications , the mda - mb-231 breast cancer cell line with high tfr1 expression was chosen based on our previous studies.10,11 cancer cells were cultured by using dulbecco s modified eagle s medium ( dmem , cat 12800 - 058 , invitrogen ) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin solution ( cat c0222 , beyotime ) at 37c in 5% co2 atmosphere . in the present study , cells were seeded into six - well polystyrene plate , and then , m - hfn nanoparticles were added until the cell attachment rate reaches about 80% , while the control group of cells was incubated in the absence of m - hfn nanoparticles . to further characterize the peroxidase - like activity of m - hfn nanoparticles , quadruple amount of sodium acetate buffer and half amount of m - hfn , h2o2 , and tmb were used in the color reaction ; mda - mb-231 tumor implantation was performed according to previously used procedure.14 the tumor tissues were harvested and kept in the form of paraffin embedded . these four sets of synthesized m - hfn nanoparticles are named here as m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 , respectively . the core structure and composition of m - hfn nanoparticles were analyzed by transmission electron microscope ( tem ) . cisowski test.22 the hysteresis loops at 5 k were also measured in fields of 3 t. the real iron content in the m - hfn nanoparticles was determined by inductively coupled plasma mass spectrometry ( icp - ms ) ( agilent , 7500a ) . in order to assess the size effect on cancer detection applications , the mda - mb-231 breast cancer cell line with high tfr1 expression was chosen based on our previous studies.10,11 cancer cells were cultured by using dulbecco s modified eagle s medium ( dmem , cat 12800 - 058 , invitrogen ) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin solution ( cat c0222 , beyotime ) at 37c in 5% co2 atmosphere . in the present study , cells were seeded into six - well polystyrene plate , and then , m - hfn nanoparticles were added until the cell attachment rate reaches about 80% , while the control group of cells was incubated in the absence of m - hfn nanoparticles . to further characterize the peroxidase - like activity of m - hfn nanoparticles , quadruple amount of sodium acetate buffer and half amount of m - hfn , h2o2 , and tmb were used in the color reaction ; mda - mb-231 tumor implantation was performed according to previously used procedure.14 the tumor tissues were harvested and kept in the form of paraffin embedded . the mean size of the magnetite cores in the m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 nanoparticles is 2.70.6 nm , 3.30.8 nm , 4.41.2 nm , and 5.31.3 nm , respectively ( figure 1d and table 1 ) , with major_axis / minor_axis ratios of 1.15 , 1.13 , 1.12 , and 1.11 , respectively . comparable protein - staining bands from lane 2 to lane 5 with native hfn protein cage in lane 1 ( figure 2b ) and the cd spectra ( figure 2c ) indicate that the protein structure of hfn cages / shells in the synthesized m - hfn nanoparticles is likely intact after the mineralization process . with the increase in core sizes , the saturation magnetization ( ms ) , coercivity ( hc ) , and blocking temperature ( tb ) of those samples measured at 5 k are increasing . the r1 values of these m - hfn nanoparticles are 10.4 mm s , 14.5 mm s , 17.4 mm s , and 22.0 mm s. this strongly indicates that the transverse and longitudinal relaxivity increase with the size of m - hfn cores . peroxidase - like activity of m - hfn nanoparticles tested at constant concentration of hfn nicely increased with size of m - hfn cores ( figure 4 ) . it is clear that the m - hfn nanoparticles with larger size of cores have higher peroxidase - like activity and absorbance . in vitro mri of mda - mb-231 tumor cells after incubation with m - hfn nanoparticles shows obvious dark images compared to cells - only control ( figure 5a ) . the mda - mb-231 cancer cells incubated with m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 have t2 values equal to 110.9 ms , 99.1 ms , 90.0 ms , and 81.2 ms , respectively , while the cells - only control ( incubated without m - hfn nanoparticles ) has a t2 value of 122.5 ms . as shown in figure 5b , the t2 values of mda - mb-231 tumor cells with cell densities of 110 cells ml , 110 cells ml , 210 cells ml , and 310 cells ml incubated with m - hfn7000 reduce to 73.5% , 66.3% , 63.2% , and 57.8% , respectively , compared with the t2 value of control ( 0 cells ml ) . to further identify iron uptaken by cancer cells , we performed prussian blue staining for cancer cells after incubation with the m - hfn nanoparticles . it can be seen that there is more blue deposition in cancer cells incubated with m - hfn nanoparticles with larger core size . we also assessed the iron content in cancer cells by icp - ms and found that cancer cells accumulated 0.16 pg cell , 0.29 pg cell , 0.80 pg cell , and 1.23 pg cell of fe after being incubated with equal amount of m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 for 24 hours , respectively ( figure 6f ) . quantitative analysis of effect on staining with m - hfn nanoparticles was performed using image pro plus software , which shows that m - hfn nanoparticles with large core size generate high value of density mean . by loading 1,000 , 3,000 , 5,000 , and 7,000 iron atoms , the synthesized m - hfn nanoparticle samples m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 , respectively , in the present study are monodispersed ( figure 1a ) , having well - crystalline cores with clear lattice fringes ( figure 1b ) . the mean size of the magnetite cores in the m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 nanoparticles is 2.70.6 nm , 3.30.8 nm , 4.41.2 nm , and 5.31.3 nm , respectively ( figure 1d and table 1 ) , with major_axis / minor_axis ratios of 1.15 , 1.13 , 1.12 , and 1.11 , respectively . comparable protein - staining bands from lane 2 to lane 5 with native hfn protein cage in lane 1 ( figure 2b ) and the cd spectra ( figure 2c ) indicate that the protein structure of hfn cages / shells in the synthesized m - hfn nanoparticles is likely intact after the mineralization process . with the increase in core sizes , the saturation magnetization ( ms ) , coercivity ( hc ) , and blocking temperature ( tb ) of those samples measured at 5 k are increasing . the r1 values of these m - hfn nanoparticles are 10.4 mm s , 14.5 mm s , 17.4 mm s , and 22.0 mm s. this strongly indicates that the transverse and longitudinal relaxivity increase with the size of m - hfn cores . peroxidase - like activity of m - hfn nanoparticles tested at constant concentration of hfn nicely increased with size of m - hfn cores ( figure 4 ) . it is clear that the m - hfn nanoparticles with larger size of cores have higher peroxidase - like activity and absorbance . in vitro mri of mda - mb-231 tumor cells after incubation with m - hfn nanoparticles shows obvious dark images compared to cells - only control ( figure 5a ) . the mda - mb-231 cancer cells incubated with m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 have t2 values equal to 110.9 ms , 99.1 ms , 90.0 ms , and 81.2 ms , respectively , while the cells - only control ( incubated without m - hfn nanoparticles ) has a t2 value of 122.5 ms . as shown in figure 5b , the t2 values of mda - mb-231 tumor cells with cell densities of 110 cells ml , 110 cells ml , 210 cells ml , and 310 cells ml incubated with m - hfn7000 reduce to 73.5% , 66.3% , 63.2% , and 57.8% , respectively , compared with the t2 value of control ( 0 cells ml ) . to further identify iron uptaken by cancer cells , we performed prussian blue staining for cancer cells after incubation with the m - hfn nanoparticles . it can be seen that there is more blue deposition in cancer cells incubated with m - hfn nanoparticles with larger core size . we also assessed the iron content in cancer cells by icp - ms and found that cancer cells accumulated 0.16 pg cell , 0.29 pg cell , 0.80 pg cell , and 1.23 pg cell of fe after being incubated with equal amount of m - hfn1000 , m - hfn3000 , m - hfn5000 , and m - hfn7000 for 24 hours , respectively ( figure 6f ) . quantitative analysis of effect on staining with m - hfn nanoparticles was performed using image pro plus software , which shows that m - hfn nanoparticles with large core size generate high value of density mean . the m - hfn nanoparticles are different from previously synthesized m - hosf nanoparticles , which were reconstituted in hosf . the m - hfn nanoparticles are directly synthesized in recombinant human hfn cavity under well - controlled condition in anaerobic box with stat ph , temperature , and addition rate of fe ( ii ) and oxidant , while the m - hosf needs to remove native ferrihydrite cores before reconstitution of magnetite cores . it has been estimated that ferritin cavity can be capable of sorting up to 4,500 fe atoms as a hydrated ferrihydrite and 8,400 fe atoms as a magnetite.21,23 in this study , out of theoretically added 7,000 iron atoms , the m - hfn nanoparticle has taken 4,709 fe atoms per hfn ( determined by icp - ms ) , forming magnetite core of 5.3 nm . owing to the nano - size effect , smaller nanoparticles may have longer circulation half - life in the body and exhibit better contrast.24,25 the smaller size of nanoparticles also influences the relaxivity and contrast enhancement.2628 it is noted in this study that the m - hfn7000 with the largest core size ( 5.3 nm ) exhibits the highest value of r2 ( 320.9 mm s ) , which is ten times higher than that of the m - hfn1000 with the smallest magnetite cores ( 2.7 nm ) . the increase in transverse relaxivity with increased size of m - hfn cores is well consistent with typical behavior of relatively small particles in motional averaging regime.19 this is mainly because the transverse relaxivity is proportional to the magnetic moment;29 that is , the larger the magnetization particles are , the higher the r2 the particles exhibit.30 the saturation magnetization of m - hfn nanoparticles synthesized here increased from 5.9 am kg to 37.3 am kg with the increasing size from 2.7 nm to 5.3 nm . as seen in figure 5 , t2 maps of mda - mb-231 cancer cells incubated with larger m - hfn nanoparticles show darker images . for mri detection , enhancing the intracellular cell - labeling efficiency of iron oxide nanoparticles is desired.35 in this study , it shows that the largest m - hfn ( m - hfn7000 ) nanoparticles can sensitively detect cancer cells with very low cell density of 110 cells ml of mda - mb-231 cancer cells , suggesting a promising application in cancer detection . previous studies have shown that circulation half - life of magnetic nanoparticles can be reduced drastically if their size is below 10 nm,36,37 while the magnetic nanoparticles will easily be cleared by phagocyte systems , if the size is too large.38,39 for in vivo tumor imaging , m - hfn nanoparticles can be accumulated in tumor tissues via receptor - mediated pathway and not be cleared quickly.11 it was also found by acute toxic study on mice that there was no lethal effect with injection of the m - hfn nanoparticles at proposed high dose over 14 days.11 figure 6 shows that the quantitative analysis of fe uptaken is similar to the prussian blue staining . it can be seen that there is more blue deposition in cancer cells incubated with m - hfn nanoparticles with larger core size , indicating that more iron of m - hfn with larger size was endocytosed by cancer cells . possessing intrinsic peroxidase - like activity is one of the pronounced features of magnetic nanoparticles.4042 figures 4 and 7 show that m - hfn nanoparticles with large size of cores have better performance of the peroxidase activity . this reaction is strongly influenced by the surface fe.43 the peroxidase - like activity of nanoparticles could be used in various applications such as glucose detection and immunohistochemical detection.44,45 the results in this study also clearly demonstrate that the m - hfn nanoparticles with larger size are capable of being more efficient for immunohistochemical staining of tumor tissues . in other words , large cores can enhance the performance of immunohistochemical staining by taking advantage of peroxidase - like activity of m - hfn nanoparticles . for example , ferritin shell was reengineered by fusing tumor cell - specific peptide arginine aspartate and green fluorescent protein , forming a new nanostructure for multifunctional imaging of tumor cells.15 on the other hand , gold clusters were successfully synthesized within ferritin shells , and these nanoparticles were used for kidney and liver imaging in live animals.46 the present study demonstrated that , by simply controlling the size of magnetite core , the contrast ability of m - hfn nanoparticles can be finely tuned . finally , the peroxidase - like activity of magnetic nanoparticles can be utilized to efficiently catalyze the oxidation of phenols.47 possibly , m - hfn nanoparticles with higher peroxidase - like activity could also be applied in environmental studies . the m - hfn nanoparticles with different magnetite core sizes can be synthesized by controlling the quantity of iron loading . lines of evidences have demonstrated that the magnetic properties , relaxivity , and peroxidase - like activity of m - hfn nanoparticles are clearly size dependent . the m - hfn nanoparticles with larger magnetite cores have superior performance in mri and immunoassay .

milk is very often described as the ultimate food which fulfills the nutritional needs of the mammal newborn and ensures safe development and growth during the first stages of its life ( mills et al . milk proteins , for instance , are known to exert various actions that promote human health . these actions include stimulation of the immune system , digestion and absorption of nutritional elements , development of endogenous defense mechanisms against bacteria , fungi and viruses , prebiotic effects , and others ( lnnerdal 2003 ) . recently , milk proteins have attracted extensive interest in terms of their bioavailability following ingestion . in vivo digestion of milk proteins within the gastrointestinal tract results in the release of peptides with new biological properties compared to the ones exhibited by the intact , precursor molecule ( chatterton et al . many of these protein fragments , also known as bioactive peptides , consist of approximately 320 amino acids , which are encrypted within the parental protein and become activated following their release , induced by the action of gastric and pancreatic enzymes ( kekkonen 2009 ; korhonen 2009 ) . hydrolysis of milk proteins is a common method for producing hydrolyzed peptide fragments used for hypoallergenic infant formulas ( korhonen et al . the peptides generated either in vitro or in vivo differ markedly with respect to their biological activities depending on the digestion process and/or the parental protein from which they are released . the identification of bioactive milk peptides was first reported in 1950 ( mellander 1950 ) . since then , numerous reports document the presence of peptides which exert some physiological effect on the human body in milk or milk - based products ( beaulieu et al . ; leblanc et al . 2002 ; meisel and fitzgerald 2000 ; nagpal et al . 2011 ) . however , to the best of our knowledge , limited studies exist which focus on the health - promoting properties of milk peptides detected in infant feeding regimens . this paper reviews recent research on the formation and fate of bioactive peptides from milk feeds intended for infants . furthermore , suggestions based on recently published research for improvement of the nutritional value of infant formulas through the inclusion of milk - derived peptides serving as functional ingredients are provided . thus , the latter are designed to simulate the content and the performance of the former in an attempt to mimic the effect of breast milk on metabolic and other physiologic functions . in addition to standard formulas , hydrolysates of cows ' milk are also available for infant nutrition usually supplied as a hypoallergenic therapeutic or preventive formulation for allergenized or sensitized newborns ( caffarelli et al . qualitative and quantitative differences exist between human milk and infant formulas , and these include protein compositional dissimilarities . for instance , cows ' milk - based infant formulas contain three to four times higher protein content compared to human milk ( rudloff and kunz 1997 ) . furthermore , caseins dominate the protein fraction in bovine milk ( 80% ) , whereas the principal class of protein in human milk is whey , particularly during the first days of lactation ( kunz and lnnerdal 1989 ; kunz and lnnerdal 1990 ) . proteomic analysis indicates that human milk does not contain -lactoglobulin and s2-casein , whereas lactoferrin and -lactalbumin are the main protein components ( armaforte et al . the same study provides further evidence on qualitative similarities in terms of the protein profile of infant formula and bovine milk with s1-casein , s2-casein , -casein , and -lactoglobulin being the most abundant proteins . other major differences between human and bovine milk proteins such as the different glycosylation degree of immunoglobulins , lactoferrin , and casein have been documented ( nwosu et al . 2012 ) . commercially available products include whey - enriched infants formulas based on cow 's milk . in this case , formulations include a casein - to - whey protein ratio of approximately 40:60 , which is somewhat closer to the one encountered in human milk . whey - dominant formulas which resemble the human milk compositional standard seem to a have the same impact on growth of premature infants as compared to casein - dominant formulations ( bernbaum et al . however , other advantages particularly for low - birth weight and preterm infants may be related to whey - dominant formulas , which is an expected finding as milk is a biological fluid that has evolved to meet the nutritional demands of the human species ( rudloff and kunz 1997 ) . other attempts to narrow the gap between the protein content of human milk and infant formulas involve the fortification of infant formulas with purified proteins of bovine origin , which are abundant in human milk such as -lactalbumin , lactoferrin , and others ( chatterton et al . 2004 ) . despite the efforts to replicate the protein compositional recipe of human milk , infant formulas and protein hydrolysate products display qualitative and quantitative differences in terms of protein content that may affect physiological process in the newborn . in general , formulas provide a different profile of amino acids in addition to more protein content . differences in the parental ( intact ) proteins result most likely in the generation of peptides with different biological activities following the process of digestion . it has been documented that hydrolysis products of the native human -caseins generated by the proteolytic activity of plasmin can not be detected in commercial formulations intended for infants ( armaforte et al . this is because bovine plasmin preferentially acts upon -casein ( fox and kelly 2005 ) . as a result , considerable differences are expected in terms of bioavailability and metabolic effects between the biologically active fragments generated following ingestion of human milk , infant formulas , and hydrolyzed products , which may affect infant growth . first , milk proteins may be digested in vitro during food processing by maturation with selected enzymes . in this case , where milk is fermented with a proteolytic culture , the process is carefully controlled ( enzyme specificity , temperature , incubation time , etc . ) , and thus the release of bioactive peptides may be predicted ( exl 2001 ) . common enzymes in the commercial production of bioactive peptides via in vitro digestion include pepsin , trypsin , and chymotrypsin ( korhonen et al . 1998 ) . bioactive peptides may also be generated following ingestion in vivo by either digestive enzymes or by microbial enzymes ( kekkonen 2009 ) . thus milk proteins during their passage through the digestive system may be the target of enzymes produced either by the human body or by microorganisms which normally inhabit the gastrointestinal tract . a wide range of enzymes with different cleavage sites may act throughout the whole gastrointestinal tract and under conditions which may differ depending on various physiological factors ( antalis et al . 2007 ) . in this case , the process is far less easy to control and to monitor . this is the reason why in vitro digestion , also known as simulated gastrointestinal digestion ( sgid ) , is often employed to investigate the stability of milk proteins and the fate of bioactive peptides . sgid takes into account key factors affecting the digestion process such as the rates of gastric emptying , which are used to determine the period of incubation with gastric proteases , and changes in gastric ph value which determine to a large extent the enzyme activity ( de noni 2008 ) . nevertheless , sgid may be regarded as an oversimplified method to determine the fate of precursor milk proteins . in reality , things are more complicated as physiological processes in vivo are not static , and the environment within the gastrointestinal tract of an infant is subject to changes with time or cause . gastric ph , for instance , is known to be high in early neonatal life and has been determined to be 6.0 in term newborn infants with the tendency to decrease sharply during the first 24 h of life ( widstrom et al . ( 2004 ) , proteins in human milk and infant formula showed a ph - dependent extent of hydrolysis when incubated with gastric juice in a ph range between 6.5 and 2.0 . furthermore , the concentration of milk proteases ( e.g. , plasmin ) present in breast milk is increased for premature infants , which results in extensive hydrolysis of milk proteins to facilitate the digestion process ( armaforte et al . this could in turn affect the biological activity of the digests , as peptides have to be absorbed from the intestine and reach their target cells in adequate concentrations . as a result , one may conclude that the digestion process of milk proteins in vivo is a complex process and depends on various factors which can not be adequately simulated in vitro . in vivo studies are more reliable in order to draw safe conclusions with respect to the formation of bioactive peptides following the ingestion of human milk and infant formulas . the formation of bioactive peptides in vivo following the digestion of milk proteins has been documented in the past both in human ( boutrou et al . 2013 ; foltz et al . 2007 ) and animal ( bouzerzour et al . 2012 ) models . according to these studies , although evidence exists to support their presence in the human and piglet gastrointestinal tract , further research is required to investigate the physiologic conditions which would allow the bioactive peptides to exert their bioactivity . peptides released from in vivo or in vitro digestion of milk proteins are known to affect major systems such as the cardiovascular , nervous , digestive , and immune system . according to hayes et al . ( 2007 ) , bioactive peptides from milk having a physiological impact on human body can be categorized as antihypertensive , antithrombotic , opioid , casein phosphopeptides , antimicrobial , cytomodulatory , immunomodulatory , and miscellaneous . recent findings indicate that milk peptides can have additional physiological effects such as antioxidant activity or may even possess functional significance which is not yet clearly understood . therefore , the consideration of milk peptides as dietary supplements or food additives could be promising for the food industry in the future . table 1 summarizes the bioactive peptides identified in both human and bovine milk with potential beneficial effect on human health.table 1biofunctional peptides derived from human and bovine milk proteins with effects on human healthfunctionprecursor proteinfragmentpeptide sequenceoriginreferenceantihypertensive-lactoglobulinf ( 4042)rvybovine(hernndez - ledesma et al . 1995)opioid-caseinf ( 6066)ypfpgpibovine(de noni , 2008 ; de noni and cattaneo , 2010)opioid-caseinf ( 6064)ypfpgbovine(jarmolowska et al . 2003 ) biofunctional peptides derived from human and bovine milk proteins with effects on human health the release of angiotensin converting enzymes ( ace)-inhibitory peptides by sgid of infant formulas has been documented by hernndez - ledesma et al . according to this study , enzymatically hydrolyzed casein and whey formulas exhibited higher ace - inhibitory activities than non - hydrolyzed milk protein formulations . it was hypothesized that low molecular weight peptides ( < 3,000 da ) were mainly responsible for this activity with several potent peptides being identified ( -lg : rvy and lvr ; -casein : ypfpgpi and hlplp ) . the size of the hydrolyzed fragments could be critical in terms of the antihypertensive activity as di- and tripeptides are easily absorbed in the intestine and may reach the blood and other target sites ( hara et al . 1984 ) . furthermore , sgid had a positive impact on the unhydrolyzed samples and did not affect the hydrolyzed whey - based formulas with respect to ace inhibition . 2008 ) who compared the ace - inhibitory activity of hypoallergenic formulas and breast milk . the dipeptide ile - trp ( iw ) , present at positions 5960 in bovine -lactalbumin from hypoallergenic formulas , was identified as the most potent ace inhibitor in this study . according to the authors , conventional milk - based formulas and breast milk samples did not exhibit ace inhibition . in agreement with the previous studies , sgid did not affect the overall inhibitory effect of the hydrolysate formula , indicating that the peptides survive gastrointestinal hydrolysis . the presence of peptides with potent ace - inhibitory activity was also confirmed in more recent studies with hypoallergenic formulas containing partially hydrolyzed whey , highly hydrolyzed whey and casein , and highly hydrolyzed casein bovine proteins ( catal - clariana et al . 2010 ) . in this case , five bioactive peptides namely lkp , ipy , alpm , pgpihn , and vagtwy were detected by means of capillary electrophoresis time - of - flight mass spectrometry . in other studies , breast milk and bovine milk protein - based infant formulas were digested with pepsin and pancreatin and the contribution of the fragments generated to ace - inhibitory activity was determined ( hernndez - ledesma et al . this study revealed that the peptide fraction obtained from human breast milk showed limited ace - inhibition compared with the one exhibited by the corresponding fraction obtained from infant formulas . according to the authors , this is due to the different peptide fragments obtained from human milk samples and infant formulas following hydrolysis with the specific enzymes . interestingly , only one peptide ( hlplpl ) was common between the digests of human and bovine origin . nevertheless , a potent ace inhibitor was identified in human milk and corresponded to the -casein fragment f(125129 ) with the sequence hlplp . in this case , it was hypothesized that the presence of the amino acid proline at the c - terminal end could be crucial in terms of the activity observed ( cheung et al . 1980 ) . finally , the peptide f 80 - 90 ( tpvvvppflqp ) from -casein which is known to possess antihypertensive activities was detected in animal models fed with infant formulas ( bouzerzour et al . , piglets were used as a model to study aspects of protein digestion and absorption in human infants . the production of reactive oxygen species ( ros ) is part of normal cell metabolism . in some cases , such as in preterm infants , high levels of ros are produced that disturb the redox balance and result in a state formerly known as oxidative stress ( buonocore et al . oxidative stress induced by free radicals is linked to the pathogenesis of various diseases including heart disease ( aruoma 1998 ) . the antioxidant potential of human milk has been documented in the past ( friel et al . according to samaranayaka and li - chan ( 2011 ) , peptides and proteins are implicated in the overall antioxidative status of cells and contribute towards maintaining the health of biological tissue . recent findings indicate that peptides released from human milk after digestion with pepsin and pancreatin could be beneficial with respect to antioxidant activity ( tsopmo et al . the peptides ygytga and iselgw showed high radical scavenging activity , and this property was attributed to the presence of the amino acid tryptophan which can break radical chain reactions thanks to the donation of the hydrogen attached to the nitrogen of its indole ring . the same hypothesis was made by other researchers who reported moderate antioxidant activity by peptides derived from digested human samples with pepsin and pancreatin ( hernndez - ledesma et al . 2007 ) . in this case , the peptide with the highest antioxidant activity was wsvpqpk , which also contains tryptophan and , thus , may serve as hydrogen donor and/or free radical scavenger . peptides of human ( iaippkkiqdk ) and bovine ( maippkknqdk ) origin with antithrombotic effects were detected at physiologically active levels in the plasma of newborn infants after breastfeeding or ingestion of cow milk - based formula ( chabance et al . 1995 ) . these peptides , known as -caseinoglycopeptides , exert their biological function by reducing platelet aggregation and enhancing fibrinolysis , which results in the prevention of thrombosis development . opioid peptides are small molecules which are synthesized in vivo and may function both as hormones and neurotransmitters . -casomorphins derived from -casein , and particularly -casomorphin-5 ( bcm5 ) and -casomorphin-7 ( bcm7 ) , are the best - known opioid peptides ( schlimme et al . the release of bcm7 from commercial milk - based infant formulas following sgid was confirmed ( de noni 2008 ) . the peptides identified were released from genetic variants a1 and b of -casein . according to the european food safety authority , there is no established relationship between the dietary intake of bcm7 and non - communicable diseases ( efsa european food safety authority 2009 ) . although no safe conclusions can be drawn at the moment regarding the minimum amount of bcm7 capable to elicit pharmacological effects in vivo or ex vivo , further studies confirm its release by the action of gastrointestinal proteases during in vitro digestion in commercial infant formulas at concentrations between 0.04 and 0.21 mg.l ( de noni and cattaneo 2010 ) . the presence of four opioid peptides in the commercially available humana formula ( humana gmbh , germany ) for newborns after digestion with pepsin and trypsin has been documented ( jarmolowska et al . both opioid agonists ( bcm5 ) and antagonists ( casoxin c , casoxin 6 , and lactoferroxin a ) deriving from - , -casein , and lactoferrin were identified . all four opioid peptides were detected in the formulas prior to enzymatic hydrolysis at concentrations between 0.075 to 0.39 g.ml with their concentration to rise sharply after enzyme hydrolysis . the presence of bcm5 and bcm7 in human milk has also been confirmed with the content of the opioid peptides being dependent on the lactation stage ( jarmolowska et al . bcm7 and related compounds have been linked in the past with the development of human diseases such as juvenile diabetes , heart disease , autism , and schizophrenia ( mclachlan , 2001 ) . although evidence regarding the abundance of such peptides in infant formulas is inconclusive , the formation of bcms in infant feeds following sgid has been demonstrated . the in vivo effects of these peptides on the human physiology have not yet been fully described . it has been demonstrated that protein peptides exert biological action on the metabolism of minerals . they participate in bone structure and teeth and are involved in several metabolic processes such as energy production , nutrient absorption , protein building , and blood formation ( soetan et al . casein phosphopeptides ( cpps ) deriving from the digestion of milk proteins may act as mineral carriers and affect their bioavailability in the human body ( meisel and fitzgerald 2003 ) . recent studies document the presence of bioactive peptides with mineral carrier properties in infant formulas ( miquel et al . the peptides originating from s1 and s2-casein survived sgid with pepsin and pancreatin , and their identified molecular masses were in the range of 1,400 to 9,600 da , which makes them good candidates for intestinal absorption . according to the authors , calcium could be bound preferentially to cpps with the cluster sequence spspspee and iron and zinc to cpps with the phosphorylated cluster and phosphoserine residues . the release of cpps with metal - chelating properties following sgid of infant formulas was also reported in previous studies ( miquel et al . 2005 ) . many of these cpps were only detected in probiotic formulas indicating that they are formed by the action of bifidobacteria . dietary protein is known to play a major role in the human body 's defense mechanism against infection by either stimulating the immune system ( immunomodulatory ) or contributing to the inhibition of microbial growth ( antimicrobial ) . supplementation of infant formulas with -lactalbumin and glycomacropeptide ( gmp ) , a derivative of -casein , which were digested with pepsin and pancreatin , resulted in decreased counts of bacteroides ( brck et al . this was attributed to the ability of the supplemented formula to mimic the fermentative qualities of breast milk and induce the growth of protective microflora in the gastrointestinal tract . furthermore , the bacteriostatic and bactericidal effects of -lactalbumin and gmp have been demonstrated when a significant reduction in enteropathogenic bacteria including enteropathogenic escherichia coli and salmonella was observed following supplementation of the infant formulas with the specific milk substrates ( brck et al . the release of bioactive peptides with immunomodulatory activities from infant formulas is also documented by more recent studies . in this case , the peptide f 60 - 66 ( ypfpgpi ) from -casein was detected following in vivo digestion by piglets which were fed with infant formulas ( bouzerzour et al . according to the findings of this study , whey proteins were more resistant to enzymatic digestion in the stomach of piglets compared to caseins with the latter being hydrolyzed to a large extent just 30 min after meal ingestion . the release of angiotensin converting enzymes ( ace)-inhibitory peptides by sgid of infant formulas has been documented by hernndez - ledesma et al . according to this study , enzymatically hydrolyzed casein and whey formulas exhibited higher ace - inhibitory activities than non - hydrolyzed milk protein formulations . it was hypothesized that low molecular weight peptides ( < 3,000 da ) were mainly responsible for this activity with several potent peptides being identified ( -lg : rvy and lvr ; -casein : ypfpgpi and hlplp ) . the size of the hydrolyzed fragments could be critical in terms of the antihypertensive activity as di- and tripeptides are easily absorbed in the intestine and may reach the blood and other target sites ( hara et al . 1984 ) . furthermore , sgid had a positive impact on the unhydrolyzed samples and did not affect the hydrolyzed whey - based formulas with respect to ace inhibition . 2008 ) who compared the ace - inhibitory activity of hypoallergenic formulas and breast milk . the dipeptide ile - trp ( iw ) , present at positions 5960 in bovine -lactalbumin from hypoallergenic formulas , was identified as the most potent ace inhibitor in this study . according to the authors , conventional milk - based formulas and breast milk samples did not exhibit ace inhibition . in agreement with the previous studies , sgid did not affect the overall inhibitory effect of the hydrolysate formula , indicating that the peptides survive gastrointestinal hydrolysis . the presence of peptides with potent ace - inhibitory activity was also confirmed in more recent studies with hypoallergenic formulas containing partially hydrolyzed whey , highly hydrolyzed whey and casein , and highly hydrolyzed casein bovine proteins ( catal - clariana et al . , five bioactive peptides namely lkp , ipy , alpm , pgpihn , and vagtwy were detected by means of capillary electrophoresis time - of - flight mass spectrometry . in other studies , breast milk and bovine milk protein - based infant formulas were digested with pepsin and pancreatin and the contribution of the fragments generated to ace - inhibitory activity was determined ( hernndez - ledesma et al . 2007 ) . this study revealed that the peptide fraction obtained from human breast milk showed limited ace - inhibition compared with the one exhibited by the corresponding fraction obtained from infant formulas . according to the authors , this is due to the different peptide fragments obtained from human milk samples and infant formulas following hydrolysis with the specific enzymes . interestingly , only one peptide ( hlplpl ) was common between the digests of human and bovine origin . nevertheless , a potent ace inhibitor was identified in human milk and corresponded to the -casein fragment f(125129 ) with the sequence hlplp . in this case , it was hypothesized that the presence of the amino acid proline at the c - terminal end could be crucial in terms of the activity observed ( cheung et al . 1980 ) . finally , the peptide f 80 - 90 ( tpvvvppflqp ) from -casein which is known to possess antihypertensive activities was detected in animal models fed with infant formulas ( bouzerzour et al . 2012 ) . in this study , piglets were used as a model to study aspects of protein digestion and absorption in human infants . the production of reactive oxygen species ( ros ) is part of normal cell metabolism . in some cases , such as in preterm infants , high levels of ros are produced that disturb the redox balance and result in a state formerly known as oxidative stress ( buonocore et al . oxidative stress induced by free radicals is linked to the pathogenesis of various diseases including heart disease ( aruoma 1998 ) . the antioxidant potential of human milk has been documented in the past ( friel et al . according to samaranayaka and li - chan ( 2011 ) , peptides and proteins are implicated in the overall antioxidative status of cells and contribute towards maintaining the health of biological tissue . recent findings indicate that peptides released from human milk after digestion with pepsin and pancreatin could be beneficial with respect to antioxidant activity ( tsopmo et al . 2011 ) . in this study , the peptides ygytga and iselgw showed high radical scavenging activity , and this property was attributed to the presence of the amino acid tryptophan which can break radical chain reactions thanks to the donation of the hydrogen attached to the nitrogen of its indole ring . the same hypothesis was made by other researchers who reported moderate antioxidant activity by peptides derived from digested human samples with pepsin and pancreatin ( hernndez - ledesma et al . , the peptide with the highest antioxidant activity was wsvpqpk , which also contains tryptophan and , thus , may serve as hydrogen donor and/or free radical scavenger . peptides of human ( iaippkkiqdk ) and bovine ( maippkknqdk ) origin with antithrombotic effects were detected at physiologically active levels in the plasma of newborn infants after breastfeeding or ingestion of cow milk - based formula ( chabance et al . these peptides , known as -caseinoglycopeptides , exert their biological function by reducing platelet aggregation and enhancing fibrinolysis , which results in the prevention of thrombosis development . opioid peptides are small molecules which are synthesized in vivo and may function both as hormones and neurotransmitters . these peptides depending on their bioactivity -casomorphins derived from -casein , and particularly -casomorphin-5 ( bcm5 ) and -casomorphin-7 ( bcm7 ) , are the best - known opioid peptides ( schlimme et al . the release of bcm7 from commercial milk - based infant formulas following sgid was confirmed ( de noni 2008 ) . the peptides identified were released from genetic variants a1 and b of -casein . according to the european food safety authority , there is no established relationship between the dietary intake of bcm7 and non - communicable diseases ( efsa european food safety authority 2009 ) . although no safe conclusions can be drawn at the moment regarding the minimum amount of bcm7 capable to elicit pharmacological effects in vivo or ex vivo , further studies confirm its release by the action of gastrointestinal proteases during in vitro digestion in commercial infant formulas at concentrations between 0.04 and 0.21 mg.l ( de noni and cattaneo 2010 ) . the presence of four opioid peptides in the commercially available humana formula ( humana gmbh , germany ) for newborns after digestion with pepsin and trypsin has been documented ( jarmolowska et al . both opioid agonists ( bcm5 ) and antagonists ( casoxin c , casoxin 6 , and lactoferroxin a ) deriving from - , -casein , and lactoferrin were identified . all four opioid peptides were detected in the formulas prior to enzymatic hydrolysis at concentrations between 0.075 to 0.39 g.ml with their concentration to rise sharply after enzyme hydrolysis . the presence of bcm5 and bcm7 in human milk has also been confirmed with the content of the opioid peptides being dependent on the lactation stage ( jarmolowska et al . bcm7 and related compounds have been linked in the past with the development of human diseases such as juvenile diabetes , heart disease , autism , and schizophrenia ( mclachlan , 2001 ) . although evidence regarding the abundance of such peptides in infant formulas is inconclusive , the formation of bcms in infant feeds following sgid has been demonstrated . the in vivo effects of these peptides on the human physiology have not yet been fully described . it has been demonstrated that protein peptides exert biological action on the metabolism of minerals . they participate in bone structure and teeth and are involved in several metabolic processes such as energy production , nutrient absorption , protein building , and blood formation ( soetan et al . casein phosphopeptides ( cpps ) deriving from the digestion of milk proteins may act as mineral carriers and affect their bioavailability in the human body ( meisel and fitzgerald 2003 ) . recent studies document the presence of bioactive peptides with mineral carrier properties in infant formulas ( miquel et al . 2006 ) . the peptides originating from s1 and s2-casein survived sgid with pepsin and pancreatin , and their identified molecular masses were in the range of 1,400 to 9,600 da , which makes them good candidates for intestinal absorption . according to the authors , calcium could be bound preferentially to cpps with the cluster sequence spspspee and iron and zinc to cpps with the phosphorylated cluster and phosphoserine residues . the release of cpps with metal - chelating properties following sgid of infant formulas was also reported in previous studies ( miquel et al . 2005 ) . many of these cpps were only detected in probiotic formulas indicating that they are formed by the action of bifidobacteria . dietary protein is known to play a major role in the human body 's defense mechanism against infection by either stimulating the immune system ( immunomodulatory ) or contributing to the inhibition of microbial growth ( antimicrobial ) . supplementation of infant formulas with -lactalbumin and glycomacropeptide ( gmp ) , a derivative of -casein , which were digested with pepsin and pancreatin , resulted in decreased counts of bacteroides ( brck et al . this was attributed to the ability of the supplemented formula to mimic the fermentative qualities of breast milk and induce the growth of protective microflora in the gastrointestinal tract . furthermore , the bacteriostatic and bactericidal effects of -lactalbumin and gmp have been demonstrated when a significant reduction in enteropathogenic bacteria including enteropathogenic escherichia coli and salmonella was observed following supplementation of the infant formulas with the specific milk substrates ( brck et al . the release of bioactive peptides with immunomodulatory activities from infant formulas is also documented by more recent studies . in this case , the peptide f 60 - 66 ( ypfpgpi ) from -casein was detected following in vivo digestion by piglets which were fed with infant formulas ( bouzerzour et al . according to the findings of this study , whey proteins were more resistant to enzymatic digestion in the stomach of piglets compared to caseins with the latter being hydrolyzed to a large extent just 30 min after meal ingestion . extensive research has underlined the important contribution of breastfeeding in promoting general health , growth , and development of the human species and the significant reduction in the risk of acute and chronic diseases ( gartner et al . proteins contribute to the nutritional value of milk and also have other biological roles as intact molecules , as many of them are resistant to the effects of digestion ( chatterton et al . 2004 ) . on the other hand , digestion of milk proteins in the gastrointestinal tract results in the release of bioactive agents which may affect several physiological processes . despite the fact that many of these bioactive peptides have been identified as integral parts of milk proteins , furthermore , limited information is available with respect to their passage through the human , and most importantly the infant 's digestive tract . up - to - date , bioactive peptides have been identified in both human milk and cow milk - based infant formulas , which differ in their amino acid profiles and properties . modern methods of analysis enable fast and reliable separation and identification of bioactive peptides in milk - derived products ( catal - clariana et al . the peptides identified following sgid have antihypertensive , antioxidant , antithrombotic , opioid , mineral carrier , and immunogenic effects on human physiology . further research in this field is required to establish the beneficial role of human or bovine milk from that perspective . it seems likely though that peptides from milk protein of bovine origin could be a valuable supplement to human milk . the manufacture of chimeric infant formulas with multiple health - promoting effects could be used as a model example in the future for the manufacture of ultimately functional foods .

milk proteins have attracted extensive interest in terms of their bioavailability following ingestion . enzymatic digestion of dairy products generates numerous peptides with various biological activities . both human milk and infant formulas based on cow 's milk are potential sources of bioactive peptides . this review aims to present current knowledge on the formation and fate of bioactive peptides from milk feeds intended for infants . emphasis is placed on the source of the bioactive peptides with the nutritional impact of human milk and cow milk - based formulas on infant health being critically discussed from that perspective . furthermore , the effect of processing and in vitro or in vivo digestion on the release and availability of peptides with bioactive sequences is evaluated . considerable differences with respect to bioavailability and metabolic effects between the biologically active fragments generated following ingestion of human milk and infant formulas are documented . peptides from milk protein of bovine origin could be a valuable supplement to human milk as multiple health - promoting properties are attributed to peptide fractions identified in standard cow milk - based infant formulas .

Introduction Human milk versus infant formula as a potential source of bioactive peptides Aspects of in vivo and in vitro generation of bioactive peptides Bioactive peptides in human milk and infant formulas Effects on the cardiovascular system Effects on the nervous system Other health-promoting effects Conclusions

recently , milk proteins have attracted extensive interest in terms of their bioavailability following ingestion . in vivo digestion of milk proteins within the gastrointestinal tract results in the release of peptides with new biological properties compared to the ones exhibited by the intact , precursor molecule ( chatterton et al . hydrolysis of milk proteins is a common method for producing hydrolyzed peptide fragments used for hypoallergenic infant formulas ( korhonen et al . the peptides generated either in vitro or in vivo differ markedly with respect to their biological activities depending on the digestion process and/or the parental protein from which they are released . since then , numerous reports document the presence of peptides which exert some physiological effect on the human body in milk or milk - based products ( beaulieu et al . however , to the best of our knowledge , limited studies exist which focus on the health - promoting properties of milk peptides detected in infant feeding regimens . this paper reviews recent research on the formation and fate of bioactive peptides from milk feeds intended for infants . furthermore , suggestions based on recently published research for improvement of the nutritional value of infant formulas through the inclusion of milk - derived peptides serving as functional ingredients are provided . thus , the latter are designed to simulate the content and the performance of the former in an attempt to mimic the effect of breast milk on metabolic and other physiologic functions . qualitative and quantitative differences exist between human milk and infant formulas , and these include protein compositional dissimilarities . for instance , cows ' milk - based infant formulas contain three to four times higher protein content compared to human milk ( rudloff and kunz 1997 ) . furthermore , caseins dominate the protein fraction in bovine milk ( 80% ) , whereas the principal class of protein in human milk is whey , particularly during the first days of lactation ( kunz and lnnerdal 1989 ; kunz and lnnerdal 1990 ) . the same study provides further evidence on qualitative similarities in terms of the protein profile of infant formula and bovine milk with s1-casein , s2-casein , -casein , and -lactoglobulin being the most abundant proteins . commercially available products include whey - enriched infants formulas based on cow 's milk . however , other advantages particularly for low - birth weight and preterm infants may be related to whey - dominant formulas , which is an expected finding as milk is a biological fluid that has evolved to meet the nutritional demands of the human species ( rudloff and kunz 1997 ) . other attempts to narrow the gap between the protein content of human milk and infant formulas involve the fortification of infant formulas with purified proteins of bovine origin , which are abundant in human milk such as -lactalbumin , lactoferrin , and others ( chatterton et al . despite the efforts to replicate the protein compositional recipe of human milk , infant formulas and protein hydrolysate products display qualitative and quantitative differences in terms of protein content that may affect physiological process in the newborn . differences in the parental ( intact ) proteins result most likely in the generation of peptides with different biological activities following the process of digestion . it has been documented that hydrolysis products of the native human -caseins generated by the proteolytic activity of plasmin can not be detected in commercial formulations intended for infants ( armaforte et al . as a result , considerable differences are expected in terms of bioavailability and metabolic effects between the biologically active fragments generated following ingestion of human milk , infant formulas , and hydrolyzed products , which may affect infant growth . first , milk proteins may be digested in vitro during food processing by maturation with selected enzymes . , and thus the release of bioactive peptides may be predicted ( exl 2001 ) . common enzymes in the commercial production of bioactive peptides via in vitro digestion include pepsin , trypsin , and chymotrypsin ( korhonen et al . bioactive peptides may also be generated following ingestion in vivo by either digestive enzymes or by microbial enzymes ( kekkonen 2009 ) . this is the reason why in vitro digestion , also known as simulated gastrointestinal digestion ( sgid ) , is often employed to investigate the stability of milk proteins and the fate of bioactive peptides . nevertheless , sgid may be regarded as an oversimplified method to determine the fate of precursor milk proteins . ( 2004 ) , proteins in human milk and infant formula showed a ph - dependent extent of hydrolysis when incubated with gastric juice in a ph range between 6.5 and 2.0 . furthermore , the concentration of milk proteases ( e.g. as a result , one may conclude that the digestion process of milk proteins in vivo is a complex process and depends on various factors which can not be adequately simulated in vitro . in vivo studies are more reliable in order to draw safe conclusions with respect to the formation of bioactive peptides following the ingestion of human milk and infant formulas . the formation of bioactive peptides in vivo following the digestion of milk proteins has been documented in the past both in human ( boutrou et al . according to these studies , although evidence exists to support their presence in the human and piglet gastrointestinal tract , further research is required to investigate the physiologic conditions which would allow the bioactive peptides to exert their bioactivity . peptides released from in vivo or in vitro digestion of milk proteins are known to affect major systems such as the cardiovascular , nervous , digestive , and immune system . ( 2007 ) , bioactive peptides from milk having a physiological impact on human body can be categorized as antihypertensive , antithrombotic , opioid , casein phosphopeptides , antimicrobial , cytomodulatory , immunomodulatory , and miscellaneous . therefore , the consideration of milk peptides as dietary supplements or food additives could be promising for the food industry in the future . table 1 summarizes the bioactive peptides identified in both human and bovine milk with potential beneficial effect on human health.table 1biofunctional peptides derived from human and bovine milk proteins with effects on human healthfunctionprecursor proteinfragmentpeptide sequenceoriginreferenceantihypertensive-lactoglobulinf ( 4042)rvybovine(hernndez - ledesma et al . 2003 ) biofunctional peptides derived from human and bovine milk proteins with effects on human health the release of angiotensin converting enzymes ( ace)-inhibitory peptides by sgid of infant formulas has been documented by hernndez - ledesma et al . the size of the hydrolyzed fragments could be critical in terms of the antihypertensive activity as di- and tripeptides are easily absorbed in the intestine and may reach the blood and other target sites ( hara et al . furthermore , sgid had a positive impact on the unhydrolyzed samples and did not affect the hydrolyzed whey - based formulas with respect to ace inhibition . according to the authors , conventional milk - based formulas and breast milk samples did not exhibit ace inhibition . in agreement with the previous studies , sgid did not affect the overall inhibitory effect of the hydrolysate formula , indicating that the peptides survive gastrointestinal hydrolysis . in other studies , breast milk and bovine milk protein - based infant formulas were digested with pepsin and pancreatin and the contribution of the fragments generated to ace - inhibitory activity was determined ( hernndez - ledesma et al . this study revealed that the peptide fraction obtained from human breast milk showed limited ace - inhibition compared with the one exhibited by the corresponding fraction obtained from infant formulas . according to the authors , this is due to the different peptide fragments obtained from human milk samples and infant formulas following hydrolysis with the specific enzymes . interestingly , only one peptide ( hlplpl ) was common between the digests of human and bovine origin . nevertheless , a potent ace inhibitor was identified in human milk and corresponded to the -casein fragment f(125129 ) with the sequence hlplp . in this case , it was hypothesized that the presence of the amino acid proline at the c - terminal end could be crucial in terms of the activity observed ( cheung et al . finally , the peptide f 80 - 90 ( tpvvvppflqp ) from -casein which is known to possess antihypertensive activities was detected in animal models fed with infant formulas ( bouzerzour et al . the antioxidant potential of human milk has been documented in the past ( friel et al . recent findings indicate that peptides released from human milk after digestion with pepsin and pancreatin could be beneficial with respect to antioxidant activity ( tsopmo et al . the peptides ygytga and iselgw showed high radical scavenging activity , and this property was attributed to the presence of the amino acid tryptophan which can break radical chain reactions thanks to the donation of the hydrogen attached to the nitrogen of its indole ring . in this case , the peptide with the highest antioxidant activity was wsvpqpk , which also contains tryptophan and , thus , may serve as hydrogen donor and/or free radical scavenger . peptides of human ( iaippkkiqdk ) and bovine ( maippkknqdk ) origin with antithrombotic effects were detected at physiologically active levels in the plasma of newborn infants after breastfeeding or ingestion of cow milk - based formula ( chabance et al . the release of bcm7 from commercial milk - based infant formulas following sgid was confirmed ( de noni 2008 ) . although no safe conclusions can be drawn at the moment regarding the minimum amount of bcm7 capable to elicit pharmacological effects in vivo or ex vivo , further studies confirm its release by the action of gastrointestinal proteases during in vitro digestion in commercial infant formulas at concentrations between 0.04 and 0.21 mg.l ( de noni and cattaneo 2010 ) . the presence of bcm5 and bcm7 in human milk has also been confirmed with the content of the opioid peptides being dependent on the lactation stage ( jarmolowska et al . bcm7 and related compounds have been linked in the past with the development of human diseases such as juvenile diabetes , heart disease , autism , and schizophrenia ( mclachlan , 2001 ) . although evidence regarding the abundance of such peptides in infant formulas is inconclusive , the formation of bcms in infant feeds following sgid has been demonstrated . the in vivo effects of these peptides on the human physiology have not yet been fully described . casein phosphopeptides ( cpps ) deriving from the digestion of milk proteins may act as mineral carriers and affect their bioavailability in the human body ( meisel and fitzgerald 2003 ) . recent studies document the presence of bioactive peptides with mineral carrier properties in infant formulas ( miquel et al . the release of cpps with metal - chelating properties following sgid of infant formulas was also reported in previous studies ( miquel et al . this was attributed to the ability of the supplemented formula to mimic the fermentative qualities of breast milk and induce the growth of protective microflora in the gastrointestinal tract . furthermore , the bacteriostatic and bactericidal effects of -lactalbumin and gmp have been demonstrated when a significant reduction in enteropathogenic bacteria including enteropathogenic escherichia coli and salmonella was observed following supplementation of the infant formulas with the specific milk substrates ( brck et al . the release of bioactive peptides with immunomodulatory activities from infant formulas is also documented by more recent studies . in this case , the peptide f 60 - 66 ( ypfpgpi ) from -casein was detected following in vivo digestion by piglets which were fed with infant formulas ( bouzerzour et al . according to the findings of this study , whey proteins were more resistant to enzymatic digestion in the stomach of piglets compared to caseins with the latter being hydrolyzed to a large extent just 30 min after meal ingestion . the release of angiotensin converting enzymes ( ace)-inhibitory peptides by sgid of infant formulas has been documented by hernndez - ledesma et al . the size of the hydrolyzed fragments could be critical in terms of the antihypertensive activity as di- and tripeptides are easily absorbed in the intestine and may reach the blood and other target sites ( hara et al . furthermore , sgid had a positive impact on the unhydrolyzed samples and did not affect the hydrolyzed whey - based formulas with respect to ace inhibition . according to the authors , conventional milk - based formulas and breast milk samples did not exhibit ace inhibition . in agreement with the previous studies , sgid did not affect the overall inhibitory effect of the hydrolysate formula , indicating that the peptides survive gastrointestinal hydrolysis . the presence of peptides with potent ace - inhibitory activity was also confirmed in more recent studies with hypoallergenic formulas containing partially hydrolyzed whey , highly hydrolyzed whey and casein , and highly hydrolyzed casein bovine proteins ( catal - clariana et al . in other studies , breast milk and bovine milk protein - based infant formulas were digested with pepsin and pancreatin and the contribution of the fragments generated to ace - inhibitory activity was determined ( hernndez - ledesma et al . this study revealed that the peptide fraction obtained from human breast milk showed limited ace - inhibition compared with the one exhibited by the corresponding fraction obtained from infant formulas . according to the authors , this is due to the different peptide fragments obtained from human milk samples and infant formulas following hydrolysis with the specific enzymes . interestingly , only one peptide ( hlplpl ) was common between the digests of human and bovine origin . nevertheless , a potent ace inhibitor was identified in human milk and corresponded to the -casein fragment f(125129 ) with the sequence hlplp . in this case , it was hypothesized that the presence of the amino acid proline at the c - terminal end could be crucial in terms of the activity observed ( cheung et al . finally , the peptide f 80 - 90 ( tpvvvppflqp ) from -casein which is known to possess antihypertensive activities was detected in animal models fed with infant formulas ( bouzerzour et al . the antioxidant potential of human milk has been documented in the past ( friel et al . recent findings indicate that peptides released from human milk after digestion with pepsin and pancreatin could be beneficial with respect to antioxidant activity ( tsopmo et al . in this study , the peptides ygytga and iselgw showed high radical scavenging activity , and this property was attributed to the presence of the amino acid tryptophan which can break radical chain reactions thanks to the donation of the hydrogen attached to the nitrogen of its indole ring . , the peptide with the highest antioxidant activity was wsvpqpk , which also contains tryptophan and , thus , may serve as hydrogen donor and/or free radical scavenger . peptides of human ( iaippkkiqdk ) and bovine ( maippkknqdk ) origin with antithrombotic effects were detected at physiologically active levels in the plasma of newborn infants after breastfeeding or ingestion of cow milk - based formula ( chabance et al . the release of bcm7 from commercial milk - based infant formulas following sgid was confirmed ( de noni 2008 ) . although no safe conclusions can be drawn at the moment regarding the minimum amount of bcm7 capable to elicit pharmacological effects in vivo or ex vivo , further studies confirm its release by the action of gastrointestinal proteases during in vitro digestion in commercial infant formulas at concentrations between 0.04 and 0.21 mg.l ( de noni and cattaneo 2010 ) . the presence of bcm5 and bcm7 in human milk has also been confirmed with the content of the opioid peptides being dependent on the lactation stage ( jarmolowska et al . bcm7 and related compounds have been linked in the past with the development of human diseases such as juvenile diabetes , heart disease , autism , and schizophrenia ( mclachlan , 2001 ) . although evidence regarding the abundance of such peptides in infant formulas is inconclusive , the formation of bcms in infant feeds following sgid has been demonstrated . the in vivo effects of these peptides on the human physiology have not yet been fully described . casein phosphopeptides ( cpps ) deriving from the digestion of milk proteins may act as mineral carriers and affect their bioavailability in the human body ( meisel and fitzgerald 2003 ) . recent studies document the presence of bioactive peptides with mineral carrier properties in infant formulas ( miquel et al . according to the authors , calcium could be bound preferentially to cpps with the cluster sequence spspspee and iron and zinc to cpps with the phosphorylated cluster and phosphoserine residues . the release of cpps with metal - chelating properties following sgid of infant formulas was also reported in previous studies ( miquel et al . this was attributed to the ability of the supplemented formula to mimic the fermentative qualities of breast milk and induce the growth of protective microflora in the gastrointestinal tract . furthermore , the bacteriostatic and bactericidal effects of -lactalbumin and gmp have been demonstrated when a significant reduction in enteropathogenic bacteria including enteropathogenic escherichia coli and salmonella was observed following supplementation of the infant formulas with the specific milk substrates ( brck et al . the release of bioactive peptides with immunomodulatory activities from infant formulas is also documented by more recent studies . in this case , the peptide f 60 - 66 ( ypfpgpi ) from -casein was detected following in vivo digestion by piglets which were fed with infant formulas ( bouzerzour et al . proteins contribute to the nutritional value of milk and also have other biological roles as intact molecules , as many of them are resistant to the effects of digestion ( chatterton et al . on the other hand , digestion of milk proteins in the gastrointestinal tract results in the release of bioactive agents which may affect several physiological processes . despite the fact that many of these bioactive peptides have been identified as integral parts of milk proteins , furthermore , limited information is available with respect to their passage through the human , and most importantly the infant 's digestive tract . up - to - date , bioactive peptides have been identified in both human milk and cow milk - based infant formulas , which differ in their amino acid profiles and properties . modern methods of analysis enable fast and reliable separation and identification of bioactive peptides in milk - derived products ( catal - clariana et al . further research in this field is required to establish the beneficial role of human or bovine milk from that perspective . it seems likely though that peptides from milk protein of bovine origin could be a valuable supplement to human milk . the manufacture of chimeric infant formulas with multiple health - promoting effects could be used as a model example in the future for the manufacture of ultimately functional foods .

human activities have caused phenotypic changes in many ecosystems ( palumbi 2001 ; smith and bernatchez 2008 ) . these changes can be rapid , with large modifications occurring within decades only ( thompson 1998 ; hendry and kinnison 1999 ; stockwell et al . 2005 ) . in many fish populations , for instance , significant shifts in life - history traits have been described . 2003 ) , elevated reproductive effort ( yoneda and wright 2004 ) and changes in individual growth rate ( handford et al . 1977 ; ricker 1981 ; thomas and eckmann 2007 ) . many of these phenotypic changes may be linked to fishery - induced evolution . in experiments , the systematic removal of larger fish indeed decreases the mean weight of descendants ( conover and munch 2002 ) and impacts various life - history traits ( walsh et al . ( 2007 ) for a review on phenotypic traits for which evolutionary changes are likely , and hard et al . several conditions are mandatory for evolution to occur , and fishing on wild populations usually fulfils all these conditions . first , fishing - induced mortality can be very high and may exceed natural mortality by far more than 100% ( rijnsdorp 1993 ; mertz and myers 1998 ; jackson et al . 2001 ) . second , fishing is typically selective with regard to size ( myers and hoenig 1997 ; fukuwaka and morita 2008 ) . third , heritable variance has been found for many life - history traits in fish and can be as large as 0.5 ( theriault et al . 2007 ) . large - scale experiment in life - history evolution ( rijnsdorp 1993 ; law 2000 ; stokes and law 2000 ) . there is , however , much controversy regarding the relative importance of fishery - induced evolution as compared to the impact of phenotypic plasticity in response to environmental change ( hilborn 2006 ) . it is often questioned whether significant genetic changes over conservation - relevant periods of time are frequent , as discussed in smith and bernatchez ( 2008 ) . 2008 ) , and many alleged adaptations could indeed be environmentally induced phenotypic responses rather than genetic changes ( gienapp et al . changes in eutrophication ( gerdeaux and perga 2006 ) , salinity ( ricker 1981 ) , temperature ( thresher et al . 2007 ) , competition ( lorenzen and enberg 2002 ) , or large - scale ocean regime shifts ( pearcy 1992 ; thresher et al . 2007 ) can have an immediate impact on phenotypic traits , particularly life - history traits , without necessarily changing the genetics of a population . the relative importance of both fishery - induced evolution and phenotypic plasticity is thus a key issue that needs to be addressed ( law 2000 , 2007 ; smith and bernatchez 2008 ) . to date , only few studies have tried to separate the effects of fishery - induced evolution and environment - induced changes on individual growth rate . a major problem in such studies is that additive genetic effects can be correlated with long - term changes in , for example , population density , water temperature , or phosphorus concentration and hence productivity of typical freshwater habitats ( hutchings and fraser 2008 ) . ( 2007 ) used back - calculated length at age 4 ( from otolith measurements ) to determine the difference in growth between parental and offspring generations of atlantic cod ( gadus morhua ) in the gulf of st .- lawrence . the authors found significant length at age differences between the generations and concluded that these differences indicate genetic change in growth , i.e. that they may reveal genetic effects of size - selective fishing . ( 2008 ) discuss several potential limitations to swain et al.s approach , especially that their approach did not account for potential changes in reproductive strategies ( 2008 ) for a further discussion. growth rate is indeed influenced by at least three different life - history traits ( heino et al . 2008 ) : ( i ) growth capacity , i.e. the ability of fish to transform energy intake into body mass , ( ii ) the maturation schedule , and ( iii ) the reproductive investment , i.e. the ratio of gonad mass to somatic mass . in the case of selection against fast growers , resource reallocation from growth capacity to reproductive investment is likely ( gadgil and bossert 1970 ; heino et al . 2008 ) , and any observed change in growth rate could therefore be linked to a change in reproductive investment or in maturation schedule rather than , or in addition to , selection against fast growers . in this study , we applied the method of swain et al . ( 2007 ) to estimate selection differential in a population of the alpine whitefish coregonus palaea , fatio 1890 ( a freshwater salmonid ) . we also determined potential indices of resource reallocation over an observational period of 25 years , and we used a growth metric that takes the whole lifespan of the fish into account ( i.e. the whole period under fishing - induced selection ) . the population is isolated , i.e. no fish from other populations have been introduced into the lake during the observational period , and migration , which can also potentially affect the estimation of the selection differentials , is impossible . fishing mortality is very high , i.e. most fish are harvested in their first years of life and old individuals are scarce ( 95% of the fish where caught before the age of 8 years , whereas the oldest individual in the sample was 13 + ) . moreover , fishing effort can be considered constant and uniform over the study period : for the duration of the study , two fishermen have been harvesting , following regulations that have not significantly changed since 1960 . fisheries data on yield since the late fifties do not show any directional trend with regard to the total whitefish yield . heritability of growth has been studied in various other salmonids and found to be significant ( theriault et al . the specific situation of our study population therefore allows us to estimate the evolutionary consequences of fishing - induced selection on growth within the range of the existing heritability estimates . the study population is confined to the lake joux , switzerland ( lat = 46.63n , long = 6.28e , 9.5 km , maximum depth = 32 m ) . in the course of a monitoring programme that started in 1980 , a total of 1654 fish were sampled from large catches ( on average 75 fish caught each year , 37 sd ) . fish were sampled each year except between 1997 and 2002 when fishing occurred but no monitoring was done . the catches were taken during the spawning season ( november and december ) at the spawning site with nylon gill nets of 40 , 45 and 50 mm mesh size . the total number of eggs that were collected for supplementary breeding was recorded every year . for size measurement , males and females were pooled because no sexual size dimorphism seems to exist in this species . total body length was measured in millimetres and scales were taken from above the lateral line between the dorsal and adipose fins for subsequent age determination and back - calculation of previous body lengths . on 719 fish , scale radius and annulus radii , i.e. the distances from the nucleus to the subsequent annuli , were measured using an ocular micrometre for length measurements . probably due to the high altitude of lake joux and the marked temperature differences between summer and winter , annuli on scales we back - calculated the length at previous ages of each fish according to the method of finstad ( 2003 ) . this method is based on a multiple regression of fish scale including the age and length of the fish . we used a logarithm transformation of fish length and annulus length . from the resulting length - at - age back - calculations , we computed the following two - parameter logarithmic growth curve for each fish : where li(t ) is the back - calculated length of each fish at age t , 0i the back - calculated length at age 1 , and ti the logarithmic growth of each fish . we estimated the parameters 0i and ti for each fish from the back - calculated lengths : where ti is the age of each fish at capture . for each fish , we calculated the length - at - age with two different methods : with the back - calculations and with our logarithmic model . we then assessed the goodness - of - fit of our growth model with an analysis of variance of all the back - calculated lengths as dependant variable and the theoretical lengths fitted with the two - parameter logarithmic model as independent variable ( anova : d.f . this model has several advantages : first , it has fewer parameters than other growth models such as a three - parameter von bertalanffy model . second , the interpretation of the two parameters is very intuitive : 0 represents the length at age 1 ( i.e. can be understood as juvenile growth ) and t represents growth after age 1 , i.e. approximates adult growth . third , all the sampled fish are taken into account . with a single size - at - age measure as used in swain et al . ( 2007 ) , all the fish younger than the reference age are discarded from the analysis . this can result in a biased estimation of selection differentials that is linked to size - selective fishing , especially if growth varies among cohorts . moreover a single size - at - age measure is more subject to environmental influence in particular years . this problem is probably less significant in our model as the parameter t takes into account growth over several years , i.e. we expect the variance in our growth measure to be smaller than with a single size - at - age measure . finally it has been shown for arctic charr ( salvelinus alpinus ) , a freshwater salmonid , that a two - parameter log - linear growth model provides a fit that is at least as good as the von bertalanffy growth model ( rubin and perrin 1990 ) . to detect a potential change in growth parameters over time , 0 and t were averaged for each cohort and a linear regression was calculated . average growth parameters were the dependant variables and the birth year of the cohort the independent variable . we were interested in the relative growth difference ( in % ) between two generations and therefore calculated the average relative change over the generations as the observed change in both parameters divided by the average growth parameter and multiplied by the generation time . the average generation time over the generations was estimated according to stearns ( 1992 ) and was calculated over the whole sample for simplicity , without taking the cohorts into account : where x is the age class of the fish , lx the probability of survival to age x , and mx the fecundity of age class x. fecundity was estimated as the probability ( p ) of being mature at age x times the mean length ( l ) of the fish in the age class x cubed ( mx = pl ) , assuming that fecundity is proportional to the length cubed of the fish ( clark and bernard 1992 ) . as a potential indicator for resource reallocation from growth to reproduction , we estimated the average reproductive investment of all the females captured during the spawning season as the proportional volume of egg per female . some females had already partially spawned , i.e. our measure of reproductive investment underestimates the total eggs production . we used the average volume of eggs per reproducing female of each spawning season and divided this value by the mean length cubed of the fish the allometric relationship between weight and length was : weight = exp(12.04 + 3.06*ln(length)). we also estimated the age at maturation for each fish according to rijnsdorp and storbeck ( 1995 ) . this method assumes that growth , i.e. the yearly size increment , is maximal and linear when the fish is immature and decreases after the fish becomes mature because some resources are invested into reproduction instead of growth . we therefore interpret the gap between large and small yearly length increases as the timing of maturation . we used linear regressions to test for linear trends over time in these two measures . the expected response to selection ( r ) , i.e. the change in growth rate expected if only selection by fishing is occurring under a constant environment , was estimated from the breeder 's equation ( falconer and mackay 1996 ) : where h is the heritability of growth traits and s the selection differential , i.e. the mean difference of a trait between the actual reproducers ( the fish surviving to reproduction ) , and the whole population . heritability estimates for growth rate in fish range approximately from 0.1 to 0.5 ( law 2000 ; garcia de leaniz et al . 2007 ; , we used an intermediate heritability ( h = 0.3 ) and two extreme ones ( h = 0.1 and h = 0.5 ) . the selection differential s was determined for each age class within each cohort by comparing the reproducers , i.e. the fish caught in subsequent years and at older age , with all the fish of that particular age class . we then estimated the average selection differential in every single cohort born in year k , , as the mean of the selection differentials calculated for each age class . this mean took into account the number of fish in each age class and the relative contribution of each age class to reproduction , i.e. the average fecundity of the age class . where pjk is the proportion of fish born in year k reproducing at age j and wj is a weighting parameter for each age class j. to account for the differential contribution of each age class within each cohort due to differences in fecundity , the weighting parameter was set as the cube of the average length of the fish within the age class . sjk is the selection differential for age class j within cohort k and is estimated as : where njk is the number of mature fish born in year k reproducing at age j , m is the maturation status and equals 1 if j age at maturation and 0 otherwise , r is the reproductive status and equals 1 if age at maturation < j age at capture , and 0 otherwise , and is the average growth parameter ( 0 or t ) of all the fish born in year k. the size selectivity of nonfishing induced mortality was considered negligible compared to fishing selection for the estimation of selection differential . to disentangle the change in growth due to fishery - induced selection from the change due to environmental variation , we postulated that both factors were additive , i.e. that the observed growth change was equal to the sum of genetic ( estimated by hs ) and phenotypic plasticity . to simplify the fraction of change due to fishery - induced selection was finally calculated as hs divided by the total observed change in growth . we did not observe any significant linear change in resource reallocation from growth to reproduction over the observational period . neither the maturation schedule , estimated by the mean age at maturation , nor the fecundity , estimated by the proportional volume of eggs per fish , seems to change consistently over time ( maturation schedule : t21 = 0.08 , p = 0.94 , fig . 1b ) may be linked to intra - specific competition between age classes ( naceur and bttiker 1999 ) . indicators of resource reallocation from growth to reproduction : ( a ) reproduction schedule as the mean age at maturation for each cohort and ( b ) reproductive investment as the mean proportion ( in % ) of egg volume per female each year of capture during spawning season . length at age 1 ( 0 ) did not linearly change over time ( t18 = 0.34 , p = 0.74 , fig . however , logarithmic growth ( t ) declined by 0.94 0.36% per year ( t18 = 2.6 , p = 0.017 , fig . the average generation time , i.e. the average age difference between parents and offspring was estimated to be 4.67 years . growth parameters over time : ( a ) average length at age 1 ( 0 ) and ( b ) average logarithmic growth ( t ) . selection differentials on parameter 0 , i.e. the difference in growth between reproducers and the whole population , did not change linearly over time ( linear regression : t21 = 0.50 , p = 0.62 ) , neither did the selection differentials on parameter t ( linear regression : t21 = 1.02 , p = 0.32 ) . moreover , no clear trend was found with these parameters . we therefore considered each sk as an independent estimation of an average selection differential s over the whole period with a precision that depends on the number of fish on which the estimation is based . as the number of observations per cohort varied , a weighted t - test , with a weighting proportional to the number of fish in each cohort , was used to test whether s was significantly different from zero . the selection differential for length at age 1 ( 0 ) was not significantly different from zero ( t22 = 0.87 , p = 0.39 , fig . 3a ) . however , the selection differential for the logarithmic growth ( t ) was significantly negative : s = 4.93 1.23% ( t22 = 4.02 , p < 0.001 , fig . ( a ) s for length at age 1 ( 0 ) , and ( b ) s for logarithmic growth ( t ) . assuming that the heritability of growth is h = 0.3 , with r = 4.37 1.67% , and s = 4.93 1.23% , the proportion of logarithmic growth decrease ( t ) due to fishery - induced selection was estimated to be 33.8% . with the two extreme scenarios ( i.e. heritability h = 0.1 and 0.5 ) , this proportion would be 11.3% and 56.2% respectively . the population is closed to migration and under a fishing pressure that can be considered constant over the observational period . the fishing pressure is strong and most fish that reach maturity seem to be eventually harvested . we therefore consider this population ideal for testing the potential effects of fishery - induced evolution of individual growth rates , a topic that has received much attention recently . the first parameter 0 describes juvenile growth in the first year of life when the fish are under no direct fishing pressure , whereas the second parameter t describes the growth trajectories at later ages and at times when selection by fishing is relevant . changes in individual growth rates over time can be due to fishing - induced evolution , to ecological changes ( e.g. temperature , water phosphorus concentration , population density ) , to a change in life history such as reallocation of resources from growth to reproduction , or to any combination of these possible causes ( heino et al . obviously , any increase in energy allocation to reproduction is expected to slow down growth ( heino and kaitala 1999 ) . a change in the timing of maturation or in fecundity will therefore change individual growth rates ( stearns 1992 ) . however , we found no evidence for a change in maturation schedule or reproductive strategies in our study population . we therefore concentrate our discussion on the importance of fishing - induced evolution relative to ecological changes over time . to study fishing - induced evolution , we need to understand the selection induced by fishing , i.e. we need good estimates of the selection differentials . selection differentials measure the difference in a phenotypic trait between the mean of a population and the mean of the individuals selected to be parents of the next generation . such phenotypic differences are expected to have a strong genetic component if the fish share the same environmental history . immigrating individuals and fish escaping harvesting , both common in marine populations , could bias the estimation of selection differentials . in our study population this , combined with a constant fishing pressure ( see introduction ) , allows us to determine the selection differentials probably more accurately than analogous determinations in open marine populations . we found that the change in t ( the growth trajectories at later ages ) is around 1% per year or about 4% per generation , but there was no significant change in 0 ( juvenile growth in the first year of life ) . first , we assumed that genetic and ecological factors have additive effects on individual growth rates and that genotype environment interactions are negligible . although we have data of > 20 cohorts , only about 75 fish were sampled per cohort , and direct parent offspring comparisons were not possible like in , for example , grant and grant ( 1995 ) who studied micro - evolutionary responses to directional selection by sampling and assigning parentage to each individual of a population . however , we believe that our model assumptions still lead to useful results in our case because the selection differential can be assumed to vary around an average that does not change over time ( the fishing pressure and the reproductive strategies did not seem to change ) , and nonlinear responses to selection would therefore be somewhat surprising . these assumptions are supported by our data ( see fig . the evolutionary change in t that we observed may be somewhat underestimated because slow growers are more likely to reproduce and die before being caught , i.e. natural mortality may be inversely proportional to size ( conover 2007 ) and slow growing fish are harvested at an older age because fishing targets fish above a certain size . if we assume that the heritability of growth rates in our study population is about the average of what has been described for salmonids so far ( i.e. h = 0.3 ) , we conclude that about a third of the decrease in t is directly linked to fishing - induced genetic changes in the population . the systematic removal of larger and older fish therefore seems to significantly affect the evolution of individual growth rates in the whitefish of lake joux . although there is no fishing pressure on small fish , juvenile and adult growth are likely to be genetically correlated ( lande and arnold 1983 ; walsh et al . moreover , everything being equal , juveniles fish that are small may attain a lesser size than large ones and may therefore be likely to suffer less from fishing selection . a possible reason for the observed absence of a decrease might be that 0 is a single length - at - age measure and therefore more strongly influenced by environmental factors than adult growth ( t ) that is an average over several years . a possible genetic decrease could therefore be masked by a plastic response to a changing environment . temperature is known to have a significant impact on juvenile growth ( malzahn et al . finally , there could also be an adaptive response linked to resources reallocation , with more energy invested for juvenile growth to increase juvenile survival and less in adult growth , the status quo in juvenile growth could be the maximal viable growth . the marked decrease in the growth parameter t could potentially have negative consequences for the population . there is now mounting evidence that artificial selection such as size - selective harvesting reduces the average viability in some populations ( fenberg and roy 2008 ) . several specific consequences may arise from the removal of large fish , and even if these issues are controversial ( carlson et al . 2008 ) , a precautionary approach should be taken when managing evolving fish stocks ( francis and shotton 1997 ) . first , large and fast - growing individuals may be of higher genetic quality than small and slow - growing individuals ( birkeland and dayton 2005 ) . a systematic removal of high quality adults could therefore result in an increase of the average genetic load in a population . second , as large females usually produce larger offspring of higher viability ( trippel 1995 ; walsh et al . 2006 ) , a decrease in growth could impair the recruitment and consequently the long - term yield of the population . third , as females in some species prefer to mate with large males ( hutchings and rowe 2008 ) , increased mortality of large fish could have an impact on sexual selection and therefore on mating behaviour . fourth , nonrandom mortality could decrease the genetic diversity of the population and make it more vulnerable to environmental changes or diseases ( jones et al . , we found that the large selection differentials imposed by size - selective fishing can significantly change the genetics of a population . this needs to be taken into account by population managers ( stokes and law 2000 ; ashley et al .

size - selective fishing , environmental changes and reproductive strategies are expected to affect life - history traits such as the individual growth rate . the relative contribution of these factors is not clear , particularly whether size - selective fishing can have a substantial impact on the genetics and hence on the evolution of individual growth rates in wild populations . we analysed a 25-year monitoring survey of an isolated population of the alpine whitefish coregonus palaea . we determined the selection differentials on growth rate , the actual change of growth rate over time and indicators of reproductive strategies that may potentially change over time . the selection differential can be reliably estimated in our study population because almost all the fish are harvested within their first years of life , i.e. few fish escape fishing mortality . we found a marked decline in average adult growth rate over the 25 years and a significant selection differential for adult growth , but no evidence for any linear change in reproductive strategies over time . assuming that the heritability of growth in this whitefish corresponds to what was found in other salmonids , about a third of the observed decline in growth rate would be linked to fishery - induced evolution . size - selective fishing seems to affect substantially the genetics of individual growth in our study population .

Introduction Methods Results Discussion

these changes can be rapid , with large modifications occurring within decades only ( thompson 1998 ; hendry and kinnison 1999 ; stockwell et al . 2005 ) . in many fish populations , for instance , significant shifts in life - history traits have been described . 2003 ) , elevated reproductive effort ( yoneda and wright 2004 ) and changes in individual growth rate ( handford et al . many of these phenotypic changes may be linked to fishery - induced evolution . in experiments , the systematic removal of larger fish indeed decreases the mean weight of descendants ( conover and munch 2002 ) and impacts various life - history traits ( walsh et al . several conditions are mandatory for evolution to occur , and fishing on wild populations usually fulfils all these conditions . first , fishing - induced mortality can be very high and may exceed natural mortality by far more than 100% ( rijnsdorp 1993 ; mertz and myers 1998 ; jackson et al . third , heritable variance has been found for many life - history traits in fish and can be as large as 0.5 ( theriault et al . large - scale experiment in life - history evolution ( rijnsdorp 1993 ; law 2000 ; stokes and law 2000 ) . there is , however , much controversy regarding the relative importance of fishery - induced evolution as compared to the impact of phenotypic plasticity in response to environmental change ( hilborn 2006 ) . 2007 ) , competition ( lorenzen and enberg 2002 ) , or large - scale ocean regime shifts ( pearcy 1992 ; thresher et al . 2007 ) can have an immediate impact on phenotypic traits , particularly life - history traits , without necessarily changing the genetics of a population . the relative importance of both fishery - induced evolution and phenotypic plasticity is thus a key issue that needs to be addressed ( law 2000 , 2007 ; smith and bernatchez 2008 ) . to date , only few studies have tried to separate the effects of fishery - induced evolution and environment - induced changes on individual growth rate . a major problem in such studies is that additive genetic effects can be correlated with long - term changes in , for example , population density , water temperature , or phosphorus concentration and hence productivity of typical freshwater habitats ( hutchings and fraser 2008 ) . ( 2007 ) used back - calculated length at age 4 ( from otolith measurements ) to determine the difference in growth between parental and offspring generations of atlantic cod ( gadus morhua ) in the gulf of st .- lawrence . the authors found significant length at age differences between the generations and concluded that these differences indicate genetic change in growth , i.e. that they may reveal genetic effects of size - selective fishing . ( 2008 ) discuss several potential limitations to swain et al.s approach , especially that their approach did not account for potential changes in reproductive strategies ( 2008 ) for a further discussion. growth rate is indeed influenced by at least three different life - history traits ( heino et al . 2008 ) : ( i ) growth capacity , i.e. the ability of fish to transform energy intake into body mass , ( ii ) the maturation schedule , and ( iii ) the reproductive investment , i.e. 2008 ) , and any observed change in growth rate could therefore be linked to a change in reproductive investment or in maturation schedule rather than , or in addition to , selection against fast growers . in this study , we applied the method of swain et al . ( 2007 ) to estimate selection differential in a population of the alpine whitefish coregonus palaea , fatio 1890 ( a freshwater salmonid ) . we also determined potential indices of resource reallocation over an observational period of 25 years , and we used a growth metric that takes the whole lifespan of the fish into account ( i.e. the whole period under fishing - induced selection ) . the population is isolated , i.e. no fish from other populations have been introduced into the lake during the observational period , and migration , which can also potentially affect the estimation of the selection differentials , is impossible . fishing mortality is very high , i.e. most fish are harvested in their first years of life and old individuals are scarce ( 95% of the fish where caught before the age of 8 years , whereas the oldest individual in the sample was 13 + ) . moreover , fishing effort can be considered constant and uniform over the study period : for the duration of the study , two fishermen have been harvesting , following regulations that have not significantly changed since 1960 . fisheries data on yield since the late fifties do not show any directional trend with regard to the total whitefish yield . heritability of growth has been studied in various other salmonids and found to be significant ( theriault et al . the specific situation of our study population therefore allows us to estimate the evolutionary consequences of fishing - induced selection on growth within the range of the existing heritability estimates . the study population is confined to the lake joux , switzerland ( lat = 46.63n , long = 6.28e , 9.5 km , maximum depth = 32 m ) . fish were sampled each year except between 1997 and 2002 when fishing occurred but no monitoring was done . for size measurement , males and females were pooled because no sexual size dimorphism seems to exist in this species . on 719 fish , scale radius and annulus radii , i.e. this method is based on a multiple regression of fish scale including the age and length of the fish . we then assessed the goodness - of - fit of our growth model with an analysis of variance of all the back - calculated lengths as dependant variable and the theoretical lengths fitted with the two - parameter logarithmic model as independent variable ( anova : d.f . this model has several advantages : first , it has fewer parameters than other growth models such as a three - parameter von bertalanffy model . second , the interpretation of the two parameters is very intuitive : 0 represents the length at age 1 ( i.e. can be understood as juvenile growth ) and t represents growth after age 1 , i.e. approximates adult growth . third , all the sampled fish are taken into account . with a single size - at - age measure as used in swain et al . ( 2007 ) , all the fish younger than the reference age are discarded from the analysis . this can result in a biased estimation of selection differentials that is linked to size - selective fishing , especially if growth varies among cohorts . moreover a single size - at - age measure is more subject to environmental influence in particular years . this problem is probably less significant in our model as the parameter t takes into account growth over several years , i.e. we expect the variance in our growth measure to be smaller than with a single size - at - age measure . finally it has been shown for arctic charr ( salvelinus alpinus ) , a freshwater salmonid , that a two - parameter log - linear growth model provides a fit that is at least as good as the von bertalanffy growth model ( rubin and perrin 1990 ) . to detect a potential change in growth parameters over time , 0 and t were averaged for each cohort and a linear regression was calculated . average growth parameters were the dependant variables and the birth year of the cohort the independent variable . we were interested in the relative growth difference ( in % ) between two generations and therefore calculated the average relative change over the generations as the observed change in both parameters divided by the average growth parameter and multiplied by the generation time . the average generation time over the generations was estimated according to stearns ( 1992 ) and was calculated over the whole sample for simplicity , without taking the cohorts into account : where x is the age class of the fish , lx the probability of survival to age x , and mx the fecundity of age class x. fecundity was estimated as the probability ( p ) of being mature at age x times the mean length ( l ) of the fish in the age class x cubed ( mx = pl ) , assuming that fecundity is proportional to the length cubed of the fish ( clark and bernard 1992 ) . as a potential indicator for resource reallocation from growth to reproduction , we estimated the average reproductive investment of all the females captured during the spawning season as the proportional volume of egg per female . some females had already partially spawned , i.e. our measure of reproductive investment underestimates the total eggs production . we used the average volume of eggs per reproducing female of each spawning season and divided this value by the mean length cubed of the fish the allometric relationship between weight and length was : weight = exp(12.04 + 3.06*ln(length)). this method assumes that growth , i.e. the yearly size increment , is maximal and linear when the fish is immature and decreases after the fish becomes mature because some resources are invested into reproduction instead of growth . we therefore interpret the gap between large and small yearly length increases as the timing of maturation . we used linear regressions to test for linear trends over time in these two measures . the expected response to selection ( r ) , i.e. the change in growth rate expected if only selection by fishing is occurring under a constant environment , was estimated from the breeder 's equation ( falconer and mackay 1996 ) : where h is the heritability of growth traits and s the selection differential , i.e. the mean difference of a trait between the actual reproducers ( the fish surviving to reproduction ) , and the whole population . heritability estimates for growth rate in fish range approximately from 0.1 to 0.5 ( law 2000 ; garcia de leaniz et al . the selection differential s was determined for each age class within each cohort by comparing the reproducers , i.e. the fish caught in subsequent years and at older age , with all the fish of that particular age class . we then estimated the average selection differential in every single cohort born in year k , , as the mean of the selection differentials calculated for each age class . this mean took into account the number of fish in each age class and the relative contribution of each age class to reproduction , i.e. the average fecundity of the age class . where pjk is the proportion of fish born in year k reproducing at age j and wj is a weighting parameter for each age class j. to account for the differential contribution of each age class within each cohort due to differences in fecundity , the weighting parameter was set as the cube of the average length of the fish within the age class . sjk is the selection differential for age class j within cohort k and is estimated as : where njk is the number of mature fish born in year k reproducing at age j , m is the maturation status and equals 1 if j age at maturation and 0 otherwise , r is the reproductive status and equals 1 if age at maturation < j age at capture , and 0 otherwise , and is the average growth parameter ( 0 or t ) of all the fish born in year k. the size selectivity of nonfishing induced mortality was considered negligible compared to fishing selection for the estimation of selection differential . to disentangle the change in growth due to fishery - induced selection from the change due to environmental variation , we postulated that both factors were additive , i.e. that the observed growth change was equal to the sum of genetic ( estimated by hs ) and phenotypic plasticity . to simplify the fraction of change due to fishery - induced selection was finally calculated as hs divided by the total observed change in growth . we did not observe any significant linear change in resource reallocation from growth to reproduction over the observational period . neither the maturation schedule , estimated by the mean age at maturation , nor the fecundity , estimated by the proportional volume of eggs per fish , seems to change consistently over time ( maturation schedule : t21 = 0.08 , p = 0.94 , fig . 1b ) may be linked to intra - specific competition between age classes ( naceur and bttiker 1999 ) . indicators of resource reallocation from growth to reproduction : ( a ) reproduction schedule as the mean age at maturation for each cohort and ( b ) reproductive investment as the mean proportion ( in % ) of egg volume per female each year of capture during spawning season . length at age 1 ( 0 ) did not linearly change over time ( t18 = 0.34 , p = 0.74 , fig . the average generation time , i.e. growth parameters over time : ( a ) average length at age 1 ( 0 ) and ( b ) average logarithmic growth ( t ) . selection differentials on parameter 0 , i.e. the difference in growth between reproducers and the whole population , did not change linearly over time ( linear regression : t21 = 0.50 , p = 0.62 ) , neither did the selection differentials on parameter t ( linear regression : t21 = 1.02 , p = 0.32 ) . moreover , no clear trend was found with these parameters . we therefore considered each sk as an independent estimation of an average selection differential s over the whole period with a precision that depends on the number of fish on which the estimation is based . as the number of observations per cohort varied , a weighted t - test , with a weighting proportional to the number of fish in each cohort , was used to test whether s was significantly different from zero . the selection differential for length at age 1 ( 0 ) was not significantly different from zero ( t22 = 0.87 , p = 0.39 , fig . however , the selection differential for the logarithmic growth ( t ) was significantly negative : s = 4.93 1.23% ( t22 = 4.02 , p < 0.001 , fig . assuming that the heritability of growth is h = 0.3 , with r = 4.37 1.67% , and s = 4.93 1.23% , the proportion of logarithmic growth decrease ( t ) due to fishery - induced selection was estimated to be 33.8% . with the two extreme scenarios ( i.e. heritability h = 0.1 and 0.5 ) , this proportion would be 11.3% and 56.2% respectively . the population is closed to migration and under a fishing pressure that can be considered constant over the observational period . we therefore consider this population ideal for testing the potential effects of fishery - induced evolution of individual growth rates , a topic that has received much attention recently . the first parameter 0 describes juvenile growth in the first year of life when the fish are under no direct fishing pressure , whereas the second parameter t describes the growth trajectories at later ages and at times when selection by fishing is relevant . changes in individual growth rates over time can be due to fishing - induced evolution , to ecological changes ( e.g. temperature , water phosphorus concentration , population density ) , to a change in life history such as reallocation of resources from growth to reproduction , or to any combination of these possible causes ( heino et al . obviously , any increase in energy allocation to reproduction is expected to slow down growth ( heino and kaitala 1999 ) . a change in the timing of maturation or in fecundity will therefore change individual growth rates ( stearns 1992 ) . however , we found no evidence for a change in maturation schedule or reproductive strategies in our study population . we therefore concentrate our discussion on the importance of fishing - induced evolution relative to ecological changes over time . to study fishing - induced evolution , we need to understand the selection induced by fishing , i.e. we need good estimates of the selection differentials . selection differentials measure the difference in a phenotypic trait between the mean of a population and the mean of the individuals selected to be parents of the next generation . such phenotypic differences are expected to have a strong genetic component if the fish share the same environmental history . immigrating individuals and fish escaping harvesting , both common in marine populations , could bias the estimation of selection differentials . in our study population this , combined with a constant fishing pressure ( see introduction ) , allows us to determine the selection differentials probably more accurately than analogous determinations in open marine populations . we found that the change in t ( the growth trajectories at later ages ) is around 1% per year or about 4% per generation , but there was no significant change in 0 ( juvenile growth in the first year of life ) . first , we assumed that genetic and ecological factors have additive effects on individual growth rates and that genotype environment interactions are negligible . although we have data of > 20 cohorts , only about 75 fish were sampled per cohort , and direct parent offspring comparisons were not possible like in , for example , grant and grant ( 1995 ) who studied micro - evolutionary responses to directional selection by sampling and assigning parentage to each individual of a population . however , we believe that our model assumptions still lead to useful results in our case because the selection differential can be assumed to vary around an average that does not change over time ( the fishing pressure and the reproductive strategies did not seem to change ) , and nonlinear responses to selection would therefore be somewhat surprising . these assumptions are supported by our data ( see fig . the evolutionary change in t that we observed may be somewhat underestimated because slow growers are more likely to reproduce and die before being caught , i.e. natural mortality may be inversely proportional to size ( conover 2007 ) and slow growing fish are harvested at an older age because fishing targets fish above a certain size . if we assume that the heritability of growth rates in our study population is about the average of what has been described for salmonids so far ( i.e. h = 0.3 ) , we conclude that about a third of the decrease in t is directly linked to fishing - induced genetic changes in the population . the systematic removal of larger and older fish therefore seems to significantly affect the evolution of individual growth rates in the whitefish of lake joux . although there is no fishing pressure on small fish , juvenile and adult growth are likely to be genetically correlated ( lande and arnold 1983 ; walsh et al . moreover , everything being equal , juveniles fish that are small may attain a lesser size than large ones and may therefore be likely to suffer less from fishing selection . a possible reason for the observed absence of a decrease might be that 0 is a single length - at - age measure and therefore more strongly influenced by environmental factors than adult growth ( t ) that is an average over several years . temperature is known to have a significant impact on juvenile growth ( malzahn et al . finally , there could also be an adaptive response linked to resources reallocation , with more energy invested for juvenile growth to increase juvenile survival and less in adult growth , the status quo in juvenile growth could be the maximal viable growth . there is now mounting evidence that artificial selection such as size - selective harvesting reduces the average viability in some populations ( fenberg and roy 2008 ) . a systematic removal of high quality adults could therefore result in an increase of the average genetic load in a population . 2006 ) , a decrease in growth could impair the recruitment and consequently the long - term yield of the population . third , as females in some species prefer to mate with large males ( hutchings and rowe 2008 ) , increased mortality of large fish could have an impact on sexual selection and therefore on mating behaviour . fourth , nonrandom mortality could decrease the genetic diversity of the population and make it more vulnerable to environmental changes or diseases ( jones et al . , we found that the large selection differentials imposed by size - selective fishing can significantly change the genetics of a population .

stria vascularis and the spiral ganglion cells are also reportedly affected by the drug.2 , 3 after cisplatin administration , ohcs are initially damaged in the base of the cochlea , leading to a highfrequency hearing loss.4 there is an individual susceptibility to cisplatin ototoxicity and no otoprotective treatments are clinically proven.1 nephrotoxic side effects resulting from damage to the proximal tubule cells can be alleviated by prehydration and forced diuresis.5 it is not known how cisplatin is transported from the systemic circulation to the target cells in the inner ear , but active transport mechanisms are suspected.6 , 7 organic cation transporters ( octs ) located in kidney and liver are known to be involved in the excretion of drugs from the systemic circulation.8 , 9 various isoforms of octs have specific species and tissuedistribution patterns.10 , 11 organic cations , such as the antidiabetic drugs metformin12 , 13 and phenformin14 , 15 and the histamine receptor antagonists cimetidine and ranitidine,16 are transported by octs in the renal endothelial cells . in previous studies , organic transport protein 2 ( oct2 ) was found to be involved in cisplatininduced nephro17 , 18 and ototoxicity.6 organic transport protein 2 has been shown to transport cisplatin to the renal proximal tubule cells,17 , 19 , 20 , 21 , 22 and immunostaining has localized oct2 to the cochlea of the murine inner ear.6 , 23 depleted ototoxic and nephrotoxic side effects of cisplatin were demonstrated in oct2deficient mice and mice treated with cimetidine.6 , 17 we have studied oct2 because of its suggested causative involvement in the ototoxic side effect of cisplatin . organic transport protein 2 is not reported to be a transport protein for cisplatin into cancer cells.24 therefore , a protective therapy utilizing transport pathways would not counteract the antitumour effect of cisplatin . in this study , phenformin was chosen as an otoprotector because previous studies have shown a competitive inhibitory effect of oct2.14 , 25 hence , the aim of the study was twofold , namely to locate oct2 by immunohistochemical means in the rat , guinea pig , and pig cochlea and to establish if pretreatment with phenformin reduces cisplatininduced ototoxicity in the guinea pig in vivo . guinea pigs ( duncunhartley , lidkpings kaninfarm ab , sweden ) and rats ( spraguedawley , scanbur ab , sollenuna , sweden ) were housed at the animal facility at karolinska university hospital in uppsala , sweden , and kept in enrichment cages at a room temperature of 21c , with a 12hour light dark cycle and free access to food and tap water . a pig cochlea ( sus scrofa ) care and use of the animals reported in this study were approved in accordance with ethical standards ( ethical permits n135/11 and c315/5 ) . for immunohistochemical staining , tissues from four untreated female albino rats , four untreated female albino guinea pigs , and an untreated pig were used . in the in vivo study , 15 female albino guinea pigs ( range 261337 g ) were used , anesthetized with an intramuscular injection of ketamine ( 40 mg / kg ) and xylazine ( 10 mg / kg ) . the weight of the animals was monitored daily , and they were given a daily subcutaneous injection of 5 ml saline . the end of the experiment was set to 96 hours after cisplatin was given ; thereafter , the animals were sacrificed with an overdose of pentobarbiturate and cochlea were harvested . parvalbumin is known to exert both a cytoplasmatic and a nuclear immunoreactivity in type i spiral ganglion cells and in hair cells . costaining with parvalbumin and oct2 was used to identify inner hair cells ( ihcs ) and ohcs.26 , 27 kidney served as positive control for oct2 , as shown in previous studies.20 , 28 the perilymphatic space of rat , guinea pig , and pig cochlea was perfused with 4% paraformaldehyde after decapitation , followed by decalcification . rat and guinea pig cochlea and kidney were embedded in paraffin , sectioned 5m thick , and mounted . the tissue sections were deparaffinized and boiled for 10 minutes in an antigen unmasking solution ( vector laboratories , burlingame , ca ) . a pig cochlea was embedded in tissuetek ( oct polysciences , warrington , pa ) , rapidly frozen , and sectioned at 8 to 10 m with a cryostat.29 the frozen sections were collected into gelatin / chrome alumcoated slides and stored below 70c prior to immunohistochemistry . all sections were incubated with blocking serum ( 5% goat serum ) for 30 minutes at room temperature . rabbit antirat organic cation transporter 2 ( oct2 ) polyclonal antibodies , diluted 1:100 ( alpha diagnostic international , san antonio , tx ) , and mouse antiparvalbumin , diluted 1:250 ( chemicon international , hants , united kingdom ) were used as primary antibodies . one separate slide and one section on each slide were used as a negative control . organic transport protein 2 antibodies were visualized by means of indocarbocyanine cy3 ( red dye ) conjugated goat antirabbit antibody , diluted 1:200 ( jackson immuno research laboratories inc . , parvalbumin antibodies were visualized by means of alexa fluor 488 ( green dye ) conjugated goat antimouse antibody , diluted 1:400 ( invitrogen , carlsbad , cal . all slides were mounted with vecta shield mounting medium with 4,6diamidino2phenylindole ( dapi ) ( vector laboratories , burlingame , ca ) , and nuclei of all sections were then counterstained with dapi ( blue color ) . fluorescent signals were analyzed with a light microscope ( zeiss axio scope ; carl zeiss , jena , germany ) or with a laser confocal microscope ( nikon te2000 ; nikon co , tokyo , japan ) . the right internal jugular vein was catheterized for drug administration along the venous flow , that is , against the heart . the study group ( n = 8) were given phenformin 20 mg / kg intravenously ( phenformin hydrochloride 7.5 mg / ml ; sigmaaldrich , saint louis , mo ) 30 minutes prior to intravenous administration of cisplatin 8 mg / kg ( platinol 1 mg / ml ; bristolmyers squibb pharmaceuticals , new york , ny ) . control animals ( n = 7 ) were given only cisplatin 8 mg / kg intravenously ototoxicity was evaluated by measuring the electrophysiological hearing thresholds with auditory brainstem response ( abr ) and assessing the morphological loss of ihcs and ohcs . the levels of platinum in cochlear tissue were assessed and compared between the two groups . tuckerdavis technologies ( gainesville , fl ) equipment was used to measure the electrophysiological hearing thresholds of anesthetized animals . auditory threshold shifts were determined in the left ear at 6.3 khz , 12.5 khz , and 20 khz and calculated as the difference between thresholds obtained before and 96 hours after drug treatment , expressed as db change.30 the temporal bones were immediately removed after the animals were euthanized . for morphological calculation of the loss of ihcs and ohcs , after the bone was removed , the hair cells were labeled with phalloidin ( diluted 1:200 ) . hair cells ( scar formations ) were counted along the cochlea in a zeiss fluorescence microscope ( carl zeiss ) . the percentage of missing hair cells per mm was calculated.31 before cisplatin administration , one 0.25ml blood sample was aspirated from the left jugular vein of control group animals . after 96 hours , 0.25 ml blood was aspirated from the heart from all animals before euthanization . the right cochlea was used to determine the total platinum content in the tissue . from all cochlea , entire basilar membranes , including the organ of corti and stria vascularis , were pooled together to form one sample . inductively coupled plasma mass spectrometry ( analytica ab , lule , sweden ) was used for the analytical procedure.32 for descriptive data , median ( maximum minimum ) values were used . guinea pigs ( duncunhartley , lidkpings kaninfarm ab , sweden ) and rats ( spraguedawley , scanbur ab , sollenuna , sweden ) were housed at the animal facility at karolinska university hospital in uppsala , sweden , and kept in enrichment cages at a room temperature of 21c , with a 12hour light dark cycle and free access to food and tap water . a pig cochlea ( sus scrofa ) care and use of the animals reported in this study were approved in accordance with ethical standards ( ethical permits n135/11 and c315/5 ) . for immunohistochemical staining , tissues from four untreated female albino rats , four untreated female albino guinea pigs , and an untreated pig were used . in the in vivo study , 15 female albino guinea pigs ( range 261337 g ) were used , anesthetized with an intramuscular injection of ketamine ( 40 mg / kg ) and xylazine ( 10 mg / kg ) . the weight of the animals was monitored daily , and they were given a daily subcutaneous injection of 5 ml saline . the end of the experiment was set to 96 hours after cisplatin was given ; thereafter , the animals were sacrificed with an overdose of pentobarbiturate and cochlea were harvested . parvalbumin is known to exert both a cytoplasmatic and a nuclear immunoreactivity in type i spiral ganglion cells and in hair cells . costaining with parvalbumin and oct2 was used to identify inner hair cells ( ihcs ) and ohcs.26 , 27 kidney served as positive control for oct2 , as shown in previous studies.20 , 28 the perilymphatic space of rat , guinea pig , and pig cochlea was perfused with 4% paraformaldehyde after decapitation , followed by decalcification . rat and guinea pig cochlea and kidney were embedded in paraffin , sectioned 5m thick , and mounted . the tissue sections were deparaffinized and boiled for 10 minutes in an antigen unmasking solution ( vector laboratories , burlingame , ca ) . a pig cochlea was embedded in tissuetek ( oct polysciences , warrington , pa ) , rapidly frozen , and sectioned at 8 to 10 m with a cryostat.29 the frozen sections were collected into gelatin / chrome alumcoated slides and stored below 70c prior to immunohistochemistry . all sections were incubated with blocking serum ( 5% goat serum ) for 30 minutes at room temperature . rabbit antirat organic cation transporter 2 ( oct2 ) polyclonal antibodies , diluted 1:100 ( alpha diagnostic international , san antonio , tx ) , and mouse antiparvalbumin , diluted 1:250 ( chemicon international , hants , united kingdom ) were used as primary antibodies . one separate slide and one section on each slide were used as a negative control . organic transport protein 2 antibodies were visualized by means of indocarbocyanine cy3 ( red dye ) conjugated goat antirabbit antibody , diluted 1:200 ( jackson immuno research laboratories inc . parvalbumin antibodies were visualized by means of alexa fluor 488 ( green dye ) conjugated goat antimouse antibody , diluted 1:400 ( invitrogen , carlsbad , cal . all slides were mounted with vecta shield mounting medium with 4,6diamidino2phenylindole ( dapi ) ( vector laboratories , burlingame , ca ) , and nuclei of all sections were then counterstained with dapi ( blue color ) . fluorescent signals were analyzed with a light microscope ( zeiss axio scope ; carl zeiss , jena , germany ) or with a laser confocal microscope ( nikon te2000 ; nikon co , tokyo , japan ) . the right internal jugular vein was catheterized for drug administration along the venous flow , that is , against the heart . the study group ( n = 8) were given phenformin 20 mg / kg intravenously ( phenformin hydrochloride 7.5 mg / ml ; sigmaaldrich , saint louis , mo ) 30 minutes prior to intravenous administration of cisplatin 8 mg / kg ( platinol 1 mg / ml ; bristolmyers squibb pharmaceuticals , new york , ny ) . control animals ( n = 7 ) were given only cisplatin 8 mg / kg intravenously ototoxicity was evaluated by measuring the electrophysiological hearing thresholds with auditory brainstem response ( abr ) and assessing the morphological loss of ihcs and ohcs . the levels of platinum in cochlear tissue were assessed and compared between the two groups . tuckerdavis technologies ( gainesville , fl ) equipment was used to measure the electrophysiological hearing thresholds of anesthetized animals . auditory threshold shifts were determined in the left ear at 6.3 khz , 12.5 khz , and 20 khz and calculated as the difference between thresholds obtained before and 96 hours after drug treatment , expressed as db change.30 the temporal bones were immediately removed after the animals were euthanized . for morphological calculation of the loss of ihcs and ohcs , after the bone was removed , the hair cells were labeled with phalloidin ( diluted 1:200 ) . hair cells ( scar formations ) were counted along the cochlea in a zeiss fluorescence microscope ( carl zeiss ) . the percentage of missing hair cells per mm was calculated.31 before cisplatin administration , one 0.25ml blood sample was aspirated from the left jugular vein of control group animals . after 96 hours , 0.25 ml blood was aspirated from the heart from all animals before euthanization . the right cochlea was used to determine the total platinum content in the tissue . from all cochlea , entire basilar membranes , including the organ of corti and stria vascularis , were pooled together to form one sample . inductively coupled plasma mass spectrometry ( analytica ab , lule , sweden ) was used for the analytical procedure.32 positive immunolabeling for oct2 ( red ) was seen in endothelial tubular cells from renal tissue ( fig . this was found in previous studies and served as a positive control.20 , 28 intense cytoplasmic immunoreactivity for oct2 ( red ) was observed in the supporting cells of organ of corti and in type i spiral ganglion cells . similar immunolabeling for oct2 was identified in the rat , guinea pig , and pig ( fig . positive staining for oct2 was seen in deiters cells , hensens cells , outer sulcus ( claudius cells ) and inner sulcus cells , outer and inner pillar cells , and in the tympanic covering layer localized under the basilar membrane ( fig . guinea pig and pig cochlea showed positive cytoplasmic and nuclear immunoreactivity to parvalbumin ( green ) in the ihcs with the connecting axons . weak staining was also seen in the three rows of ohcs ( fig 2c and e ) . type i spiral ganglion cells showed both nuclear and cytoplasmic immunoreactivity to parvalbumin ( fig . organic cation transporter 2 ( red ) and nuclear 4,6diamidino2phenylindole ( dapi ) staining ( blue ) . positive immunoreactivity to organic cation transporter 2 ( oct2 ) ( long arrows ) is evident in the endothelial tubular cells . nuclear staining with dapi ( short arrows ) confirms that oct2 is localized in the cell cytoplasm . intense immunoreactivity to oct2 is evident in the organ of corti ( long arrows ) and in type i spiral ganglion cells ( short arrows ) . organic cation transporter 2 ( red ) , parvalbumin ( green ) , and nuclear 4,6diamidino2phenylindole ( dapi ) staining ( blue ) . scala media ( sm ) , scala tympani ( st ) , and scala vestibuli ( sv ) . asterisks identify the inner hair cells ( ihc ) , first row of outer hair cells ( ohc1 ) , second row of outer hair cells ( ohc2 ) , and third row of outer hair cells ( ohc3 ) . organic cation transporter 2 immunoreactivity is visualized in deiters cells ( short arrows ) , inner sulcus cells ( opened arrowhead ) , outer sulcus cells , cells of claudius ( opened long arrow ) , hensens cells ( short opened arrow ) , inner and outer pillar cells ( arrowheads ) , and the tympanic covering layer ( long arrow ) . organic cation transporter 2 immunoreactivity is evident in deiters cells ( short arrows ) and inner sulcus cells ( long arrow ) . positive immunoreactivity to parvalbumin is seen both in cytoplasm and in nuclei of type i spiral ganglion cells ( arrowheads ) . cytoplasmic staining of oct2 is evident in type i spiral ganglion cells ( long arrows ) . asterisks identify the inner hair cells ( ihc ) , first row of outer hair cells ( ohc1 ) , second row of outer hair cells ( ohc2 ) , and third row of outer hair cells ( ohc3 ) . organic cation transporter 2 immunoreactivity is shown in the deiters cells ( short arrows ) . dapi identifies the nuclei of type i and ii spiral ganglion cells ( asterisks ) . positive immunoreactivity to parvalbumin is identified both in the cytoplasm and in the nuclei of type i spiral ganglion cells ( arrowheads ) . cytoplasmic staining of oct2 is identified in type i spiral ganglion cells ( long arrows ) . the guinea pigs had electrophysiological hearing thresholds in the normal range before treatment . after the intravenous injections of drugs , two animals in the study group died the day after treatment and were excluded . no weight differences were noted between the two groups over the study period ( p > 0.05 ) . generally , there was considerable individual variability of ototoxicity in both groups following cisplatin treatment ( table 1 ) . morphological evaluation revealed a pronounced loss of ohcs , whereas the ihcs were preserved in both groups . no difference in ohcs loss was seen between the two groups ( p > 0.05 ) . the platinum content in cochlear tissues did not differ between the groups ( p > 0.05 ) ( table 1 ) . abr = auditory brainstem response ; ohc 1 = first row of outer hair cells ; ohc 2 = second row of outer hair cells ; ohc3 = third row of outer hair cells ; platina = concentration in cochlea tissue 96 hours after drug treatment . positive immunolabeling for oct2 ( red ) was seen in endothelial tubular cells from renal tissue ( fig . this was found in previous studies and served as a positive control.20 , 28 intense cytoplasmic immunoreactivity for oct2 ( red ) was observed in the supporting cells of organ of corti and in type i spiral ganglion cells . similar immunolabeling for oct2 was identified in the rat , guinea pig , and pig ( fig . positive staining for oct2 was seen in deiters cells , hensens cells , outer sulcus ( claudius cells ) and inner sulcus cells , outer and inner pillar cells , and in the tympanic covering layer localized under the basilar membrane ( fig . guinea pig and pig cochlea showed positive cytoplasmic and nuclear immunoreactivity to parvalbumin ( green ) in the ihcs with the connecting axons . weak staining was also seen in the three rows of ohcs ( fig 2c and e ) . type i spiral ganglion cells showed both nuclear and cytoplasmic immunoreactivity to parvalbumin ( fig . organic cation transporter 2 ( red ) and nuclear 4,6diamidino2phenylindole ( dapi ) staining ( blue ) . positive immunoreactivity to organic cation transporter 2 ( oct2 ) ( long arrows ) is evident in the endothelial tubular cells . nuclear staining with dapi ( short arrows ) confirms that oct2 is localized in the cell cytoplasm . intense immunoreactivity to oct2 is evident in the organ of corti ( long arrows ) and in type i spiral ganglion cells ( short arrows ) . organic cation transporter 2 ( red ) , parvalbumin ( green ) , and nuclear 4,6diamidino2phenylindole ( dapi ) staining ( blue ) . scala media ( sm ) , scala tympani ( st ) , and scala vestibuli ( sv ) . asterisks identify the inner hair cells ( ihc ) , first row of outer hair cells ( ohc1 ) , second row of outer hair cells ( ohc2 ) , and third row of outer hair cells ( ohc3 ) . organic cation transporter 2 immunoreactivity is visualized in deiters cells ( short arrows ) , inner sulcus cells ( opened arrowhead ) , outer sulcus cells , cells of claudius ( opened long arrow ) , hensens cells ( short opened arrow ) , inner and outer pillar cells ( arrowheads ) , and the tympanic covering layer ( long arrow ) . organic cation transporter 2 immunoreactivity is evident in deiters cells ( short arrows ) and inner sulcus cells ( long arrow ) . positive immunoreactivity to parvalbumin is seen both in cytoplasm and in nuclei of type i spiral ganglion cells ( arrowheads ) . cytoplasmic staining of oct2 is evident in type i spiral ganglion cells ( long arrows ) . asterisks identify the inner hair cells ( ihc ) , first row of outer hair cells ( ohc1 ) , second row of outer hair cells ( ohc2 ) , and third row of outer hair cells ( ohc3 ) . organic cation transporter 2 immunoreactivity is shown in the deiters cells ( short arrows ) . dapi identifies the nuclei of type i and ii spiral ganglion cells ( asterisks ) . positive immunoreactivity to parvalbumin is identified both in the cytoplasm and in the nuclei of type i spiral ganglion cells ( arrowheads ) . cytoplasmic staining of oct2 is identified in type i spiral ganglion cells ( long arrows ) . the guinea pigs had electrophysiological hearing thresholds in the normal range before treatment . after the intravenous injections of drugs , two animals in the study group died the day after treatment and were excluded . no weight differences were noted between the two groups over the study period ( p > 0.05 ) . generally , there was considerable individual variability of ototoxicity in both groups following cisplatin treatment ( table 1 ) . morphological evaluation revealed a pronounced loss of ohcs , whereas the ihcs were preserved in both groups . no difference in ohcs loss was seen between the two groups ( p > 0.05 ) . the platinum content in cochlear tissues did not differ between the groups ( p > 0.05 ) ( table 1 ) . abr = auditory brainstem response ; ohc 1 = first row of outer hair cells ; ohc 2 = second row of outer hair cells ; ohc3 = third row of outer hair cells ; platina = concentration in cochlea tissue 96 hours after drug treatment . organic cation transporters may play an important part in the influx of cisplatin , a highly cytotoxic drug , to cochlear target cells . organic cation transporter 2 is one transport protein that has been identified to facilitate cisplatin uptake and thereby intensifies to its ototoxic side effect . inner ears from three different species were analyzed by immunohistochemistry using confocal microscopy , particularly regarding the cellular distribution of oct2 . positive immunoreactivity to oct2 was localized in the supporting cells and in the type i spiral ganglion cells from rat , guinea pig , and pig cochlea . these findings may partly corroborate previous studies and indicate a possible involvement of oct2 in cisplatin ototoxicity.6 however , administration of phenformin could not demonstrate any reduction of cisplatin ototoxicity or drug uptake , although earlier experimental studies have shown that cisplatin is transported actively by oct2.6 , 17 , 33 the concept of cisplatin ototoxicity as a consequence of active transport is based mainly on immunohistochemistry and polymerase chain reaction in the murine cochlea.6 in previous studies , the immunoreactivity to oct2 was identified in stria vascularis , ihcs , ohcs , and in spiral ganglion cells.6 our results localize positive immunostaining of oct2 in the supporting cells without any expression in the hair cells previously described by more et al.23 species differences are less likely to explain the inconsistent findings of the cochlear expression of oct2 . a possible explanation for the discrepancy appears more to be related to differences in immunochemical staining and microscopy techniques . in an effort to improve the resolution , furthermore , costaining with parvalbumin was carried out , which made it possible to separate the positive immunoreactivity in the organ of corti between the hair cells and supporting cells , thereby excluding the presence of oct2 both in the ihcs and ohcs . no immunoreactivity to oct2 could be visualized in the vicinity of the cochlear vascular networks of the spiral ligament and stria vascularis.34 , 35 these findings casts doubt as to whether oct2 is involved in cisplatin transport across the blood perilymph or intrastrial fluid because earlier studies using the mouse model have identified oct2 expression in the stria vascularis,6 , 23 further studies are needed to clarify the role of oct2 for the uptake from the systemic circulation . nuclear immunolabeling with dapi confirmed a cytoplasmic immunostaining of oct2,6 , 23 which is known to have 12 transmembrane domains with intra and extracellular loops.36 the cytoplasmic staining seen in the present study might be due to the antibody binding to amino acids in the endoplasmic reticulum and golgi apparatus . our previous findings based on pharmacokinetic studies suggested that the ototoxic effect of cisplatin is related to the drug concentration in the perilymphatic compartments.32 , 37 the findings in this study give no support that oct2 is primarily involved in the uptake of cisplatin from the systemic circulation but does not exclude an oct2mediated transport from deeper compartments of the cochlea to the target cells for cisplatin . the complex processes that take place in cisplatininduced ototoxicity probably involve several series of events , of which influx of the drug to target cells is the crucial first step . the question arises concerning whether oct2 , by acting in the supporting cells , implies an active uptake of cisplatin at these cellular sites and thus if that could be of major importance for the distribution of the drug to the ohcs . alternatively , the uptake of cisplatin by the supporting cells could lead to apoptosis in these cells as an early event in the ototoxic process preceding injury of the ohcs . cisplatin is reported to induce toxic lesions on the supporting cells in the organ of corti , mainly on the deiters cells that connect the ohcs to the basal membrane.38 , 39 the supporting cells are important for the bolster and survival of the ohcs . several ion channels40 and gap junction proteins such as connexins are located in the supporting cells.27 they are probably involved in the transport and recycling of the potassium that is released from the hair cells and recycled to the endolymphatic compartment.41 in this study , distinct positivity for oct2 was identified in deiters cells , and immunoreactivity of oct2 was also seen in the pillar cells that form the tunnel of corti , as well in hensens and claudius cells situated more laterally in the organ of corti.42 organic cation transporter 2 immunoreactivity in the type i spiral ganglion cells also confirms a direct uptake of cisplatin by these cells . the spiral ganglion cells connect the hair cells to the central nervous system , and the relatively large type i cells comprise 95% of the spiral ganglion cells . their peripheral processes are connected to the primary sensory cells , the ihcs.43 no positive immunoreactivity could be identified in the type ii spiral ganglion cells . recent studies indicate that several protein transporters are involved in the transport mechanisms of cisplatin . the copper transporter ctr1 has been studied both in the kidney and in the inner ear in relation to the side effects of cisplatin . it has been identified immunohistochemically in the inner ear.23 an intratympanic administration of copper sulphate prior to cisplatin injection has been found to prevent hearing loss.23 because cancer cells reportedly contain ctrl,44 , 45 local administration of a competitive binder to ctr1 is preferred , with the intention of reducing the ototoxic effect while preserving antineoplastic activity . in this study , no reduction of the ototoxic side effects induced by cisplatin could be established in guinea pigs pretreated the potential oct2 antagonist phenformin . this finding was corroborated by the fact that the concentration of total platinum in cochlear tissue did not differ between the two groups . first , phenformin in the guinea pig may not reach the deeper compartments of the inner ear where oct2 was found to be localized ; thus , the passage of phenformin over the bloodperilymph and intrastrial fluidblood barrier may be restricted.46 , 47 second , due to an anticipated risk of metabolic acidosis , only a limited dose of phenformin was given in vivo48 ; hence , the concentration of phenformin that reached oct2 in the cochlea might have been too weak for a competitive antagonistic effect . and finally , oct2 in the guinea pig might not primarily be involved in cisplatin transport to the inner ear structures . one limitation of this study was that we did not evaluate the pharmacokinetics of phenformin in blood and scala tympani perilymph . more knowledge about cisplatin transport into the inner ear compartments is necessary in order to understand the evolution of the toxic process . in this study , one possible problem in our interpretation , in the light of earlier observation , concerns the localization of oct2 and possible active transport of cisplatin to the target cells via the intrastrial fluid blood barrier . in contrast to earlier findings , no oct2 expression was localized to the stria vascularis . organic cation transporter 2 was located in the supporting cells and type i spiral ganglion cells . the localization of oct2 in this study gives no support that this transport protein is involved in the uptake of cisplatin from the vascular networks in the cochlea . systemically administered phenformin did not alleviate cisplatininduced ototoxicity , possibly due to restricted passage across the intrastrial fluid blood barrier . this present findings afford new insight into the possible mechanism of active cisplatin uptake by target cells . more studies are necessary , however , to obtain a complete picture of these processes as related to various transcellular transport mechanisms in the ear .

objectiveto locate the organic cation transporter 2 ( oct2 ) in the cochlea of three different species and to modulate the ototoxicity of cisplatin in the guinea pig by pretreatment with phenformin , having a known affinity for oct2.study designimmunohistochemical and in vivo study.methodssections from the auditory end organs were subjected to immunohistochemical staining in order to identify oct2 in cochlea from untreated rats , guinea pigs , and a pig . in the in vivo study , guinea pigs were given phenformin intravenously 30 minutes before cisplatin administration . electrophysiological hearing thresholds were determined , and hair cells loss was assessed 96 hours later . the total amount of platinum in cochlear tissue was determined using mass spectrometry.resultsorganic cation transporter 2 was found in the supporting cells and in type i spiral ganglion cells in the cochlea of all species studied . pretreatment with phenformin did not reduce the ototoxic side effect of cisplatin . furthermore , the concentration of platinum in the cochlea was not affected by phenformin.conclusionsthe localization of oct2 in the supporting cells and type i spiral ganglion cells suggests that this transport protein is not primarily involved in cisplatin uptake from the systemic circulation . we hypothesize that oct2 transport intensifies cisplatin ototoxicity via transport mechanisms in alternate compartments of the cochlea.level of evidencen / a . laryngoscope , 125:e320e325 , 2015

Level of Evidence INTRODUCTION MATERIALS AND METHODS Animals Immunohistochemistry of the Cochleae of Three Different Species Modulation of Ototoxicity by OCT Inhibition: An In Vivo Study Statistical Evaluation RESULTS Immunohistochemistry In Vivo Study DISCUSSION CONCLUSION

stria vascularis and the spiral ganglion cells are also reportedly affected by the drug.2 , 3 after cisplatin administration , ohcs are initially damaged in the base of the cochlea , leading to a highfrequency hearing loss.4 there is an individual susceptibility to cisplatin ototoxicity and no otoprotective treatments are clinically proven.1 nephrotoxic side effects resulting from damage to the proximal tubule cells can be alleviated by prehydration and forced diuresis.5 it is not known how cisplatin is transported from the systemic circulation to the target cells in the inner ear , but active transport mechanisms are suspected.6 , 7 organic cation transporters ( octs ) located in kidney and liver are known to be involved in the excretion of drugs from the systemic circulation.8 , 9 various isoforms of octs have specific species and tissuedistribution patterns.10 , 11 organic cations , such as the antidiabetic drugs metformin12 , 13 and phenformin14 , 15 and the histamine receptor antagonists cimetidine and ranitidine,16 are transported by octs in the renal endothelial cells . in previous studies , organic transport protein 2 ( oct2 ) was found to be involved in cisplatininduced nephro17 , 18 and ototoxicity.6 organic transport protein 2 has been shown to transport cisplatin to the renal proximal tubule cells,17 , 19 , 20 , 21 , 22 and immunostaining has localized oct2 to the cochlea of the murine inner ear.6 , 23 depleted ototoxic and nephrotoxic side effects of cisplatin were demonstrated in oct2deficient mice and mice treated with cimetidine.6 , 17 we have studied oct2 because of its suggested causative involvement in the ototoxic side effect of cisplatin . organic transport protein 2 is not reported to be a transport protein for cisplatin into cancer cells.24 therefore , a protective therapy utilizing transport pathways would not counteract the antitumour effect of cisplatin . in this study , phenformin was chosen as an otoprotector because previous studies have shown a competitive inhibitory effect of oct2.14 , 25 hence , the aim of the study was twofold , namely to locate oct2 by immunohistochemical means in the rat , guinea pig , and pig cochlea and to establish if pretreatment with phenformin reduces cisplatininduced ototoxicity in the guinea pig in vivo . for immunohistochemical staining , tissues from four untreated female albino rats , four untreated female albino guinea pigs , and an untreated pig were used . in the in vivo study , 15 female albino guinea pigs ( range 261337 g ) were used , anesthetized with an intramuscular injection of ketamine ( 40 mg / kg ) and xylazine ( 10 mg / kg ) . the end of the experiment was set to 96 hours after cisplatin was given ; thereafter , the animals were sacrificed with an overdose of pentobarbiturate and cochlea were harvested . parvalbumin is known to exert both a cytoplasmatic and a nuclear immunoreactivity in type i spiral ganglion cells and in hair cells . costaining with parvalbumin and oct2 was used to identify inner hair cells ( ihcs ) and ohcs.26 , 27 kidney served as positive control for oct2 , as shown in previous studies.20 , 28 the perilymphatic space of rat , guinea pig , and pig cochlea was perfused with 4% paraformaldehyde after decapitation , followed by decalcification . rabbit antirat organic cation transporter 2 ( oct2 ) polyclonal antibodies , diluted 1:100 ( alpha diagnostic international , san antonio , tx ) , and mouse antiparvalbumin , diluted 1:250 ( chemicon international , hants , united kingdom ) were used as primary antibodies . the study group ( n = 8) were given phenformin 20 mg / kg intravenously ( phenformin hydrochloride 7.5 mg / ml ; sigmaaldrich , saint louis , mo ) 30 minutes prior to intravenous administration of cisplatin 8 mg / kg ( platinol 1 mg / ml ; bristolmyers squibb pharmaceuticals , new york , ny ) . control animals ( n = 7 ) were given only cisplatin 8 mg / kg intravenously ototoxicity was evaluated by measuring the electrophysiological hearing thresholds with auditory brainstem response ( abr ) and assessing the morphological loss of ihcs and ohcs . the levels of platinum in cochlear tissue were assessed and compared between the two groups . auditory threshold shifts were determined in the left ear at 6.3 khz , 12.5 khz , and 20 khz and calculated as the difference between thresholds obtained before and 96 hours after drug treatment , expressed as db change.30 the temporal bones were immediately removed after the animals were euthanized . for morphological calculation of the loss of ihcs and ohcs , after the bone was removed , the hair cells were labeled with phalloidin ( diluted 1:200 ) . the percentage of missing hair cells per mm was calculated.31 before cisplatin administration , one 0.25ml blood sample was aspirated from the left jugular vein of control group animals . the right cochlea was used to determine the total platinum content in the tissue . for immunohistochemical staining , tissues from four untreated female albino rats , four untreated female albino guinea pigs , and an untreated pig were used . in the in vivo study , 15 female albino guinea pigs ( range 261337 g ) were used , anesthetized with an intramuscular injection of ketamine ( 40 mg / kg ) and xylazine ( 10 mg / kg ) . the weight of the animals was monitored daily , and they were given a daily subcutaneous injection of 5 ml saline . parvalbumin is known to exert both a cytoplasmatic and a nuclear immunoreactivity in type i spiral ganglion cells and in hair cells . costaining with parvalbumin and oct2 was used to identify inner hair cells ( ihcs ) and ohcs.26 , 27 kidney served as positive control for oct2 , as shown in previous studies.20 , 28 the perilymphatic space of rat , guinea pig , and pig cochlea was perfused with 4% paraformaldehyde after decapitation , followed by decalcification . rabbit antirat organic cation transporter 2 ( oct2 ) polyclonal antibodies , diluted 1:100 ( alpha diagnostic international , san antonio , tx ) , and mouse antiparvalbumin , diluted 1:250 ( chemicon international , hants , united kingdom ) were used as primary antibodies . the study group ( n = 8) were given phenformin 20 mg / kg intravenously ( phenformin hydrochloride 7.5 mg / ml ; sigmaaldrich , saint louis , mo ) 30 minutes prior to intravenous administration of cisplatin 8 mg / kg ( platinol 1 mg / ml ; bristolmyers squibb pharmaceuticals , new york , ny ) . the levels of platinum in cochlear tissue were assessed and compared between the two groups . auditory threshold shifts were determined in the left ear at 6.3 khz , 12.5 khz , and 20 khz and calculated as the difference between thresholds obtained before and 96 hours after drug treatment , expressed as db change.30 the temporal bones were immediately removed after the animals were euthanized . the percentage of missing hair cells per mm was calculated.31 before cisplatin administration , one 0.25ml blood sample was aspirated from the left jugular vein of control group animals . the right cochlea was used to determine the total platinum content in the tissue . this was found in previous studies and served as a positive control.20 , 28 intense cytoplasmic immunoreactivity for oct2 ( red ) was observed in the supporting cells of organ of corti and in type i spiral ganglion cells . similar immunolabeling for oct2 was identified in the rat , guinea pig , and pig ( fig . positive staining for oct2 was seen in deiters cells , hensens cells , outer sulcus ( claudius cells ) and inner sulcus cells , outer and inner pillar cells , and in the tympanic covering layer localized under the basilar membrane ( fig . type i spiral ganglion cells showed both nuclear and cytoplasmic immunoreactivity to parvalbumin ( fig . organic cation transporter 2 ( red ) and nuclear 4,6diamidino2phenylindole ( dapi ) staining ( blue ) . positive immunoreactivity to organic cation transporter 2 ( oct2 ) ( long arrows ) is evident in the endothelial tubular cells . intense immunoreactivity to oct2 is evident in the organ of corti ( long arrows ) and in type i spiral ganglion cells ( short arrows ) . organic cation transporter 2 ( red ) , parvalbumin ( green ) , and nuclear 4,6diamidino2phenylindole ( dapi ) staining ( blue ) . organic cation transporter 2 immunoreactivity is visualized in deiters cells ( short arrows ) , inner sulcus cells ( opened arrowhead ) , outer sulcus cells , cells of claudius ( opened long arrow ) , hensens cells ( short opened arrow ) , inner and outer pillar cells ( arrowheads ) , and the tympanic covering layer ( long arrow ) . organic cation transporter 2 immunoreactivity is evident in deiters cells ( short arrows ) and inner sulcus cells ( long arrow ) . positive immunoreactivity to parvalbumin is seen both in cytoplasm and in nuclei of type i spiral ganglion cells ( arrowheads ) . cytoplasmic staining of oct2 is evident in type i spiral ganglion cells ( long arrows ) . organic cation transporter 2 immunoreactivity is shown in the deiters cells ( short arrows ) . dapi identifies the nuclei of type i and ii spiral ganglion cells ( asterisks ) . positive immunoreactivity to parvalbumin is identified both in the cytoplasm and in the nuclei of type i spiral ganglion cells ( arrowheads ) . cytoplasmic staining of oct2 is identified in type i spiral ganglion cells ( long arrows ) . the guinea pigs had electrophysiological hearing thresholds in the normal range before treatment . this was found in previous studies and served as a positive control.20 , 28 intense cytoplasmic immunoreactivity for oct2 ( red ) was observed in the supporting cells of organ of corti and in type i spiral ganglion cells . similar immunolabeling for oct2 was identified in the rat , guinea pig , and pig ( fig . positive staining for oct2 was seen in deiters cells , hensens cells , outer sulcus ( claudius cells ) and inner sulcus cells , outer and inner pillar cells , and in the tympanic covering layer localized under the basilar membrane ( fig . guinea pig and pig cochlea showed positive cytoplasmic and nuclear immunoreactivity to parvalbumin ( green ) in the ihcs with the connecting axons . type i spiral ganglion cells showed both nuclear and cytoplasmic immunoreactivity to parvalbumin ( fig . organic cation transporter 2 ( red ) and nuclear 4,6diamidino2phenylindole ( dapi ) staining ( blue ) . positive immunoreactivity to organic cation transporter 2 ( oct2 ) ( long arrows ) is evident in the endothelial tubular cells . intense immunoreactivity to oct2 is evident in the organ of corti ( long arrows ) and in type i spiral ganglion cells ( short arrows ) . organic cation transporter 2 ( red ) , parvalbumin ( green ) , and nuclear 4,6diamidino2phenylindole ( dapi ) staining ( blue ) . organic cation transporter 2 immunoreactivity is visualized in deiters cells ( short arrows ) , inner sulcus cells ( opened arrowhead ) , outer sulcus cells , cells of claudius ( opened long arrow ) , hensens cells ( short opened arrow ) , inner and outer pillar cells ( arrowheads ) , and the tympanic covering layer ( long arrow ) . positive immunoreactivity to parvalbumin is seen both in cytoplasm and in nuclei of type i spiral ganglion cells ( arrowheads ) . cytoplasmic staining of oct2 is evident in type i spiral ganglion cells ( long arrows ) . organic cation transporter 2 immunoreactivity is shown in the deiters cells ( short arrows ) . positive immunoreactivity to parvalbumin is identified both in the cytoplasm and in the nuclei of type i spiral ganglion cells ( arrowheads ) . cytoplasmic staining of oct2 is identified in type i spiral ganglion cells ( long arrows ) . the guinea pigs had electrophysiological hearing thresholds in the normal range before treatment . organic cation transporter 2 is one transport protein that has been identified to facilitate cisplatin uptake and thereby intensifies to its ototoxic side effect . positive immunoreactivity to oct2 was localized in the supporting cells and in the type i spiral ganglion cells from rat , guinea pig , and pig cochlea . these findings may partly corroborate previous studies and indicate a possible involvement of oct2 in cisplatin ototoxicity.6 however , administration of phenformin could not demonstrate any reduction of cisplatin ototoxicity or drug uptake , although earlier experimental studies have shown that cisplatin is transported actively by oct2.6 , 17 , 33 the concept of cisplatin ototoxicity as a consequence of active transport is based mainly on immunohistochemistry and polymerase chain reaction in the murine cochlea.6 in previous studies , the immunoreactivity to oct2 was identified in stria vascularis , ihcs , ohcs , and in spiral ganglion cells.6 our results localize positive immunostaining of oct2 in the supporting cells without any expression in the hair cells previously described by more et al.23 species differences are less likely to explain the inconsistent findings of the cochlear expression of oct2 . in an effort to improve the resolution , furthermore , costaining with parvalbumin was carried out , which made it possible to separate the positive immunoreactivity in the organ of corti between the hair cells and supporting cells , thereby excluding the presence of oct2 both in the ihcs and ohcs . no immunoreactivity to oct2 could be visualized in the vicinity of the cochlear vascular networks of the spiral ligament and stria vascularis.34 , 35 these findings casts doubt as to whether oct2 is involved in cisplatin transport across the blood perilymph or intrastrial fluid because earlier studies using the mouse model have identified oct2 expression in the stria vascularis,6 , 23 further studies are needed to clarify the role of oct2 for the uptake from the systemic circulation . our previous findings based on pharmacokinetic studies suggested that the ototoxic effect of cisplatin is related to the drug concentration in the perilymphatic compartments.32 , 37 the findings in this study give no support that oct2 is primarily involved in the uptake of cisplatin from the systemic circulation but does not exclude an oct2mediated transport from deeper compartments of the cochlea to the target cells for cisplatin . the question arises concerning whether oct2 , by acting in the supporting cells , implies an active uptake of cisplatin at these cellular sites and thus if that could be of major importance for the distribution of the drug to the ohcs . alternatively , the uptake of cisplatin by the supporting cells could lead to apoptosis in these cells as an early event in the ototoxic process preceding injury of the ohcs . cisplatin is reported to induce toxic lesions on the supporting cells in the organ of corti , mainly on the deiters cells that connect the ohcs to the basal membrane.38 , 39 the supporting cells are important for the bolster and survival of the ohcs . several ion channels40 and gap junction proteins such as connexins are located in the supporting cells.27 they are probably involved in the transport and recycling of the potassium that is released from the hair cells and recycled to the endolymphatic compartment.41 in this study , distinct positivity for oct2 was identified in deiters cells , and immunoreactivity of oct2 was also seen in the pillar cells that form the tunnel of corti , as well in hensens and claudius cells situated more laterally in the organ of corti.42 organic cation transporter 2 immunoreactivity in the type i spiral ganglion cells also confirms a direct uptake of cisplatin by these cells . the spiral ganglion cells connect the hair cells to the central nervous system , and the relatively large type i cells comprise 95% of the spiral ganglion cells . their peripheral processes are connected to the primary sensory cells , the ihcs.43 no positive immunoreactivity could be identified in the type ii spiral ganglion cells . recent studies indicate that several protein transporters are involved in the transport mechanisms of cisplatin . in this study , no reduction of the ototoxic side effects induced by cisplatin could be established in guinea pigs pretreated the potential oct2 antagonist phenformin . this finding was corroborated by the fact that the concentration of total platinum in cochlear tissue did not differ between the two groups . first , phenformin in the guinea pig may not reach the deeper compartments of the inner ear where oct2 was found to be localized ; thus , the passage of phenformin over the bloodperilymph and intrastrial fluidblood barrier may be restricted.46 , 47 second , due to an anticipated risk of metabolic acidosis , only a limited dose of phenformin was given in vivo48 ; hence , the concentration of phenformin that reached oct2 in the cochlea might have been too weak for a competitive antagonistic effect . and finally , oct2 in the guinea pig might not primarily be involved in cisplatin transport to the inner ear structures . in this study , one possible problem in our interpretation , in the light of earlier observation , concerns the localization of oct2 and possible active transport of cisplatin to the target cells via the intrastrial fluid blood barrier . organic cation transporter 2 was located in the supporting cells and type i spiral ganglion cells . the localization of oct2 in this study gives no support that this transport protein is involved in the uptake of cisplatin from the vascular networks in the cochlea . more studies are necessary , however , to obtain a complete picture of these processes as related to various transcellular transport mechanisms in the ear .

bisphosphonates ( bps ) are stable analogs of pyrophosphate , which are naturally occurring modulators of bone metabolism and have been synthesized and used since the 19 century , but their in - vitro ability to inhibit the precipitation of calcium phosphate was applied clinically in 1960s . oral bps are poorly absorbed by the gastrointestinal tract about 10% and excreted largely unchanged by the kidneys , but if given intravenously nitrogen containing bisphosphonates ( n - bps ) about 50% of the drugs goes to the bone . bps are commonly used to treat certain resorptive bone diseases such as osteoporosis , paget 's disease and hypercalcemia associated with certain malignancies such as multiple myeloma and bone metastasis from the breast or prostate . their principal action is to inhibit resorption of bone by inhibiting osteoclast activity and its life - span that leads to modulation of the osteoclast osteoblast inter relation , which results in an increase in the mineral density of bone and a reduction in serum calcium ; although , other actions such as inhibition of angiogenesis and anti - human endothelial cell proliferation and to modulate endothelial cell adhesion and migration have also been reported . bps has unique pharmacokinetic properties like long retention time in bone ; it is possible that beneficial effects on fracture risk may persist for some time after treatment is stopped . bps has a high affinity for exposed hydroxyapatite within bone mineral and bone is metabolically inactive . as the process of metabolic bone resorption progresses , previously bound bps is released and exerts their clinical effect . however , according to previous literature , all three above mentioned pharmacological action produced by n - bps becomes more aggressive in the presence of other risk factors ( drug related , local , demographic / systemic , genetic and preventive ) that finally leads to a rare , but serious clinical condition called n - bisphosphonate - related osteonecrosis of the jaw ( bronj ) that was first described by marx in 2003 . chemical structure of bp [ figure 1 ] has two important entity p - c - p back bone and r2 side chain that shows a strong affinity for bone mineral and provides potent inhibition of bone turn over both in - vivo and in - vitro and therapeutic potency of the bps respectively . chemically bps had two sub - divisions , which have a different mechanism of action on osteoclasts based on the presence or absence of a nitrogen side chain on the pyrophosphate group ( r2 side chain ) . n - bps had poorly absorbed by gastrointestinal tract as compare to non - n - bps , owing to this region n - bps are commonly prepared for iv administration . chemical structure of bisphosphonate bone remodeling is a physiologically coordinated process involving bone formation by osteoblasts and bone resorption by osteoclasts . imbalance between these two entities may lead to skeletal abnormalities characterized by increases or decreases in bone density . in contrast to other skeleton , jaw bones ( alveolar process and periodontium ) have relatively high vascularity , bone turn over and remodeling because of continuous mechanical stress . non - n - bps ( tiludronate , clodronate and etidronate ) is taken up by the osteoclasts and antagonized the cellular energy pathways due to intracellular liberation of methylene that contains toxic analogs of adenosine triphosphate ( atp ) , which probably inhibit atp - utilizing enzymes and induce osteoclast apoptosis , whereas n - bps ( zoledronate , pamidronate , alendronateetc . , ) has a more complex pathway of action where they inhibit the mevalonate pathway by inhibition of farnesyl - pyrophosphate synthetase leading to prenylation of small gtpase signaling proteins that are essential for osteoclast activity and survival . thus , there is no osteoclastic resorption of mineral matrix and the resultant release of bone morphogenic protein and insulin - like growth to induced stem cell production of osteoblasts , the osteon becomes a - cellular and necrotic . therefore , any mechanism that leads to the breakdown of the over laying mucosa will expose the necrotic bone , which fails to heal . according to previous literature , zoledronate has also been shown to inhibit human endothelial cell to proliferate and to modulate endothelial cell adhesion and migration . therefore , a possible association was seen between bps and two rare but serious conditions , namely atypical femoral fracture and osteonecrosis of the jaw ( onj ) with an incidence of 0.8 - 12% in iv bps and 0.01 - 0.04% in oral bps administration . although the exact pathophysiology of bps induced onj has not be completely illuminated , but according to previous literature , n - bps are potent inhibitors of osteoclastic activity , angiogenesis , human endothelial cell to proliferate and to modulate endothelial cell adhesion and migration . thus , the net result is that the jaw bone is unable to meet the peak demand for bone repair and remodeling that may finally lead to bronj . exact diagnosis criteria to distinguish bronj from other delayed healing conditions ( alveolar osteitis , osteoradionecrosis , osteomyelitis etc . , ) are not known yet today , but according to previous literature patients may be considered to have bronj if all of the following four characteristics are present : ( 1 ) current or previous treatment with a bps , ( 2 ) exposed or necrotic bone in the maxillofacial region that has persisted for more than 8 weeks , ( 3 ) no history of radiation therapy to the jaws and ( 4 ) no evidence of cancer at the site . lesions in patients who have not fulfilled above four characteristics should be excluded from the diagnosis of bronj . radiographic findings of bronj are not specific and are found in other conditions such as osteomyelitis , osteoradionecrosis and metastatic bone lesions . this can vary from subtle thickening of the lamina dura and alveolar crest to attenuated osteopetrosis like sclerosis . other findings such as osteolysis , soft - tissue swelling , periosteal new bone formation , periapical lucencies , oroantral fistula and sequestra , which are likely to correspond with the presence of infection . its imaging differential diagnosis includes chronic sclerosing osteomyelitis , osteoradionecrosis , bone metastasis and paget 's disease . if osteonecrosis is suspected , different imaging techniques may be performed to confirm the diagnosis and extent of the lesions . periapical radiograph and cone - beam computed tomography ( cbct ) reveals generalized thickening of the cortical plate and laminadura , mixed sclerotic and lytic bone destruction involving alveolar bone and basal bone , sequestrum , encroachment on the mandibular canal and maxillary antrum and pathological fracture while thickening of the cortical plate in the affected region was the only radiological findings of cbct.computed tomography images reveals sclerotic changes , osteolytic changes , periosteal bone proliferation , sequestration and inferior alveolar canal involvement while contrast enhanced magnetic resonance images reveals intensity changes of the cortical and sub cortical bone structures , contrast enhancement in necrotic bone area , soft - tissue involvement and cervical lymphadenopathy.99tcm myocardial perfusion defect reveals detection of local bone remodeling activity / high bone turn over sites , but the presence of increased uptake was conformed through the single - photon emission computed tomography because they provide a high degree of accuracy . periapical radiograph and cone - beam computed tomography ( cbct ) reveals generalized thickening of the cortical plate and laminadura , mixed sclerotic and lytic bone destruction involving alveolar bone and basal bone , sequestrum , encroachment on the mandibular canal and maxillary antrum and pathological fracture while thickening of the cortical plate in the affected region was the only radiological findings of cbct . computed tomography images reveals sclerotic changes , osteolytic changes , periosteal bone proliferation , sequestration and inferior alveolar canal involvement while contrast enhanced magnetic resonance images reveals intensity changes of the cortical and sub cortical bone structures , contrast enhancement in necrotic bone area , soft - tissue involvement and cervical lymphadenopathy . 99tcm myocardial perfusion defect reveals detection of local bone remodeling activity / high bone turn over sites , but the presence of increased uptake was conformed through the single - photon emission computed tomography because they provide a high degree of accuracy . in past 10 year , some important serological , histopathological and immunohistochemistry findings are obtained that may suggest the appearance of bronj in that patient . according to recent literature , angiogenesis suppression may play a significant role in development of bronj . on the basis of this evidence , vincenzi et al . evaluated the role of vascular endothelial growth factor ( vegf ) as a predictive marker of bronj and found decreased vegf circulating levels at day 7 and 21 after the 1 administration of n - bps ; thus , authors concluded that the anti - angiogenic properties of n - bps are directly linked to bronj pathogenesis and serum vegf levels could represent an effective early predictive marker . similarly , morning fasting serum c - terminal telopertide ( ctx ) value is less than 100pg / ml in those patients who received / receiving n - bps representing high risk of bronj development , but invasive dental procedure is only indicated if ctx value is greater than 150pg / ml to achieve , uncomplicated healing . microscopic evaluation of hard tissues reveals necrotic bone spicules with bacterial colonization and inter aspersed acute and chronic inflammatory cells while soft - tissue evaluation shows proliferating stratified squamous epithelium with arcading rete pegs and neutrophilic exocytosis and adjacent fibrous connective tissue revealed the presence of patches of plasma cells , inter aspersed neutrophils and surgical hemorrhage that was similar to osteomyelitis . immuno histo - chemistry reveals increased expression of human defensins ( hbd)-1,-2,-3 , reduced expression of transforming growth factor beta1 and increased galectin-3 expression in cases of bronj . microbial cultures may provide identification of the pathogens causing secondary infections ( actinomyces and other pathogens ) which is important for selection of appropriate antibiotics . 1 was given by recently published position paper by american association of oral and maxillofacial surgeons ( aaoms ) ( 2009 ) while 2 was given by olutayo et al . thus , bronj may remain asymptomatic for many weeks or months and may only be recognized clinically by the presence of the exposed bone in the oral cavity . these lesions are most frequently symptomatic when sites becomes secondary infected or there is a trauma to the soft - tissues through the sharp edges of the exposed bone . typical signs and symptoms include pain , soft - tissues swelling , paresthesia , suppuration , soft - tissue ulceration , loosening of teeth and drainage that is similar to osteoradionecrosis . according to lam et al . , common orofacial findings associated with bps associated onj are poor wound healing , spontaneous or post - surgical soft - tissue breakdown leading to intra oral bone exposure , bone necrosis and osteomyelitis . however , in severe cases some additional orofacial finding like intense pain , extensive sequestration of bone and cutaneous draining sinus tracts . aaoms staging for bronj on the basis of clinical findings staging for bronj on the basis of clinical and radiologic findings according to recent position paper by aaoms and nsw health guideline , risk factors for the development of bps associated onj can be grouped as drug - related , local , demographic / systematic , genetic and preventive . drug related risk factors include : potency of bps : more potent bps ( zoledronate > pamidronate > oral bps ) have more tendency to developed onj . the iv route of administration resulting greater drug exposure to the jaw than the oral route ; therefore more tendencies to developed onj . incidence rate of bronj in cancer patient is increased 2.7 - 4.2 fold if given iv bps . longer duration therapy of bps may be associated with increased risk of development of onjlocal risk factors include : dento alveolar surgery like extraction , dental implant placement , periapical surgery , periodontal surgery etc . has 7 times more likely to develop bronj than patients who are not having dentoalveolar surgery . anatomic location : bronj is more common in the mandible than in the maxilla with the ratio of 2:1 and more common in areas with thin mucosa overlaying bony prominences like tori , bony exostoses and mylohyoid ridge . concomitant oral diseases like cancer patients exposed to iv bps with a history of inflammatory ( possibly infective ) dental diseases are at a 7 fold increased risk for developing onj.demographic and systemic factors:- age : with each passing decade there is a 9% increased risk of onj in multiple myeloma patients treated with iv bps , but sex was not statically associated with onj . cancer type : multiple myeloma > > breast cancer > other cancer and osteopenia / osteoporosis concurrent with cancer are more prone to developed onj . concomitant risk factors : renal dialysis , low hemoglobin , obesity , diabetes , tobacco users , poor oral hygiene and chemotherapeutic agents such as cyclophosphamide , erythropoietin and steroids are risk factors , but no increased risk associated with alcohol exposure.genetic risk factors : like genetic perturbations , i.e. , single nucleotide polymorphisms , in the cytochrome p450 - 2c gene ( cyp2c8 ) gene were associated with an increased risk for onj among multiple myeloma patients treated with iv bps.preventive factors : the aaoms task force on bronj recommended that patients undergo dental evaluations and receive necessary treatment prior to initiating iv bps therapy to reduce the incidence of bronj . manipulation of iv bps dosing may be effective in reducing skeletal related events ( sres ) and minimizing bps associated onj . drug related risk factors include : potency of bps : more potent bps ( zoledronate > pamidronate > oral bps ) have more tendency to developed onj . the iv route of administration resulting greater drug exposure to the jaw than the oral route ; therefore more tendencies to developed onj . incidence rate of bronj in cancer patient is increased 2.7 - 4.2 fold if given iv bps . longer duration therapy of bps may be associated with increased risk of development of onj local risk factors include : dento alveolar surgery like extraction , dental implant placement , periapical surgery , periodontal surgery etc . bps therapy in association with dento alveolar surgery has 7 times more likely to develop bronj than patients who are not having dentoalveolar surgery . anatomic location : bronj is more common in the mandible than in the maxilla with the ratio of 2:1 and more common in areas with thin mucosa overlaying bony prominences like tori , bony exostoses and mylohyoid ridge . concomitant oral diseases like cancer patients exposed to iv bps with a history of inflammatory ( possibly infective ) dental diseases are at a 7 fold increased risk for developing onj . demographic and systemic factors:- age : with each passing decade there is a 9% increased risk of onj in multiple myeloma patients treated with iv bps , but sex was not statically associated with onj . cancer type : multiple myeloma > > breast cancer > other cancer and osteopenia / osteoporosis concurrent with cancer are more prone to developed onj . concomitant risk factors : renal dialysis , low hemoglobin , obesity , diabetes , tobacco users , poor oral hygiene and chemotherapeutic agents such as cyclophosphamide , erythropoietin and steroids are risk factors , but no increased risk associated with alcohol exposure . genetic risk factors : like genetic perturbations , i.e. , single nucleotide polymorphisms , in the cytochrome p450 - 2c gene ( cyp2c8 ) gene were associated with an increased risk for onj among multiple myeloma patients treated with iv bps . preventive factors : the aaoms task force on bronj recommended that patients undergo dental evaluations and receive necessary treatment prior to initiating iv bps therapy to reduce the incidence of bronj . manipulation of iv bps dosing may be effective in reducing skeletal related events ( sres ) and minimizing bps associated onj . management strategies of bronj is mostly palliative and empirical ( to eliminate clinical symptoms such as pain , infection and minimize the progression of bone necrosis ) before microbial culture report . most authors agreed that management of bronj started after advised morning fasting serum ctx test and begins palliative care . if the exposed bone is pain less , treatment started with 0.12% chlorhexidine mouth rinse , but if patient complaints pain and/or clinical evidence of infection , antibiotic therapy should be provided in addition to the 0.12% chlorhexidine . however , invasive dental procedure is indicated only when morning ctx value is greater than 150pg / ml , to achieve , uncomplicated healing . many treatment modalities are discussed for correction of bronj like cover the exposed bone areas with tissue flaps by marx . sequential removal of sequestra ( conservative approach ) and extensive involvement may necessitate large area of debridement to include segmental mandibulectomy and partial maxillectomy for correction of bronj and was reported that most patients with limited regions of exposed bone ( except those lesions that are a constant source of infection ) have been successfully managed with irrigations and antibiotic therapy while surgical management have not been completely effective in eradication of the bronj by ruggiero et al . administration of pentoxifylline with -tocopherol reduces 74% area of bony exposure and symptom control by epstein et al . transplantation of intra - lesional autologous bone marrow stem cell with complete response by cella et al . hyperbaric oxygen therapy was beneficial for patients and the outcome is increased with cessation of bps administration . no fixed treatment protocol was adopted before recent position paper published by aaoms,(2009 ) that provide new treatment strategies on the basis of staging of bronj : at risk - patients who are at risk of developing bronj by virtue of the fact that they have been exposed to a bps do not require any treatment . however , these patients should be informed of the risk of developing bronj as well as the signs and symptoms of this diseases process . stage 0-here provide symptomatic treatment and conservatively manage other local factors , such as caries and periodontal disease . systemic management may include the use of medication for chronic pain and control of infection with antibiotics , when indicated . stage 1-these patients benefit from the use of antiseptic mouth washes ( chlorhexidine gluconate 0.12% ) and/or analgesia is proposed for patients with clinical evidence of bronj , but in the absence of any evidence of infection . here , primary aim is to reduce the likelihood of further progression of bronj and avoid infection of exposed bone , but not any surgical intervention required . however , patient education and review of indications for continued bp therapy is also important . stage 2-these patients benefit from the use of oral antimicrobial rinse in combination with antibiotic therapy . in bronj most of the isolated microbes have been sensitive to penicillin group because its development is not infectious origin . if patient was allergic to above group then choice of drug is quinolones , metronidazole , clindamycin , doxycycline and erythromycin . if microbial cultures show positive evidence of actinomyces species then antibiotic regimen should be adjusted accordingly . in some refractory cases , patients may require combination antibiotic therapy , long - term antibiotic maintenance or a course of iv antibiotic therapy . stage 3-these patients benefit from debridement , including resection , in combination with antibiotic therapy , which may offer long - term palliation with resolution of acute infection and pain . regardless of the disease stage the extraction of symptomatic teeth within exposed , necrotic bone should be considered since it is unlikely that the extraction will exacerbate the established necrotic process . exact diagnosis criteria to distinguish bronj from other delayed healing conditions ( alveolar osteitis , osteoradionecrosis , osteomyelitis etc . , ) are not known yet today , but according to previous literature patients may be considered to have bronj if all of the following four characteristics are present : ( 1 ) current or previous treatment with a bps , ( 2 ) exposed or necrotic bone in the maxillofacial region that has persisted for more than 8 weeks , ( 3 ) no history of radiation therapy to the jaws and ( 4 ) no evidence of cancer at the site . lesions in patients who have not fulfilled above four characteristics should be excluded from the diagnosis of bronj . radiographic findings of bronj are not specific and are found in other conditions such as osteomyelitis , osteoradionecrosis and metastatic bone lesions . this can vary from subtle thickening of the lamina dura and alveolar crest to attenuated osteopetrosis like sclerosis . other findings such as osteolysis , soft - tissue swelling , periosteal new bone formation , periapical lucencies , oroantral fistula and sequestra , which are likely to correspond with the presence of infection . its imaging differential diagnosis includes chronic sclerosing osteomyelitis , osteoradionecrosis , bone metastasis and paget 's disease . if osteonecrosis is suspected , different imaging techniques may be performed to confirm the diagnosis and extent of the lesions . periapical radiograph and cone - beam computed tomography ( cbct ) reveals generalized thickening of the cortical plate and laminadura , mixed sclerotic and lytic bone destruction involving alveolar bone and basal bone , sequestrum , encroachment on the mandibular canal and maxillary antrum and pathological fracture while thickening of the cortical plate in the affected region was the only radiological findings of cbct.computed tomography images reveals sclerotic changes , osteolytic changes , periosteal bone proliferation , sequestration and inferior alveolar canal involvement while contrast enhanced magnetic resonance images reveals intensity changes of the cortical and sub cortical bone structures , contrast enhancement in necrotic bone area , soft - tissue involvement and cervical lymphadenopathy.99tcm myocardial perfusion defect reveals detection of local bone remodeling activity / high bone turn over sites , but the presence of increased uptake was conformed through the single - photon emission computed tomography because they provide a high degree of accuracy . periapical radiograph and cone - beam computed tomography ( cbct ) reveals generalized thickening of the cortical plate and laminadura , mixed sclerotic and lytic bone destruction involving alveolar bone and basal bone , sequestrum , encroachment on the mandibular canal and maxillary antrum and pathological fracture while thickening of the cortical plate in the affected region was the only radiological findings of cbct . computed tomography images reveals sclerotic changes , osteolytic changes , periosteal bone proliferation , sequestration and inferior alveolar canal involvement while contrast enhanced magnetic resonance images reveals intensity changes of the cortical and sub cortical bone structures , contrast enhancement in necrotic bone area , soft - tissue involvement and cervical lymphadenopathy . 99tcm myocardial perfusion defect reveals detection of local bone remodeling activity / high bone turn over sites , but the presence of increased uptake was conformed through the single - photon emission computed tomography because they provide a high degree of accuracy . in past 10 year , some important serological , histopathological and immunohistochemistry findings are obtained that may suggest the appearance of bronj in that patient . according to recent literature , angiogenesis suppression may play a significant role in development of bronj . on the basis of this evidence , vincenzi et al . evaluated the role of vascular endothelial growth factor ( vegf ) as a predictive marker of bronj and found decreased vegf circulating levels at day 7 and 21 after the 1 administration of n - bps ; thus , authors concluded that the anti - angiogenic properties of n - bps are directly linked to bronj pathogenesis and serum vegf levels could represent an effective early predictive marker . similarly , morning fasting serum c - terminal telopertide ( ctx ) value is less than 100pg / ml in those patients who received / receiving n - bps representing high risk of bronj development , but invasive dental procedure is only indicated if ctx value is greater than 150pg / ml to achieve , uncomplicated healing . microscopic evaluation of hard tissues reveals necrotic bone spicules with bacterial colonization and inter aspersed acute and chronic inflammatory cells while soft - tissue evaluation shows proliferating stratified squamous epithelium with arcading rete pegs and neutrophilic exocytosis and adjacent fibrous connective tissue revealed the presence of patches of plasma cells , inter aspersed neutrophils and surgical hemorrhage that was similar to osteomyelitis . immuno histo - chemistry reveals increased expression of human defensins ( hbd)-1,-2,-3 , reduced expression of transforming growth factor beta1 and increased galectin-3 expression in cases of bronj . microbial cultures may provide identification of the pathogens causing secondary infections ( actinomyces and other pathogens ) which is important for selection of appropriate antibiotics . 1 was given by recently published position paper by american association of oral and maxillofacial surgeons ( aaoms ) ( 2009 ) while 2 was given by olutayo et al . thus , bronj may remain asymptomatic for many weeks or months and may only be recognized clinically by the presence of the exposed bone in the oral cavity . these lesions are most frequently symptomatic when sites becomes secondary infected or there is a trauma to the soft - tissues through the sharp edges of the exposed bone . typical signs and symptoms include pain , soft - tissues swelling , paresthesia , suppuration , soft - tissue ulceration , loosening of teeth and drainage that is similar to osteoradionecrosis . according to lam et al . , common orofacial findings associated with bps associated onj are poor wound healing , spontaneous or post - surgical soft - tissue breakdown leading to intra oral bone exposure , bone necrosis and osteomyelitis . however , in severe cases some additional orofacial finding like intense pain , extensive sequestration of bone and cutaneous draining sinus tracts . aaoms staging for bronj on the basis of clinical findings staging for bronj on the basis of clinical and radiologic findings according to recent position paper by aaoms and nsw health guideline , risk factors for the development of bps associated onj can be grouped as drug - related , local , demographic / systematic , genetic and preventive . drug related risk factors include : potency of bps : more potent bps ( zoledronate > pamidronate > oral bps ) have more tendency to developed onj . the iv route of administration resulting greater drug exposure to the jaw than the oral route ; therefore more tendencies to developed onj . incidence rate of bronj in cancer patient is increased 2.7 - 4.2 fold if given iv bps . longer duration therapy of bps may be associated with increased risk of development of onjlocal risk factors include : dento alveolar surgery like extraction , dental implant placement , periapical surgery , periodontal surgery etc . has 7 times more likely to develop bronj than patients who are not having dentoalveolar surgery . anatomic location : bronj is more common in the mandible than in the maxilla with the ratio of 2:1 and more common in areas with thin mucosa overlaying bony prominences like tori , bony exostoses and mylohyoid ridge . concomitant oral diseases like cancer patients exposed to iv bps with a history of inflammatory ( possibly infective ) dental diseases are at a 7 fold increased risk for developing onj.demographic and systemic factors:- age : with each passing decade there is a 9% increased risk of onj in multiple myeloma patients treated with iv bps , but sex was not statically associated with onj . cancer type : multiple myeloma > > breast cancer > other cancer and osteopenia / osteoporosis concurrent with cancer are more prone to developed onj . concomitant risk factors : renal dialysis , low hemoglobin , obesity , diabetes , tobacco users , poor oral hygiene and chemotherapeutic agents such as cyclophosphamide , erythropoietin and steroids are risk factors , but no increased risk associated with alcohol exposure.genetic risk factors : like genetic perturbations , i.e. , single nucleotide polymorphisms , in the cytochrome p450 - 2c gene ( cyp2c8 ) gene were associated with an increased risk for onj among multiple myeloma patients treated with iv bps.preventive factors : the aaoms task force on bronj recommended that patients undergo dental evaluations and receive necessary treatment prior to initiating iv bps therapy to reduce the incidence of bronj . manipulation of iv bps dosing may be effective in reducing skeletal related events ( sres ) and minimizing bps associated onj . drug related risk factors include : potency of bps : more potent bps ( zoledronate > pamidronate > oral bps ) have more tendency to developed onj . the iv route of administration resulting greater drug exposure to the jaw than the oral route ; therefore more tendencies to developed onj . incidence rate of bronj in cancer patient is increased 2.7 - 4.2 fold if given iv bps . longer duration therapy of bps may be associated with increased risk of development of onj local risk factors include : dento alveolar surgery like extraction , dental implant placement , periapical surgery , periodontal surgery etc . has 7 times more likely to develop bronj than patients who are not having dentoalveolar surgery . anatomic location : bronj is more common in the mandible than in the maxilla with the ratio of 2:1 and more common in areas with thin mucosa overlaying bony prominences like tori , bony exostoses and mylohyoid ridge . concomitant oral diseases like cancer patients exposed to iv bps with a history of inflammatory ( possibly infective ) dental diseases are at a 7 fold increased risk for developing onj . demographic and systemic factors:- age : with each passing decade there is a 9% increased risk of onj in multiple myeloma patients treated with iv bps , but sex was not statically associated with onj . cancer type : multiple myeloma > > breast cancer > other cancer and osteopenia / osteoporosis concurrent with cancer are more prone to developed onj . concomitant risk factors : renal dialysis , low hemoglobin , obesity , diabetes , tobacco users , poor oral hygiene and chemotherapeutic agents such as cyclophosphamide , erythropoietin and steroids are risk factors , but no increased risk associated with alcohol exposure . genetic risk factors : like genetic perturbations , i.e. , single nucleotide polymorphisms , in the cytochrome p450 - 2c gene ( cyp2c8 ) gene were associated with an increased risk for onj among multiple myeloma patients treated with iv bps . preventive factors : the aaoms task force on bronj recommended that patients undergo dental evaluations and receive necessary treatment prior to initiating iv bps therapy to reduce the incidence of bronj . manipulation of iv bps dosing may be effective in reducing skeletal related events ( sres ) and minimizing bps associated onj . management strategies of bronj is mostly palliative and empirical ( to eliminate clinical symptoms such as pain , infection and minimize the progression of bone necrosis ) before microbial culture report . most authors agreed that management of bronj started after advised morning fasting serum ctx test and begins palliative care . if the exposed bone is pain less , treatment started with 0.12% chlorhexidine mouth rinse , but if patient complaints pain and/or clinical evidence of infection , antibiotic therapy should be provided in addition to the 0.12% chlorhexidine . however , invasive dental procedure is indicated only when morning ctx value is greater than 150pg / ml , to achieve , uncomplicated healing . many treatment modalities are discussed for correction of bronj like cover the exposed bone areas with tissue flaps by marx . sequential removal of sequestra ( conservative approach ) and extensive involvement may necessitate large area of debridement to include segmental mandibulectomy and partial maxillectomy for correction of bronj and was reported that most patients with limited regions of exposed bone ( except those lesions that are a constant source of infection ) have been successfully managed with irrigations and antibiotic therapy while surgical management have not been completely effective in eradication of the bronj by ruggiero et al . administration of pentoxifylline with -tocopherol reduces 74% area of bony exposure and symptom control by epstein et al . transplantation of intra - lesional autologous bone marrow stem cell with complete response by cella et al . hyperbaric oxygen therapy was beneficial for patients and the outcome is increased with cessation of bps administration . no fixed treatment protocol was adopted before recent position paper published by aaoms,(2009 ) that provide new treatment strategies on the basis of staging of bronj : at risk - patients who are at risk of developing bronj by virtue of the fact that they have been exposed to a bps do not require any treatment . however , these patients should be informed of the risk of developing bronj as well as the signs and symptoms of this diseases process . stage 0-here provide symptomatic treatment and conservatively manage other local factors , such as caries and periodontal disease . systemic management may include the use of medication for chronic pain and control of infection with antibiotics , when indicated . stage 1-these patients benefit from the use of antiseptic mouth washes ( chlorhexidine gluconate 0.12% ) and/or analgesia is proposed for patients with clinical evidence of bronj , but in the absence of any evidence of infection . here , primary aim is to reduce the likelihood of further progression of bronj and avoid infection of exposed bone , but not any surgical intervention required . however , patient education and review of indications for continued bp therapy is also important . stage 2-these patients benefit from the use of oral antimicrobial rinse in combination with antibiotic therapy . in bronj most of the isolated microbes have been sensitive to penicillin group because its development is not infectious origin . if patient was allergic to above group then choice of drug is quinolones , metronidazole , clindamycin , doxycycline and erythromycin . if microbial cultures show positive evidence of actinomyces species then antibiotic regimen should be adjusted accordingly . in some refractory cases , patients may require combination antibiotic therapy , long - term antibiotic maintenance or a course of iv antibiotic therapy . stage 3-these patients benefit from debridement , including resection , in combination with antibiotic therapy , which may offer long - term palliation with resolution of acute infection and pain . regardless of the disease stage , mobile segments of bony sequestrum should be removed without exposing uninvolved bone . the extraction of symptomatic teeth within exposed , necrotic bone should be considered since it is unlikely that the extraction will exacerbate the established necrotic process . bps associated onj is a rare , but serious clinical condition caused by anti - osteoclastic , anti - angiogenic and anti - human endothelial cell proliferation effects of bps , which inhibit bone turnover . they are commonly developed in those patients who are receiving either long term nitrogen containing iv bps therapy alone or associated with invasive dental procedure . therefore , proper dental evaluations and receiving necessary treatment prior to initiating iv bps therapy is recommended .. manipulation of iv bps dosing may be effective in reducing sres and minimizing bps associated onj . cbct and morning fasting ctx level are the useful assessment tool to predict risk and to make appropriate line of treatment . in cases of established disease management strategies

nitrogen containing bisphosphonate ( n - bp ) therapy is used extensively to treat osteoporosis and osteolytic bone lesions . recently , a special form of osteonecrosis limited to the maxillofacial skeleton has been discovered especially within those patients who are receiving either long - term n - bp therapy alone and/or associated with invasive dental procedure . bisphosphonates accumulate almost exclusively in maxillofacial skeleton owing to high bone turn over remodeling to maintain the mechanical competence . the pathogenesis and why it commonly appears in maxillofacial skeleton and the fixed treatment strategies remains unknown . the aim of this study was to improve the clinician understanding of n - bps associated osteonecrosis of maxillofacial skeleton by reviewing the past 10 year literature .

INTRODUCTION ETIOPATHOGENESIS OF BRONJ Diagnosis, staging and clinical presentation of BRONJ Risk factors of BRONJ Management of BRONJ CONCLUSION

oral bps are poorly absorbed by the gastrointestinal tract about 10% and excreted largely unchanged by the kidneys , but if given intravenously nitrogen containing bisphosphonates ( n - bps ) about 50% of the drugs goes to the bone . bps are commonly used to treat certain resorptive bone diseases such as osteoporosis , paget 's disease and hypercalcemia associated with certain malignancies such as multiple myeloma and bone metastasis from the breast or prostate . however , according to previous literature , all three above mentioned pharmacological action produced by n - bps becomes more aggressive in the presence of other risk factors ( drug related , local , demographic / systemic , genetic and preventive ) that finally leads to a rare , but serious clinical condition called n - bisphosphonate - related osteonecrosis of the jaw ( bronj ) that was first described by marx in 2003 . chemical structure of bp [ figure 1 ] has two important entity p - c - p back bone and r2 side chain that shows a strong affinity for bone mineral and provides potent inhibition of bone turn over both in - vivo and in - vitro and therapeutic potency of the bps respectively . n - bps had poorly absorbed by gastrointestinal tract as compare to non - n - bps , owing to this region n - bps are commonly prepared for iv administration . in contrast to other skeleton , jaw bones ( alveolar process and periodontium ) have relatively high vascularity , bone turn over and remodeling because of continuous mechanical stress . non - n - bps ( tiludronate , clodronate and etidronate ) is taken up by the osteoclasts and antagonized the cellular energy pathways due to intracellular liberation of methylene that contains toxic analogs of adenosine triphosphate ( atp ) , which probably inhibit atp - utilizing enzymes and induce osteoclast apoptosis , whereas n - bps ( zoledronate , pamidronate , alendronateetc . thus , there is no osteoclastic resorption of mineral matrix and the resultant release of bone morphogenic protein and insulin - like growth to induced stem cell production of osteoblasts , the osteon becomes a - cellular and necrotic . therefore , any mechanism that leads to the breakdown of the over laying mucosa will expose the necrotic bone , which fails to heal . therefore , a possible association was seen between bps and two rare but serious conditions , namely atypical femoral fracture and osteonecrosis of the jaw ( onj ) with an incidence of 0.8 - 12% in iv bps and 0.01 - 0.04% in oral bps administration . although the exact pathophysiology of bps induced onj has not be completely illuminated , but according to previous literature , n - bps are potent inhibitors of osteoclastic activity , angiogenesis , human endothelial cell to proliferate and to modulate endothelial cell adhesion and migration . , ) are not known yet today , but according to previous literature patients may be considered to have bronj if all of the following four characteristics are present : ( 1 ) current or previous treatment with a bps , ( 2 ) exposed or necrotic bone in the maxillofacial region that has persisted for more than 8 weeks , ( 3 ) no history of radiation therapy to the jaws and ( 4 ) no evidence of cancer at the site . lesions in patients who have not fulfilled above four characteristics should be excluded from the diagnosis of bronj . radiographic findings of bronj are not specific and are found in other conditions such as osteomyelitis , osteoradionecrosis and metastatic bone lesions . periapical radiograph and cone - beam computed tomography ( cbct ) reveals generalized thickening of the cortical plate and laminadura , mixed sclerotic and lytic bone destruction involving alveolar bone and basal bone , sequestrum , encroachment on the mandibular canal and maxillary antrum and pathological fracture while thickening of the cortical plate in the affected region was the only radiological findings of cbct.computed tomography images reveals sclerotic changes , osteolytic changes , periosteal bone proliferation , sequestration and inferior alveolar canal involvement while contrast enhanced magnetic resonance images reveals intensity changes of the cortical and sub cortical bone structures , contrast enhancement in necrotic bone area , soft - tissue involvement and cervical lymphadenopathy.99tcm myocardial perfusion defect reveals detection of local bone remodeling activity / high bone turn over sites , but the presence of increased uptake was conformed through the single - photon emission computed tomography because they provide a high degree of accuracy . 99tcm myocardial perfusion defect reveals detection of local bone remodeling activity / high bone turn over sites , but the presence of increased uptake was conformed through the single - photon emission computed tomography because they provide a high degree of accuracy . in past 10 year , some important serological , histopathological and immunohistochemistry findings are obtained that may suggest the appearance of bronj in that patient . on the basis of this evidence , vincenzi et al . evaluated the role of vascular endothelial growth factor ( vegf ) as a predictive marker of bronj and found decreased vegf circulating levels at day 7 and 21 after the 1 administration of n - bps ; thus , authors concluded that the anti - angiogenic properties of n - bps are directly linked to bronj pathogenesis and serum vegf levels could represent an effective early predictive marker . similarly , morning fasting serum c - terminal telopertide ( ctx ) value is less than 100pg / ml in those patients who received / receiving n - bps representing high risk of bronj development , but invasive dental procedure is only indicated if ctx value is greater than 150pg / ml to achieve , uncomplicated healing . immuno histo - chemistry reveals increased expression of human defensins ( hbd)-1,-2,-3 , reduced expression of transforming growth factor beta1 and increased galectin-3 expression in cases of bronj . these lesions are most frequently symptomatic when sites becomes secondary infected or there is a trauma to the soft - tissues through the sharp edges of the exposed bone . , common orofacial findings associated with bps associated onj are poor wound healing , spontaneous or post - surgical soft - tissue breakdown leading to intra oral bone exposure , bone necrosis and osteomyelitis . aaoms staging for bronj on the basis of clinical findings staging for bronj on the basis of clinical and radiologic findings according to recent position paper by aaoms and nsw health guideline , risk factors for the development of bps associated onj can be grouped as drug - related , local , demographic / systematic , genetic and preventive . the iv route of administration resulting greater drug exposure to the jaw than the oral route ; therefore more tendencies to developed onj . longer duration therapy of bps may be associated with increased risk of development of onjlocal risk factors include : dento alveolar surgery like extraction , dental implant placement , periapical surgery , periodontal surgery etc . has 7 times more likely to develop bronj than patients who are not having dentoalveolar surgery . concomitant oral diseases like cancer patients exposed to iv bps with a history of inflammatory ( possibly infective ) dental diseases are at a 7 fold increased risk for developing onj.demographic and systemic factors:- age : with each passing decade there is a 9% increased risk of onj in multiple myeloma patients treated with iv bps , but sex was not statically associated with onj . concomitant risk factors : renal dialysis , low hemoglobin , obesity , diabetes , tobacco users , poor oral hygiene and chemotherapeutic agents such as cyclophosphamide , erythropoietin and steroids are risk factors , but no increased risk associated with alcohol exposure.genetic risk factors : like genetic perturbations , i.e. , single nucleotide polymorphisms , in the cytochrome p450 - 2c gene ( cyp2c8 ) gene were associated with an increased risk for onj among multiple myeloma patients treated with iv bps.preventive factors : the aaoms task force on bronj recommended that patients undergo dental evaluations and receive necessary treatment prior to initiating iv bps therapy to reduce the incidence of bronj . manipulation of iv bps dosing may be effective in reducing skeletal related events ( sres ) and minimizing bps associated onj . the iv route of administration resulting greater drug exposure to the jaw than the oral route ; therefore more tendencies to developed onj . longer duration therapy of bps may be associated with increased risk of development of onj local risk factors include : dento alveolar surgery like extraction , dental implant placement , periapical surgery , periodontal surgery etc . bps therapy in association with dento alveolar surgery has 7 times more likely to develop bronj than patients who are not having dentoalveolar surgery . demographic and systemic factors:- age : with each passing decade there is a 9% increased risk of onj in multiple myeloma patients treated with iv bps , but sex was not statically associated with onj . concomitant risk factors : renal dialysis , low hemoglobin , obesity , diabetes , tobacco users , poor oral hygiene and chemotherapeutic agents such as cyclophosphamide , erythropoietin and steroids are risk factors , but no increased risk associated with alcohol exposure . , single nucleotide polymorphisms , in the cytochrome p450 - 2c gene ( cyp2c8 ) gene were associated with an increased risk for onj among multiple myeloma patients treated with iv bps . manipulation of iv bps dosing may be effective in reducing skeletal related events ( sres ) and minimizing bps associated onj . if the exposed bone is pain less , treatment started with 0.12% chlorhexidine mouth rinse , but if patient complaints pain and/or clinical evidence of infection , antibiotic therapy should be provided in addition to the 0.12% chlorhexidine . however , invasive dental procedure is indicated only when morning ctx value is greater than 150pg / ml , to achieve , uncomplicated healing . hyperbaric oxygen therapy was beneficial for patients and the outcome is increased with cessation of bps administration . no fixed treatment protocol was adopted before recent position paper published by aaoms,(2009 ) that provide new treatment strategies on the basis of staging of bronj : at risk - patients who are at risk of developing bronj by virtue of the fact that they have been exposed to a bps do not require any treatment . however , these patients should be informed of the risk of developing bronj as well as the signs and symptoms of this diseases process . however , patient education and review of indications for continued bp therapy is also important . in some refractory cases , patients may require combination antibiotic therapy , long - term antibiotic maintenance or a course of iv antibiotic therapy . stage 3-these patients benefit from debridement , including resection , in combination with antibiotic therapy , which may offer long - term palliation with resolution of acute infection and pain . , ) are not known yet today , but according to previous literature patients may be considered to have bronj if all of the following four characteristics are present : ( 1 ) current or previous treatment with a bps , ( 2 ) exposed or necrotic bone in the maxillofacial region that has persisted for more than 8 weeks , ( 3 ) no history of radiation therapy to the jaws and ( 4 ) no evidence of cancer at the site . lesions in patients who have not fulfilled above four characteristics should be excluded from the diagnosis of bronj . radiographic findings of bronj are not specific and are found in other conditions such as osteomyelitis , osteoradionecrosis and metastatic bone lesions . periapical radiograph and cone - beam computed tomography ( cbct ) reveals generalized thickening of the cortical plate and laminadura , mixed sclerotic and lytic bone destruction involving alveolar bone and basal bone , sequestrum , encroachment on the mandibular canal and maxillary antrum and pathological fracture while thickening of the cortical plate in the affected region was the only radiological findings of cbct.computed tomography images reveals sclerotic changes , osteolytic changes , periosteal bone proliferation , sequestration and inferior alveolar canal involvement while contrast enhanced magnetic resonance images reveals intensity changes of the cortical and sub cortical bone structures , contrast enhancement in necrotic bone area , soft - tissue involvement and cervical lymphadenopathy.99tcm myocardial perfusion defect reveals detection of local bone remodeling activity / high bone turn over sites , but the presence of increased uptake was conformed through the single - photon emission computed tomography because they provide a high degree of accuracy . 99tcm myocardial perfusion defect reveals detection of local bone remodeling activity / high bone turn over sites , but the presence of increased uptake was conformed through the single - photon emission computed tomography because they provide a high degree of accuracy . in past 10 year , some important serological , histopathological and immunohistochemistry findings are obtained that may suggest the appearance of bronj in that patient . on the basis of this evidence , vincenzi et al . evaluated the role of vascular endothelial growth factor ( vegf ) as a predictive marker of bronj and found decreased vegf circulating levels at day 7 and 21 after the 1 administration of n - bps ; thus , authors concluded that the anti - angiogenic properties of n - bps are directly linked to bronj pathogenesis and serum vegf levels could represent an effective early predictive marker . similarly , morning fasting serum c - terminal telopertide ( ctx ) value is less than 100pg / ml in those patients who received / receiving n - bps representing high risk of bronj development , but invasive dental procedure is only indicated if ctx value is greater than 150pg / ml to achieve , uncomplicated healing . these lesions are most frequently symptomatic when sites becomes secondary infected or there is a trauma to the soft - tissues through the sharp edges of the exposed bone . , common orofacial findings associated with bps associated onj are poor wound healing , spontaneous or post - surgical soft - tissue breakdown leading to intra oral bone exposure , bone necrosis and osteomyelitis . aaoms staging for bronj on the basis of clinical findings staging for bronj on the basis of clinical and radiologic findings according to recent position paper by aaoms and nsw health guideline , risk factors for the development of bps associated onj can be grouped as drug - related , local , demographic / systematic , genetic and preventive . the iv route of administration resulting greater drug exposure to the jaw than the oral route ; therefore more tendencies to developed onj . longer duration therapy of bps may be associated with increased risk of development of onjlocal risk factors include : dento alveolar surgery like extraction , dental implant placement , periapical surgery , periodontal surgery etc . has 7 times more likely to develop bronj than patients who are not having dentoalveolar surgery . concomitant oral diseases like cancer patients exposed to iv bps with a history of inflammatory ( possibly infective ) dental diseases are at a 7 fold increased risk for developing onj.demographic and systemic factors:- age : with each passing decade there is a 9% increased risk of onj in multiple myeloma patients treated with iv bps , but sex was not statically associated with onj . concomitant risk factors : renal dialysis , low hemoglobin , obesity , diabetes , tobacco users , poor oral hygiene and chemotherapeutic agents such as cyclophosphamide , erythropoietin and steroids are risk factors , but no increased risk associated with alcohol exposure.genetic risk factors : like genetic perturbations , i.e. , single nucleotide polymorphisms , in the cytochrome p450 - 2c gene ( cyp2c8 ) gene were associated with an increased risk for onj among multiple myeloma patients treated with iv bps.preventive factors : the aaoms task force on bronj recommended that patients undergo dental evaluations and receive necessary treatment prior to initiating iv bps therapy to reduce the incidence of bronj . manipulation of iv bps dosing may be effective in reducing skeletal related events ( sres ) and minimizing bps associated onj . the iv route of administration resulting greater drug exposure to the jaw than the oral route ; therefore more tendencies to developed onj . longer duration therapy of bps may be associated with increased risk of development of onj local risk factors include : dento alveolar surgery like extraction , dental implant placement , periapical surgery , periodontal surgery etc . has 7 times more likely to develop bronj than patients who are not having dentoalveolar surgery . demographic and systemic factors:- age : with each passing decade there is a 9% increased risk of onj in multiple myeloma patients treated with iv bps , but sex was not statically associated with onj . concomitant risk factors : renal dialysis , low hemoglobin , obesity , diabetes , tobacco users , poor oral hygiene and chemotherapeutic agents such as cyclophosphamide , erythropoietin and steroids are risk factors , but no increased risk associated with alcohol exposure . , single nucleotide polymorphisms , in the cytochrome p450 - 2c gene ( cyp2c8 ) gene were associated with an increased risk for onj among multiple myeloma patients treated with iv bps . manipulation of iv bps dosing may be effective in reducing skeletal related events ( sres ) and minimizing bps associated onj . if the exposed bone is pain less , treatment started with 0.12% chlorhexidine mouth rinse , but if patient complaints pain and/or clinical evidence of infection , antibiotic therapy should be provided in addition to the 0.12% chlorhexidine . however , invasive dental procedure is indicated only when morning ctx value is greater than 150pg / ml , to achieve , uncomplicated healing . hyperbaric oxygen therapy was beneficial for patients and the outcome is increased with cessation of bps administration . no fixed treatment protocol was adopted before recent position paper published by aaoms,(2009 ) that provide new treatment strategies on the basis of staging of bronj : at risk - patients who are at risk of developing bronj by virtue of the fact that they have been exposed to a bps do not require any treatment . however , these patients should be informed of the risk of developing bronj as well as the signs and symptoms of this diseases process . however , patient education and review of indications for continued bp therapy is also important . in some refractory cases , patients may require combination antibiotic therapy , long - term antibiotic maintenance or a course of iv antibiotic therapy . stage 3-these patients benefit from debridement , including resection , in combination with antibiotic therapy , which may offer long - term palliation with resolution of acute infection and pain . bps associated onj is a rare , but serious clinical condition caused by anti - osteoclastic , anti - angiogenic and anti - human endothelial cell proliferation effects of bps , which inhibit bone turnover . they are commonly developed in those patients who are receiving either long term nitrogen containing iv bps therapy alone or associated with invasive dental procedure . therefore , proper dental evaluations and receiving necessary treatment prior to initiating iv bps therapy is recommended .. manipulation of iv bps dosing may be effective in reducing sres and minimizing bps associated onj .

we used data from the korean genome and epidemiology study on atherosclerosis risk of rural areas in the korean general population ( koges - arirang ) , a population - based prospective cohort study to assess the prevalence , incidence , and risk factors for chronic degenerative disorders such as hypertension , diabetes , osteoporosis , and cardiovascular disease ( 911 ) . koges - arirang invited all adults aged 4070 years who resided in rural areas of wonju and pyeongchang in south korea to participate in the study . demographic shifts are infrequent in this area , and the population can be followed long term . the baseline survey , carried out from november 2005 to january 2008 , included 5,178 adults ( 2,127 men and 3,051 women ) aged 4070 years . all study participants were invited to the first follow - up survey ( april 2008january 2011 ) , of whom 3,862 ( 74.6% ) attended . we then excluded 1,345 subjects with metabolic syndrome at baseline , 436 subjects without baseline adiponectin measurements , 24 subjects with any history or presence of cardiovascular disease at baseline , and 13 subjects with incomplete data . the final sample size for the present analysis was 2,044 participants ( 831 men and 1,213 women ) without metabolic syndrome at baseline ( supplementary fig . all participants provided written informed consent . at baseline and at the follow - up examination , study participants completed a standardized medical history and lifestyle questionnaire and underwent a comprehensive health examination according to standard procedures . body weight and height were measured while participants were wearing light indoor clothing without shoes . waist circumference was measured in a horizontal plane midway between the inferior margin of the ribs and the superior border of the iliac crest using a tape measure ( seca-200 ; seca , hamburg , germany ) . systolic and diastolic blood pressure were measured twice in the right arm using a standard mercury sphygmomanometer ( baumanometer , copiague , ny ) . never smokers were defined as participants who had smoked < 100 cigarettes ( < 5 packs of cigarettes ) in their lifetime . current smokers were defined as participants who had smoked 100 cigarettes in their lifetime and who reported currently smoking in the questionnaire . former smokers were defined as participants who had smoked 100 cigarettes in their lifetime but who reported abstain from smoking in the questionnaire . subjects who answered yes to the question , do you perform physical exercise regularly enough to make you sweat ? were assigned to the regular exercise group . a venous blood sample was drawn from study participants after fasting for > 12 h or overnight . serum aliquots were stored at 80c until thawed for adiponectin analysis within 1 week after blood extraction . charles , mo ) with intra - assay and interassay coefficients of variation ranging between 2.9 and 6.6% . fasting insulin was determined by a double - antibody radioimmunoassay ( biosource europe sa , nivelles , belgium ) . serum concentrations of ldl cholesterol , hdl cholesterol , and triglycerides were determined by enzymatic methods ( advia 1650 ; siemens , tarrytown , ny ) . high - sensitivity c - reactive protein ( hs - crp ) was measured by the denka seiken ( tokyo , japan ) assay , which has been validated against the dade behring method . insulin resistance was calculated using the homeostasis model assessment of insulin resistance ( homa - ir ) model using the following formula : fasting insulin ( iu / ml ) fasting plasma glucose ( mg / dl)/405 . the study end point was the development of metabolic syndrome at the follow - up visit , defined following the harmonized definition for metabolic syndrome ( 12 ) as the presence of at least three of the following criteria : 1 ) abdominal obesity , defined as a waist circumference 90 cm for men or 85 cm for women ( following korean - specific cutoffs for abdominal obesity defined by the korean society of obesity ) ( 13 ) ; 2 ) hypertriglyceridemia , defined as a serum triglyceride concentration 150 mg / dl ( 1.69 mmol / l ) ; 3 ) low hdl cholesterol , defined as a serum hdl cholesterol concentration < 40 mg / dl ( 1.04 mmol / l ) for men or < 50 mg / dl ( 1.29 mmol / l ) for women ; 4 ) high blood pressure , defined as a systolic blood pressure 130 mmhg , a diastolic blood pressure 85mmhg , or treatment with antihypertensive agents ; and 5 ) high fasting glucose , defined as a fasting serum glucose 100 mg / dl or previously diagnosed type 2 diabetes . since women have higher levels of adiponectin compared with men , we performed all analyses separately for men and women . we divided the study population into sex - specific quartiles of serum adiponectin levels with cut points 5.94 , 8.26 , and 11.22 g / ml for men and 8.91 , 11.90 , and 15.24 g / ml for women . we evaluated the association of baseline adiponectin levels with the incidence of new cases of metabolic syndrome and with the incidence of new cases of each component of the metabolic syndrome at the follow - up visit . to evaluate the incidence of new cases of each component , we excluded subjects with the presence of that specific component at baseline . multivariable logistic regression was used to assess the independent association of baseline adiponectin levels with incident metabolic syndrome . second , we adjusted for age ( continuous variable ) , bmi ( continuous variable ) , smoking ( current , former , or never ) , ldl cholesterol ( continuous variable ) , and regular exercise ( yes or no ) . finally , we further adjusted for baseline levels of hs - crp ( log - transformed continuous variable ) , homa - ir ( log - transformed continuous variable ) , and follow - up bmi ( continuous variable ) . to evaluate the added discrimination provided by adiponectin levels to predict incident cases of metabolic syndrome beyond the information provided by the components of the metabolic syndrome , we compared the areas under the receiver operating characteristic ( roc ) curve in models that included waist circumference , hdl cholesterol , systolic blood pressure , triglycerides , and glucose levels with and without adiponectin levels . in addition , we calculated reclassification tables for models with and without serum adiponectin four categories of predicted risk ( cutoffs at 5 , 10 , and 20% ) , as well as the continuous ( or category - less ) net reclassification improvement ( nri ) ( 14 ) and the integrated discrimination improvement ( idi ) ( 15 ) . the nri compared classifications from the model with and without serum adiponectin for changes by incident metabolic syndrome for a net calculation of changes in the right direction . it represents the sum of the percentage of participants with an improvement in sensitivity after considering the new marker among those who develop the event plus the percentage of participants with an improvement in specificity after considering the new marker among those who do not develop the event . the idi represents the sum of the average increase in sensitivity after considering the new marker among those who develop the event plus the increase in specificity after considering the new marker among those who do not develop the event . all analyses were performed using sas , version 9.2 ( sas institute , cary , nc ) . at baseline and at the follow - up examination , study participants completed a standardized medical history and lifestyle questionnaire and underwent a comprehensive health examination according to standard procedures . body weight and height were measured while participants were wearing light indoor clothing without shoes . waist circumference was measured in a horizontal plane midway between the inferior margin of the ribs and the superior border of the iliac crest using a tape measure ( seca-200 ; seca , hamburg , germany ) . systolic and diastolic blood pressure were measured twice in the right arm using a standard mercury sphygmomanometer ( baumanometer , copiague , ny ) . never smokers were defined as participants who had smoked < 100 cigarettes ( < 5 packs of cigarettes ) in their lifetime . current smokers were defined as participants who had smoked 100 cigarettes in their lifetime and who reported currently smoking in the questionnaire . former smokers were defined as participants who had smoked 100 cigarettes in their lifetime but who reported abstain from smoking in the questionnaire . subjects who answered yes to the question , do you perform physical exercise regularly enough to make you sweat ? were assigned to the regular exercise group . a venous blood sample was drawn from study participants after fasting for > 12 h or overnight . serum aliquots were stored at 80c until thawed for adiponectin analysis within 1 week after blood extraction . charles , mo ) with intra - assay and interassay coefficients of variation ranging between 2.9 and 6.6% . fasting insulin was determined by a double - antibody radioimmunoassay ( biosource europe sa , nivelles , belgium ) . serum concentrations of ldl cholesterol , hdl cholesterol , and triglycerides were determined by enzymatic methods ( advia 1650 ; siemens , tarrytown , ny ) . high - sensitivity c - reactive protein ( hs - crp ) was measured by the denka seiken ( tokyo , japan ) assay , which has been validated against the dade behring method . insulin resistance was calculated using the homeostasis model assessment of insulin resistance ( homa - ir ) model using the following formula : fasting insulin ( iu / ml ) fasting plasma glucose ( mg / dl)/405 . the study end point was the development of metabolic syndrome at the follow - up visit , defined following the harmonized definition for metabolic syndrome ( 12 ) as the presence of at least three of the following criteria : 1 ) abdominal obesity , defined as a waist circumference 90 cm for men or 85 cm for women ( following korean - specific cutoffs for abdominal obesity defined by the korean society of obesity ) ( 13 ) ; 2 ) hypertriglyceridemia , defined as a serum triglyceride concentration 150 mg / dl ( 1.69 mmol / l ) ; 3 ) low hdl cholesterol , defined as a serum hdl cholesterol concentration < 40 mg / dl ( 1.04 mmol / l ) for men or < 50 mg / dl ( 1.29 mmol / l ) for women ; 4 ) high blood pressure , defined as a systolic blood pressure 130 mmhg , a diastolic blood pressure 85mmhg , or treatment with antihypertensive agents ; and 5 ) high fasting glucose , defined as a fasting serum glucose 100 mg / dl or previously diagnosed type 2 diabetes . since women have higher levels of adiponectin compared with men , we performed all analyses separately for men and women . we divided the study population into sex - specific quartiles of serum adiponectin levels with cut points 5.94 , 8.26 , and 11.22 g / ml for men and 8.91 , 11.90 , and 15.24 g / ml for women . we evaluated the association of baseline adiponectin levels with the incidence of new cases of metabolic syndrome and with the incidence of new cases of each component of the metabolic syndrome at the follow - up visit . to evaluate the incidence of new cases of each component , we excluded subjects with the presence of that specific component at baseline . multivariable logistic regression was used to assess the independent association of baseline adiponectin levels with incident metabolic syndrome . second , we adjusted for age ( continuous variable ) , bmi ( continuous variable ) , smoking ( current , former , or never ) , ldl cholesterol ( continuous variable ) , and regular exercise ( yes or no ) . finally , we further adjusted for baseline levels of hs - crp ( log - transformed continuous variable ) , homa - ir ( log - transformed continuous variable ) , and follow - up bmi ( continuous variable ) . results are expressed as odds ratios ( ors ) ( 95% ci ) . to evaluate the added discrimination provided by adiponectin levels to predict incident cases of metabolic syndrome beyond the information provided by the components of the metabolic syndrome , we compared the areas under the receiver operating characteristic ( roc ) curve in models that included waist circumference , hdl cholesterol , systolic blood pressure , triglycerides , and glucose levels with and without adiponectin levels . in addition , we calculated reclassification tables for models with and without serum adiponectin four categories of predicted risk ( cutoffs at 5 , 10 , and 20% ) , as well as the continuous ( or category - less ) net reclassification improvement ( nri ) ( 14 ) and the integrated discrimination improvement ( idi ) ( 15 ) . the nri compared classifications from the model with and without serum adiponectin for changes by incident metabolic syndrome for a net calculation of changes in the right direction . it represents the sum of the percentage of participants with an improvement in sensitivity after considering the new marker among those who develop the event plus the percentage of participants with an improvement in specificity after considering the new marker among those who do not develop the event . the idi represents the sum of the average increase in sensitivity after considering the new marker among those who develop the event plus the increase in specificity after considering the new marker among those who do not develop the event . all analyses were performed using sas , version 9.2 ( sas institute , cary , nc ) . during an average of 2.6 years of follow - up , 153 men ( 18.4% ) and 199 women ( 16.4% ) developed metabolic syndrome . baseline bmi , waist circumference , systolic and diastolic blood pressures , fasting glucose , triglyceride , homa - ir , and high - sensitivity c - reactive protein ( hs - crp ) were significantly higher and hdl cholesterol was significantly lower in men and women who developed metabolic syndrome compared with those who did not ( table 1 ) . baseline characteristics of study population by incident metabolic syndrome median adiponectin levels at baseline were significantly lower in subjects who developed metabolic syndrome compared with those who did not in both men ( 7.09 vs. 8.63 g / ml , p < 0.001 ) and women ( 10.96 vs. 12.16 g / ml , p < a similar association was observed between adiponectin and each component of the metabolic syndrome except for high blood pressure . furthermore , serum adiponectin levels progressively decreased with the number of metabolic syndrome components developed by study participants over follow - up ( p for trend < 0.001 in both men and women ) ( table 2 ) . baseline serum adiponectin levels according to presence or absence of components of new - onset metabolic syndrome after an average of 2.6 years of follow - up , the proportions of men who developed new - onset metabolic syndrome , high waist circumference , low hdl cholesterol , high triglycerides , high blood pressure , and high blood glucose were 18.4 , 14.3 , 21.9 , 20.7 , 18.9 , and 15.5% , respectively . in multivariable - adjusted models [ model 3 ( table 3 ) ] , the or for developing metabolic syndrome comparing men in the highest with those in the lowest quartile of adiponectin was 0.25 ( 95% ci 0.140.47 ; p for trend < 0.001 ) . the corresponding ors for high waist circumference , low hdl cholesterol , high triglycerides , high blood pressure , and high blood glucose were 0.54 ( 0.251.20 ) , 0.54 ( 0.300.94 ) , 0.27 ( 0.140.50 ) , 0.46 ( 0.191.11 ) , and 0.77 ( 0.411.43 ) , respectively . among women , the proportions of participants who developed new - onset metabolic syndrome , high waist circumference , low hdl cholesterol , high triglycerides , high blood pressure , and high blood glucose were 16.4 , 21.1 , 32.2 , 14.1 , 14.4 , and 8.4% , respectively . ors for new - onset metabolic syndrome and its components according to baseline serum adiponectin in men in multivariable - adjusted models [ model 3 ( table 4 ) ] , the or for developing metabolic syndrome comparing women in the highest with those in the lowest quartile of adiponectin was 0.45 ( 95% ci 0.280.74 ; p for trend = 0.001 ) . the corresponding ors for new - onset high waist circumference , low hdl cholesterol , high triglycerides , high blood pressure , and high blood glucose were 0.50 ( 0.280.88 ) , 0.93 ( 0.551.56 ) , 0.28 ( 0.160.49 ) , 1.08 ( 0.572.06 ) , and 0.70 ( 0.381.29 ) , respectively . participants in the highest quartile of baseline adiponectin had the smallest increase in waist circumference , triglycerides , and systolic blood pressure in men and the smallest increase of triglycerides in women ( supplementary table 1 ) . ors for new - onset metabolic syndrome and its components according to baseline serum adiponectin in women we then evaluated how well baseline adiponectin levels predict incident metabolic syndrome beyond the information provided by baseline levels of metabolic syndrome components . the area under the roc curve to predict incident metabolic syndrome using waist circumference , hdl cholesterol , blood pressure , triglycerides , and blood glucose levels was 0.766 ( 95% ci 0.7260.805 ) in men and 0.783 ( 0.7520.814 ) in women . after adiponectin levels were added to the model , the corresponding areas under the roc curve were 0.772 ( 0.7340.811 ) and 0.783 ( 0.7520.815 ) , respectively . the p values for the comparison in areas under the roc curve for the models with and without adiponectin levels were 0.29 and 0.88 in men and women , respectively . the reclassification tables for improved prediction after adding serum adiponectin to a model including the components of the metabolic syndrome , separated for participants with and without new - onset metabolic syndrome , are shown in supplementary tables 2 and 3 . for men , the category - free nri was 0.21 ( 95% ci 0.040.38 ; p = 0.02 ) , and the idi was 0.010 ( 0.0010.018 ; p = 0.025 ) . the increases in nri and idi in men were derived primarily from an increase in sensitivity ( 20% more cases were reclassified in the correct direction using adiponectin , and the point estimate for the increase in integrated sensitivity was 0.008 ) . for women , the nri was 0.04 ( 0.11 to 0.20 ; p = 0.18 ) and the idi was 0.002 ( 0.001 to 0.004 ; p = 0.13 ) . the p value for the difference in nri and idi between men and women was 0.001 . finally , we also evaluated the prospective association between baseline adiponectin levels and incidence of diabetes among participants with metabolic syndrome and with impaired fasting glucose at baseline . among 595 men and 748 women with metabolic syndrome but no diabetes at baseline , 37 men and 29 women developed new cases of diabetes during follow - up . baseline adiponectin levels were inversely associated with the risk of developing diabetes in both men and women , although the association was stronger in women ( supplementary table 4 ) . among 318 men and 235 women with impaired fasting glucose but no diabetes at baseline , 39 men and 25 women developed diabetes during follow - up . in this subgroup , baseline adiponectin levels were also inversely associated with the risk of developing diabetes ( supplementary table 5 ) . in this prospective cohort study in a general asian population , serum adiponectin was a strong negative predictor of incident metabolic syndrome in both men and women . serum adiponectin levels were also a strong negative predictor of the total number of components of the metabolic syndrome developed and of each individual component of the metabolic syndrome , except for elevated blood pressure . the prospective design , the strength of the associations , and the graded dose - response relationships suggest that adiponectin levels may play a key role in the development of metabolic syndrome and its components . furthermore , our analyses indicate that in men , serum adiponectin may increase the predictive ability for identification of subjects at risk for developing new - onset metabolic syndrome beyond that of the information provided by the components of the metabolic syndrome and open the possibility of using serum adiponectin in clinical settings as a prognostic tool in men . adiponectin , a 30-kda protein , is the most abundant protein secreted mainly by visceral and subcutaneous adipose tissue . the binding of adiponectin to these receptors mediates increased amp - activated protein kinase and peroxisome proliferator activated receptor- activity resulting in increased insulin sensitivity , glucose utilization , and fatty acid oxidation in the liver and skeletal muscle ( 1618 ) . adiponectin also increases nitric oxide ( no ) synthesis in endothelial cells via amp - activated protein kinase mediated phosphorylation of endothelial no synthase ( 19 ) and inhibits inflammatory responses ( 20 ) and the production of reactive oxygen species ( 21,22 ) . additionally , the collagen - like domain of adiponectin allows oligomerization of the protein and the formation of high molecular weight ( hmw ) adiponectin ( 23 ) . while hmw adiponectin may be a more active form of adiponectin in vitro , both total and hmw adiponectin were inversely associated with progression to metabolic syndrome in two longitudinal cohort studies in japan ( 6,8 ) . additional studies in larger samples are needed to assess the role of hmw adiponectin in the prediction of metabolic syndrome . excess adiposity in obesity is associated with downregulation of adiponectin secretion ( hypoadiponectinemia ) and with decreased expression of adipor1 and -r2 ( adiponectin resistance ) , with both likely contributing to insulin resistance and metabolic dysregulation . hypoadiponectinemia is associated with obesity - related metabolic disorders , such as type 2 diabetes , hypertension , and dyslipidemia ( 1618,24,25 ) . adiponectin production is also reduced by inflammation , oxidative stress , insulin , growth hormone , smoking , testosterone , and glucocorticoids and increased by peroxisome proliferator activated receptor agonists , weight loss , and inhibition of the renin - angiotensin - aldosterone system ( 26,27 ) . despite strong evidence of the association between adiponectin and obesity - related metabolic abnormalities , there are limited data on the predictive value of circulating adiponectin for incident metabolic syndrome . in prior studies ( 68 ) , adiponectin levels were also inversely associated with incident metabolic syndrome , but those studies did not evaluate the additional predictive ability of adiponectin beyond the information provided by the components of the metabolic syndrome at baseline . in our study , decreasing levels of adiponectin were progressively associated with an increased incidence of metabolic syndrome as well as an increase in the number of metabolic components affected during follow - up . while we found no improvement in the area under the roc curve in models that added adiponectin to metabolic syndrome components , the nri and the idi were significantly improved in men , and the difference between men and women was statistically significant . the nri and the idi may be more sensitive than the area under the roc curve for identifying improvements in predictive value ( 28 ) . the idi measures the estimated improvement in the average sensitivity with the addition of adiponectin minus the estimated decrease in the mean specificity across all possible threshold values ( 15 ) . given the large number of people who may be potentially screened for prediction of metabolic risk , this improvement in sensitivity and specificity may result in better predictions in a large number of subjects . our data indicate that serum adiponectin may have a clinical value in screening for metabolic risk in men , but future studies with a larger sample size and validation samples need to delineate specific risk scoring tools and clinical thresholds for predicting the development of metabolic syndrome including measures of serum adiponectin . our results were analyses stratified by sex , a key determinant of serum adiponectin levels . median adiponectin levels in men were lower than those in women ( 8.26 vs. 11.90 g / ml , respectively ; p < 0.001 ) , despite higher prevalence of metabolic syndrome in women ( 10 ) . such sex differences in adiponectin levels have previously been reported , and it has been hypothesized that androgen inhibits adiponectin secretion triggering lower levels of adiponectin in males ( 2931 ) . in our study , serum adiponectin improved clinical prediction of new - onset metabolic syndrome in men but not in women . these differences were not due to a lower power of the study in women , as the number of cases of new - onset metabolic syndrome was higher in women . we could not establish the reason for this sex difference , although we note that the area under the roc curve for metabolic syndrome components was higher in women compared with men , possibly indicating that there is greater room for nontraditional markers to improve prediction among men . first , the study was restricted to middle - aged and elderly koreans living in a rural area with a relatively high baseline prevalence and high incidence of metabolic syndrome . the prevalence of metabolic syndrome in our cohort was similar to that in the korean national health and nutrition examination survey , a representative study of the korean population ( 9,32 ) . furthermore , korea seems to have experienced a rapid increase in the prevalence of metabolic syndrome during the 2000s , partly due to increasing adoption of western lifestyle patterns ( 32 ) . our cohort may therefore be reflecting a period of rapid increase in the frequency of metabolic syndrome , and our findings may not be generalizable to other populations , particularly to those with different secular trends , with higher levels of adiposity , or with younger age . serum adiponectin levels , however , have been associated with cardiometabolic factors in other races / ethnicities , suggesting that the associations that we observed also apply to other settings . second , the follow - up period of our cohort was only 2.6 years , and we could not evaluate whether the association between adiponectin and incident metabolic syndrome would persist in longer follow - up . third , we could not assess the presence of cardiometabolic abnormalities in 25% of the sample that did not complete the follow - up visit . however , we found no statistical differences in baseline demographics and laboratory findings , including serum adiponectin , between participants who attended the follow - up visit and those who did not ( data not shown ) . finally , our analyses were based on a single determination of serum adiponectin , which is subject to random measurement error and may have underestimated the strength of the associations . in conclusion , serum adiponectin was an independent protective factor for incident metabolic syndrome and its components in our longitudinal study . our findings thus support a central role of adiponectin in the development of cardiometabolic abnormalities in obesity . furthermore , our findings indicate that serum adiponectin may have a clinical role in predicting new - onset metabolic syndrome among men . additional studies should corroborate these findings and develop appropriate risk - scoring tools that incorporate serum adiponectin in cardiometabolic risk stratification tools .

objectiveincreased adiponectin levels may play a protective role in the development of metabolic abnormalities , but prospective studies of the predictive value of serum adiponectin to identify individuals at high risk of new - onset metabolic syndrome are lacking . we investigated whether serum adiponectin predicts incident cases of the metabolic syndrome in a population - based longitudinal study.research design and methodsa prospective cohort study was conducted of 2,044 adults ( 831 men and 1,213 women ) aged 4070 years without metabolic syndrome examined in 20052008 ( baseline ) and 20082011 ( follow - up ) . baseline serum adiponectin concentrations were measured by radioimmunoassay.resultsduring an average of 2.6 years of follow - up , 153 men ( 18.4% ) and 199 women ( 16.4% ) developed metabolic syndrome . in multivariable - adjusted models , the odds ratio for incident metabolic syndrome comparing the highest with the lowest quartiles of adiponectin levels was 0.25 ( 95% ci 0.140.47 ) in men and 0.45 ( 0.280.74 ) in women . while serum adiponectin did not improve the area under the roc curve for predicting new - onset metabolic syndrome based on information from metabolic syndrome components , the net reclassification improvement and the integrated discrimination improvement of prediction models including adiponectin were significantly higher compared with those of models not including adiponectin among men , with a significant difference between men and women ( p = 0.001).conclusionsincreased adiponectin is an independent protective factor for incident metabolic syndrome in men and women , and it may have a clinical role in predicting new - onset metabolic syndrome among men .

RESEARCH DESIGN AND METHODS Data collection End point definition Statistical analysis RESULTS CONCLUSIONS

we used data from the korean genome and epidemiology study on atherosclerosis risk of rural areas in the korean general population ( koges - arirang ) , a population - based prospective cohort study to assess the prevalence , incidence , and risk factors for chronic degenerative disorders such as hypertension , diabetes , osteoporosis , and cardiovascular disease ( 911 ) . koges - arirang invited all adults aged 4070 years who resided in rural areas of wonju and pyeongchang in south korea to participate in the study . the baseline survey , carried out from november 2005 to january 2008 , included 5,178 adults ( 2,127 men and 3,051 women ) aged 4070 years . the final sample size for the present analysis was 2,044 participants ( 831 men and 1,213 women ) without metabolic syndrome at baseline ( supplementary fig . the study end point was the development of metabolic syndrome at the follow - up visit , defined following the harmonized definition for metabolic syndrome ( 12 ) as the presence of at least three of the following criteria : 1 ) abdominal obesity , defined as a waist circumference 90 cm for men or 85 cm for women ( following korean - specific cutoffs for abdominal obesity defined by the korean society of obesity ) ( 13 ) ; 2 ) hypertriglyceridemia , defined as a serum triglyceride concentration 150 mg / dl ( 1.69 mmol / l ) ; 3 ) low hdl cholesterol , defined as a serum hdl cholesterol concentration < 40 mg / dl ( 1.04 mmol / l ) for men or < 50 mg / dl ( 1.29 mmol / l ) for women ; 4 ) high blood pressure , defined as a systolic blood pressure 130 mmhg , a diastolic blood pressure 85mmhg , or treatment with antihypertensive agents ; and 5 ) high fasting glucose , defined as a fasting serum glucose 100 mg / dl or previously diagnosed type 2 diabetes . since women have higher levels of adiponectin compared with men , we performed all analyses separately for men and women . we divided the study population into sex - specific quartiles of serum adiponectin levels with cut points 5.94 , 8.26 , and 11.22 g / ml for men and 8.91 , 11.90 , and 15.24 g / ml for women . we evaluated the association of baseline adiponectin levels with the incidence of new cases of metabolic syndrome and with the incidence of new cases of each component of the metabolic syndrome at the follow - up visit . to evaluate the incidence of new cases of each component , we excluded subjects with the presence of that specific component at baseline . finally , we further adjusted for baseline levels of hs - crp ( log - transformed continuous variable ) , homa - ir ( log - transformed continuous variable ) , and follow - up bmi ( continuous variable ) . to evaluate the added discrimination provided by adiponectin levels to predict incident cases of metabolic syndrome beyond the information provided by the components of the metabolic syndrome , we compared the areas under the receiver operating characteristic ( roc ) curve in models that included waist circumference , hdl cholesterol , systolic blood pressure , triglycerides , and glucose levels with and without adiponectin levels . in addition , we calculated reclassification tables for models with and without serum adiponectin four categories of predicted risk ( cutoffs at 5 , 10 , and 20% ) , as well as the continuous ( or category - less ) net reclassification improvement ( nri ) ( 14 ) and the integrated discrimination improvement ( idi ) ( 15 ) . the nri compared classifications from the model with and without serum adiponectin for changes by incident metabolic syndrome for a net calculation of changes in the right direction . waist circumference was measured in a horizontal plane midway between the inferior margin of the ribs and the superior border of the iliac crest using a tape measure ( seca-200 ; seca , hamburg , germany ) . the study end point was the development of metabolic syndrome at the follow - up visit , defined following the harmonized definition for metabolic syndrome ( 12 ) as the presence of at least three of the following criteria : 1 ) abdominal obesity , defined as a waist circumference 90 cm for men or 85 cm for women ( following korean - specific cutoffs for abdominal obesity defined by the korean society of obesity ) ( 13 ) ; 2 ) hypertriglyceridemia , defined as a serum triglyceride concentration 150 mg / dl ( 1.69 mmol / l ) ; 3 ) low hdl cholesterol , defined as a serum hdl cholesterol concentration < 40 mg / dl ( 1.04 mmol / l ) for men or < 50 mg / dl ( 1.29 mmol / l ) for women ; 4 ) high blood pressure , defined as a systolic blood pressure 130 mmhg , a diastolic blood pressure 85mmhg , or treatment with antihypertensive agents ; and 5 ) high fasting glucose , defined as a fasting serum glucose 100 mg / dl or previously diagnosed type 2 diabetes . since women have higher levels of adiponectin compared with men , we performed all analyses separately for men and women . we divided the study population into sex - specific quartiles of serum adiponectin levels with cut points 5.94 , 8.26 , and 11.22 g / ml for men and 8.91 , 11.90 , and 15.24 g / ml for women . we evaluated the association of baseline adiponectin levels with the incidence of new cases of metabolic syndrome and with the incidence of new cases of each component of the metabolic syndrome at the follow - up visit . to evaluate the incidence of new cases of each component , we excluded subjects with the presence of that specific component at baseline . multivariable logistic regression was used to assess the independent association of baseline adiponectin levels with incident metabolic syndrome . finally , we further adjusted for baseline levels of hs - crp ( log - transformed continuous variable ) , homa - ir ( log - transformed continuous variable ) , and follow - up bmi ( continuous variable ) . to evaluate the added discrimination provided by adiponectin levels to predict incident cases of metabolic syndrome beyond the information provided by the components of the metabolic syndrome , we compared the areas under the receiver operating characteristic ( roc ) curve in models that included waist circumference , hdl cholesterol , systolic blood pressure , triglycerides , and glucose levels with and without adiponectin levels . in addition , we calculated reclassification tables for models with and without serum adiponectin four categories of predicted risk ( cutoffs at 5 , 10 , and 20% ) , as well as the continuous ( or category - less ) net reclassification improvement ( nri ) ( 14 ) and the integrated discrimination improvement ( idi ) ( 15 ) . the nri compared classifications from the model with and without serum adiponectin for changes by incident metabolic syndrome for a net calculation of changes in the right direction . during an average of 2.6 years of follow - up , 153 men ( 18.4% ) and 199 women ( 16.4% ) developed metabolic syndrome . baseline bmi , waist circumference , systolic and diastolic blood pressures , fasting glucose , triglyceride , homa - ir , and high - sensitivity c - reactive protein ( hs - crp ) were significantly higher and hdl cholesterol was significantly lower in men and women who developed metabolic syndrome compared with those who did not ( table 1 ) . baseline characteristics of study population by incident metabolic syndrome median adiponectin levels at baseline were significantly lower in subjects who developed metabolic syndrome compared with those who did not in both men ( 7.09 vs. 8.63 g / ml , p < 0.001 ) and women ( 10.96 vs. 12.16 g / ml , p < a similar association was observed between adiponectin and each component of the metabolic syndrome except for high blood pressure . furthermore , serum adiponectin levels progressively decreased with the number of metabolic syndrome components developed by study participants over follow - up ( p for trend < 0.001 in both men and women ) ( table 2 ) . baseline serum adiponectin levels according to presence or absence of components of new - onset metabolic syndrome after an average of 2.6 years of follow - up , the proportions of men who developed new - onset metabolic syndrome , high waist circumference , low hdl cholesterol , high triglycerides , high blood pressure , and high blood glucose were 18.4 , 14.3 , 21.9 , 20.7 , 18.9 , and 15.5% , respectively . in multivariable - adjusted models [ model 3 ( table 3 ) ] , the or for developing metabolic syndrome comparing men in the highest with those in the lowest quartile of adiponectin was 0.25 ( 95% ci 0.140.47 ; p for trend < 0.001 ) . the corresponding ors for high waist circumference , low hdl cholesterol , high triglycerides , high blood pressure , and high blood glucose were 0.54 ( 0.251.20 ) , 0.54 ( 0.300.94 ) , 0.27 ( 0.140.50 ) , 0.46 ( 0.191.11 ) , and 0.77 ( 0.411.43 ) , respectively . among women , the proportions of participants who developed new - onset metabolic syndrome , high waist circumference , low hdl cholesterol , high triglycerides , high blood pressure , and high blood glucose were 16.4 , 21.1 , 32.2 , 14.1 , 14.4 , and 8.4% , respectively . ors for new - onset metabolic syndrome and its components according to baseline serum adiponectin in men in multivariable - adjusted models [ model 3 ( table 4 ) ] , the or for developing metabolic syndrome comparing women in the highest with those in the lowest quartile of adiponectin was 0.45 ( 95% ci 0.280.74 ; p for trend = 0.001 ) . the corresponding ors for new - onset high waist circumference , low hdl cholesterol , high triglycerides , high blood pressure , and high blood glucose were 0.50 ( 0.280.88 ) , 0.93 ( 0.551.56 ) , 0.28 ( 0.160.49 ) , 1.08 ( 0.572.06 ) , and 0.70 ( 0.381.29 ) , respectively . participants in the highest quartile of baseline adiponectin had the smallest increase in waist circumference , triglycerides , and systolic blood pressure in men and the smallest increase of triglycerides in women ( supplementary table 1 ) . ors for new - onset metabolic syndrome and its components according to baseline serum adiponectin in women we then evaluated how well baseline adiponectin levels predict incident metabolic syndrome beyond the information provided by baseline levels of metabolic syndrome components . the area under the roc curve to predict incident metabolic syndrome using waist circumference , hdl cholesterol , blood pressure , triglycerides , and blood glucose levels was 0.766 ( 95% ci 0.7260.805 ) in men and 0.783 ( 0.7520.814 ) in women . after adiponectin levels were added to the model , the corresponding areas under the roc curve were 0.772 ( 0.7340.811 ) and 0.783 ( 0.7520.815 ) , respectively . the p values for the comparison in areas under the roc curve for the models with and without adiponectin levels were 0.29 and 0.88 in men and women , respectively . the reclassification tables for improved prediction after adding serum adiponectin to a model including the components of the metabolic syndrome , separated for participants with and without new - onset metabolic syndrome , are shown in supplementary tables 2 and 3 . for men , the category - free nri was 0.21 ( 95% ci 0.040.38 ; p = 0.02 ) , and the idi was 0.010 ( 0.0010.018 ; p = 0.025 ) . the increases in nri and idi in men were derived primarily from an increase in sensitivity ( 20% more cases were reclassified in the correct direction using adiponectin , and the point estimate for the increase in integrated sensitivity was 0.008 ) . for women , the nri was 0.04 ( 0.11 to 0.20 ; p = 0.18 ) and the idi was 0.002 ( 0.001 to 0.004 ; p = 0.13 ) . the p value for the difference in nri and idi between men and women was 0.001 . among 595 men and 748 women with metabolic syndrome but no diabetes at baseline , 37 men and 29 women developed new cases of diabetes during follow - up . baseline adiponectin levels were inversely associated with the risk of developing diabetes in both men and women , although the association was stronger in women ( supplementary table 4 ) . among 318 men and 235 women with impaired fasting glucose but no diabetes at baseline , 39 men and 25 women developed diabetes during follow - up . in this subgroup , baseline adiponectin levels were also inversely associated with the risk of developing diabetes ( supplementary table 5 ) . in this prospective cohort study in a general asian population , serum adiponectin was a strong negative predictor of incident metabolic syndrome in both men and women . serum adiponectin levels were also a strong negative predictor of the total number of components of the metabolic syndrome developed and of each individual component of the metabolic syndrome , except for elevated blood pressure . the prospective design , the strength of the associations , and the graded dose - response relationships suggest that adiponectin levels may play a key role in the development of metabolic syndrome and its components . furthermore , our analyses indicate that in men , serum adiponectin may increase the predictive ability for identification of subjects at risk for developing new - onset metabolic syndrome beyond that of the information provided by the components of the metabolic syndrome and open the possibility of using serum adiponectin in clinical settings as a prognostic tool in men . the binding of adiponectin to these receptors mediates increased amp - activated protein kinase and peroxisome proliferator activated receptor- activity resulting in increased insulin sensitivity , glucose utilization , and fatty acid oxidation in the liver and skeletal muscle ( 1618 ) . additionally , the collagen - like domain of adiponectin allows oligomerization of the protein and the formation of high molecular weight ( hmw ) adiponectin ( 23 ) . while hmw adiponectin may be a more active form of adiponectin in vitro , both total and hmw adiponectin were inversely associated with progression to metabolic syndrome in two longitudinal cohort studies in japan ( 6,8 ) . additional studies in larger samples are needed to assess the role of hmw adiponectin in the prediction of metabolic syndrome . excess adiposity in obesity is associated with downregulation of adiponectin secretion ( hypoadiponectinemia ) and with decreased expression of adipor1 and -r2 ( adiponectin resistance ) , with both likely contributing to insulin resistance and metabolic dysregulation . despite strong evidence of the association between adiponectin and obesity - related metabolic abnormalities , there are limited data on the predictive value of circulating adiponectin for incident metabolic syndrome . in prior studies ( 68 ) , adiponectin levels were also inversely associated with incident metabolic syndrome , but those studies did not evaluate the additional predictive ability of adiponectin beyond the information provided by the components of the metabolic syndrome at baseline . in our study , decreasing levels of adiponectin were progressively associated with an increased incidence of metabolic syndrome as well as an increase in the number of metabolic components affected during follow - up . while we found no improvement in the area under the roc curve in models that added adiponectin to metabolic syndrome components , the nri and the idi were significantly improved in men , and the difference between men and women was statistically significant . the nri and the idi may be more sensitive than the area under the roc curve for identifying improvements in predictive value ( 28 ) . the idi measures the estimated improvement in the average sensitivity with the addition of adiponectin minus the estimated decrease in the mean specificity across all possible threshold values ( 15 ) . our data indicate that serum adiponectin may have a clinical value in screening for metabolic risk in men , but future studies with a larger sample size and validation samples need to delineate specific risk scoring tools and clinical thresholds for predicting the development of metabolic syndrome including measures of serum adiponectin . our results were analyses stratified by sex , a key determinant of serum adiponectin levels . median adiponectin levels in men were lower than those in women ( 8.26 vs. 11.90 g / ml , respectively ; p < 0.001 ) , despite higher prevalence of metabolic syndrome in women ( 10 ) . such sex differences in adiponectin levels have previously been reported , and it has been hypothesized that androgen inhibits adiponectin secretion triggering lower levels of adiponectin in males ( 2931 ) . in our study , serum adiponectin improved clinical prediction of new - onset metabolic syndrome in men but not in women . these differences were not due to a lower power of the study in women , as the number of cases of new - onset metabolic syndrome was higher in women . we could not establish the reason for this sex difference , although we note that the area under the roc curve for metabolic syndrome components was higher in women compared with men , possibly indicating that there is greater room for nontraditional markers to improve prediction among men . first , the study was restricted to middle - aged and elderly koreans living in a rural area with a relatively high baseline prevalence and high incidence of metabolic syndrome . the prevalence of metabolic syndrome in our cohort was similar to that in the korean national health and nutrition examination survey , a representative study of the korean population ( 9,32 ) . furthermore , korea seems to have experienced a rapid increase in the prevalence of metabolic syndrome during the 2000s , partly due to increasing adoption of western lifestyle patterns ( 32 ) . our cohort may therefore be reflecting a period of rapid increase in the frequency of metabolic syndrome , and our findings may not be generalizable to other populations , particularly to those with different secular trends , with higher levels of adiposity , or with younger age . second , the follow - up period of our cohort was only 2.6 years , and we could not evaluate whether the association between adiponectin and incident metabolic syndrome would persist in longer follow - up . third , we could not assess the presence of cardiometabolic abnormalities in 25% of the sample that did not complete the follow - up visit . however , we found no statistical differences in baseline demographics and laboratory findings , including serum adiponectin , between participants who attended the follow - up visit and those who did not ( data not shown ) . finally , our analyses were based on a single determination of serum adiponectin , which is subject to random measurement error and may have underestimated the strength of the associations . in conclusion , serum adiponectin was an independent protective factor for incident metabolic syndrome and its components in our longitudinal study . our findings thus support a central role of adiponectin in the development of cardiometabolic abnormalities in obesity . furthermore , our findings indicate that serum adiponectin may have a clinical role in predicting new - onset metabolic syndrome among men .

single nucleotide polymorphisms ( snps ) are currently the marker of choice due to their large numbers in virtually all populations of individuals . the applications of snp markers have clearly been demonstrated in human genomics where complete sequencing of the human genome led to the discovery of several million snps and technologies to analyze large sets of snps ( up to 1 million ) have been developed . snps have been applied in areas as diverse as human forensics and diagnostics , aquaculture , marker assisted - breeding of dairy cattle , crop improvement , conservation , and resource management in fisheries . functional genomic studies have capitalized upon snps located within regulatory genes , transcripts , and expressed sequence tags ( ests ) [ 9 , 10 ] . until recently large scale snp discovery in plants genetic applications such as linkage mapping , population structure , association studies , map - based cloning , marker - assisted plant breeding , and functional genomics continue to be enabled by access to large collections of snps . arabidopsis thaliana was the first plant genome sequenced followed soon after by rice [ 17 , 18 ] . in the year 2011 alone , the number of plant genomes sequenced doubled as compared to the number sequenced in the previous decade , resulting in currently , 31 and counting , publicly released sequenced plant genomes ( http://www.phytozome.net/ ) . with the ever increasing throughput of next - generation sequencing ( ngs ) , de novo and reference - based snp discovery and application are now feasible for numerous plant species . sequencing refers to the identification of the nucleotides in a polymer of nucleic acids , whether dna or rna . since its inception in 1977 , sequencing has brought about the field of genomics and increased our understanding of the organization and composition of plant genomes . tremendous improvements in sequencing have led to the generation of large amounts of dna information in a very short period of time . the analyses of large volumes of data generated through various ngs platforms require powerful computers and complex algorithms and have led to a recent expansion of the bioinformatics field of research . this book chapter focuses on the a priori discovery of snps through ngs , bioinformatics tools and resources , and the various downstream applications of snps . the sanger method is a sequencing - by - synthesis ( sbs ) method that relies on a combination of deoxy- and dideoxy - labeled chain terminator nucleotides . the first complete genome sequencing , that of bacteriophage phi x174 , was achieved that same year using this pioneering method . the chemical modification followed by cleavage at specific sites method also published in 1977 quickly became the less favored of the two methods because of its technical complexities , use of hazardous chemicals , and inherent difficulty in scale - up . in contrast , the sanger method , for which frederick sanger was awarded his second nobel prize in chemistry in 1980 , was quickly adopted by the biotechnology industry which implemented it using a broad array of chemistries and detection methods . in the last decade , new sequencing technologies have outperformed sanger - based sequencing in throughput and overall cost , if not quite in sequence length and error rate . this section will focus on the three main ngs platforms as well as the two main third - generation sequencing ( tgs ) platforms , their throughput and relative cost . we made every effort to ensure the accuracy of the data at the time of submission . however , the cost and throughput of these sequencing platforms change rapidly and , as such , our analysis only represents a snapshot in time . the flux of innovation in this field imposes a need for constant assessment of the technologies ' potentials and realignment of research goals . pyrosequencing was the first of the new highly parallel sequencing technologies to reach the market . it is commonly referred to as 454 sequencing after the name of the company that first commercialized it . it is an sbs method where single fragments of dna are hybridized to a capture bead array and the beads are emulsified with regents necessary to pcr amplifying the individually bound template . each bead in the emulsion acts as an independent pcr where millions of copies of the original template are produced and bound to the capture beads which then serve as the templates for the subsequent sequencing reaction . the individual beads are deposited into a picotiter plate along with dna polymerase , primers , and the enzymes necessary to create fluorescence through the consumption of inorganic phosphate produced during sequencing . as template - specific incorporation of a base by dna polymerase occurs , a pyrophosphate ( ppi ) is produced . this pyrophosphate is detected by an enzymatic luminometric inorganic pyrophosphate detection assay ( elida ) through the generation of a light signal following the conversion of ppi into atp . thus , the wells in which the current nucleotides are being incorporated by the sequencing reaction occurring on the bead emit a light signal proportional to the number of nucleotides incorporated , whereas wells in which the nucleotides are not being incorporated do not . the instrument repeats the sequential nucleotide wash cycle hundreds of times to lengthen the sequences . the 454 gs flx titanium xl platform currently generates up to 700 mb of raw 750 bp reads in a 23 hour run . the technology has difficulty quantifying homopolymers resulting in insertions / deletions and has an overall error rate of approximately 1% . reagent costs are approximately $ 6,200 per run . illumina technology , acquired by illumina from solexa , followed the release of 454 sequencing . with this sequencing approach , fragments of dna are hybridized to a solid substrate called a flow cell . in a process called bridge amplification , the bound dna template fragments are amplified in an isothermal reaction where copies of the template are created in close proximity to the original . this results in clusters of dna fragments on the flow cell creating a lawn of bound single strand dna molecules . the molecules are sequenced by flooding the flow cell with a new class of cleavable fluorescent nucleotides and the reagents necessary for dna polymerization . a complementary strand of each template is synthesized one base at a time using fluorescently labeled nucleotides . the fluorescent molecule is excited by a laser and emits light , the colour of which is different for each of the four bases . the fluorescent label is then cleaved off and a new round of polymerization occurs . unlike 454 sequencing , all four bases are present for the polymerization step and only a single molecule is incorporated per cycle . the flagship hiseq2500 sequencing instrument from illumina can generate up to 600 gb per run with a read length of 100 nt and 0.1% error rate . the illumina technique can generate sequence from opposite ends of a dna fragment , so called paired - end ( pe ) reads . the solid system was jointly developed by the harvard medical school and the howard hughes medical institute . the library preparation in solid is very similar to roche/454 in which clonal bead populations are prepared in microreactors containing dna template , beads , primers , and pcr components . beads that contain pcr products amplified by emulsion pcr are enriched by a proprietary process . the dna templates on the beads are modified at their 3 end to allow attachment to glass slides . a primer is annealed to an adapter on the dna template and a mixture of fluorescently tagged oligonucleotides is pumped into the flow cell . when the oligonucleotide matches the template sequence , it is ligated onto the primer and the unincorporated nucleotides are washed away . a charged couple device ( ccd ) camera captures the different colours attached to the primer . after image capture , the fluorescent tag is removed and new set of oligonucleotides are injected into the flow cell to begin the next round of dna ligation . this sequencing - by - ligation method in solid-5500x1 platform generates up to 1,410 million pe reads of 75 + 35 nt each with an error rate of 0.01% and reagent cost of approximately $ 10,500 per run . although widely accepted and used , the ngs platforms suffer from amplification biases introduced by pcr and dephasing due to varying extension of templates . the tgs technologies use single molecule sequencing which eliminates the need for prior amplification of dna thus overcoming the limitations imposed by ngs . the advantages offered by tgs technology are ( i ) lower cost , ( ii ) high throughput , ( iii ) faster turnaround , and ( iv ) longer reads [ 19 , 29 ] . the tgs can broadly be classified into three different categories : ( i ) sbs where individual nucleotides are observed as they incorporate ( pacific biosciences single molecule real time ( smart ) , heliscope true single molecule sequencing ( tsms ) , and life technologies / starlight and ion torrent ) , ( ii ) nanopore sequencing where single nucleotides are detected as they pass through a nanopore ( oxford / nanopore ) , and ( iii ) direct imaging of individual molecules ( ibm ) . heliscope sequencing involves dna library preparation and dna shearing followed by addition of a poly - a tail to the sheared dna fragments . these poly - a tailed dna fragments are attached to flow cells through poly - t anchors . the sequencing proceeds by dna extension with one out of 4 fluorescent tagged nucleotides incorporated followed by detection by the heliscope sequencer . the fluorescent tag on the incorporated nucleotide is then chemically cleaved to allow subsequent elongation of dna . heliscope sequencers can generate up to 28 gb of sequence data per run ( 50 channels ) with maximum read length of 55 bp at ~99% accuracy . the pacific biosciences sequencer uses glass anchored dna polymerases which are housed at the bottom of a zero - mode waveguide ( zmw ) . dna fragments are added into the zmw chamber with the anchored dna polymerase and nucleotides , each labeled with a different colour fluorophore , and are diffused from above the zmw . as the nucleotides circulate through the zmw , only the incorporated nucleotides remain at the bottom of the zmw while unincorporated nucleotides diffuse back above the zmw . a laser placed below the zmw excites only the fluorophores of the incorporated nucleotides as the zmw entraps the light and does not allow it to reach the unincorporated nucleotides above . the pacific biosciences sequencers can generate up to 140 mb of sequences per run ( per smart cell ) with reads of 2.5 kbp at ~85% accuracy . the cost per run per smart cell is approximately $ 600 . among the tgs technologies , pacific biosciences smart and heliscope tsms have been used in characterizing bacterial genomes and in human - disease - related studies ; however , tgs has yet to be capitalized upon in plant genomes . the heliscope generates short reads ( 55 bp ) which may cause ambiguous read mapping due to the presence of paralogous sequences and repetitive elements in plant genomes . the pacific biosciences reads have high error rates which limit their direct use in snp discovery . however , their long reads offer a definite advantage to fill gaps in genomic sequences and , at least in bacterial genomes , ngs reads have proven capable of correcting the base call errors of this tgs technology [ 3336 ] . hybrid assemblies incorporating short ( illumina , solid ) , medium ( 454/roche ) , and long reads ( pac - bio ) have the potential to yield better quality reference genomes and , as such , would provide an improved tool for snp discovery . the choice of a sequencing strategy must take into account the research goals , ability to store and analyze data , the ongoing changes in performance parameters , and the cost of ngs / tgs platforms . some key considerations include cost per raw base , cost per consensus base , raw and consensus accuracy of bases , read length , cost per read , and availability of pe or single end reads . the pre- and postprocessing protocols such as library construction and pipeline development and implementation for data analysis are also important . genome - wide analyses of rna sequences and their qualitative and quantitative measurements provide insights into the complex nature of regulatory networks . rna sequencing has been performed on a number of plant species including arabidopsis , soybean , rice , and maize for transcript profiling and detection of splice variants . rna sequencing has been used in de novo assemblies followed by snp discovery performed in nonmodel plants such as eucalyptus grandis , brassica napus , and medicago sativa . rna deep - sequencing technologies such as digital gene expression and illumina rnaseq are both qualitative and quantitative in nature and permit the identification of rare transcripts and splice variants . rna sequencing may be performed following its conversion into cdna that can then be sequenced as such . this method is , however , prone to error due to ( i ) the inefficient nature of reverse transcriptases ( rts ) , ( ii ) dna - dependent dna polymerase activity of rt causing spurious second strand dna , and ( iii ) artifactual cdna synthesis due to template switching . direct rna sequencing ( drs ) developed by helicos biosciences corporation is a high throughput and cost - effective method which eliminates the need for cdna synthesis and ligation / amplification leading to improved accuracy . chromatin immunoprecipitation ( chip ) is a specialized sequencing method that was specifically designed to identify dna sequences involved in in vivo protein dna interaction . chip - sequencing ( chip - seq ) is used to map the binding sites of transcription factors and other dna binding sites for proteins such as histones . as such , chip - seq does not aid snp discovery , but the availability of snp data along with chip - seq allows the study of allele - specific states of chromatin organization . deep sequence coverage leading to dense snp maps permits the identification of transcription factor binding sites and histone - mediated epigenetic modifications . chip - seq can be performed on serial analysis of gene expression ( sage ) tags or pe using sanger , 454 , and illumina platforms [ 55 , 56 ] . the dna , rna , and chip - seq data is analysed using a reference sequence if available or , in the absence of such reference , it requires de novo assembly , all of which is performed using specialized software , algorithms , pipelines , and hardware . the next - generation platforms generate a considerable amount of data and the impact of this with respect to data storage and processing time can be overlooked when designing an experiment . bioinformatics research is constantly developing new software and algorithms , data storage approaches , and even new computer architectures to better meet the computation requirements for projects incorporating ngs . this chapter describes the state - of - the - art with respect to software for ngs alignment and analysis at the time of writing . both commercial and noncommercial sequence analysis software are available for windows , macintosh , and linux operating systems . ngs companies offer proprietary software such as consensus assessment of sequence and variation ( cassava ) for illumina data and newbler for 454 data . such software tend to be optimized for their respective platform but have limited cross applicability to the others . web - based portals such as galaxy are tailored to a multitude of analyses , but the requirement to transfer multigigabyte sequence files across the internet can limit its usability to smaller datasets . commercially available software such as clc - bio ( http://www.clcbio.com/ ) and seqman ngen ( http://www.dnastar.com/t-sub-products-genomics-seqman-ngen.aspx ) provide a friendly user interface , are compatible with different operating systems , require minimal computing knowledge , and are capable of performing multiple downstream analyses . however , they tend to be relatively expensive , have narrow customizability , and require locally available high computing power . a recent review by wang et al . recommends linux - based programs because they are often free , not specific to any sequencing platform , and less computing power hungry and , as a consequence , tend to perform faster . however , most biologists are unfamiliar with linux operating systems , its structure and command lines , thereby imposing a steep learning curve for adoption . linux - based software such as bowtie , bwa , and soap2/3 have been used widely for the analysis of ngs data . other software may not have gained broad acceptance but may have unique features worth noting . for reviews on ngs software , see li and homer , wang et al . , and treangen and salzberg . characteristics of the most common ngs software and their attributes are listed in table 1 , and their download information can be found in table 4 . in selecting software for ngs data analysis one must consider , among other things , the sequencing platform , the availability of a reference genome , the computing and storage resources necessary , and the bioinformatics expertise available . algorithms used for sequence analysis have matured significantly but may still require computing power beyond what is currently available in most genomics facilities and/or long processing time . for example , in aligning 2 13,326,195 paired - end reads ( 76 bp ) from the cancer genome atlas project ( srr018643 ) , shrimp took 1,065 hrs with a peak memory footprint of 12 gigabytes to achieve the mapping of 81% of the reads to the human genome reference whereas bowtie used 2.9 gigabytes of memory , a run time of 2.2 hrs but only achieved a 67% mapping rate . both time and memory sequence mapping seems to be preferred over slower , memory demanding software even at the cost of a reduced mapping rate . it should be noted that a higher percentage of mapped reads is not a strict measure of quality because it may be indicative of a higher level of misaligned reads or reads aligned against repetitive elements , features that are not desirable . in the absence of a reference genome , de novo assembly of a plant genome is achieved using sequence information obtained through a combination of sanger and/or ngs of bacterial artificial chromosome ( bac ) clones , or by whole genome shotgun ( wgs ) with ngs . de novo assemblies are time consuming and require much greater computing power than read mapping onto a reference genome . the assembly accuracy depends in part on the read length and depth as well as the nature of the sequenced genome . the genomes of arabidopsis thaliana , rice , and maize were generated using a bac - by - bac approach while poplar , grape , and sorghum genomic sequences were obtained through wgs . all genomes sequenced to date are fragmented to varying degrees because of the inability of sequencing technologies and bioinformatics algorithms to assemble through highly conserved repetitive elements . a list of current plant genome sequencing projects , their sequencing strategies , and status from standard draft to finished can be found in the review by feuillet et al . . software programs such as mira , soapdenovo , abyss , and velvet have been used for de novo assembly . mira is well documented and can be readily customized , but it requires substantial computing memory and is not suited for large complex genomes . of the freely available software , soapdenovo is one of the fastest read assembly programs and it uses a comparatively moderate amount of computing memory . the assembly generated by soapdenovo can be used for snp discovery using soapsnp as implemented for the apple genome . the most common application of ngs is snp discovery , whose downstream usefulness in linkage map construction , genetic diversity analyses , association mapping , and marker - assisted selection has been demonstrated in several species . ngs - derived snps have been reported in humans , drosophila , wheat [ 81 , 82 ] , eggplant , rice [ 8486 ] , arabidopsis [ 87 , 88 ] , barley [ 14 , 89 ] , sorghum , cotton , common beans , soybean , potato , flax , aegilops tauschii , alfalfa , oat , and maize to name a few . snp discovery using ngs is readily accomplished in small plant genomes for which good reference genomes are available such as rice and arabidopsis [ 86 , 99 ] . although snp discovery in complex genomes without a reference genome such as wheat [ 81 , 82 ] , barley [ 14 , 89 ] , oat , and beans can be achieved through ngs , several challenges remain in other nonmodel but economically important crops . the presence of repeat elements , paralogs , and incomplete or inaccurate reference genome sequences can create ambiguities in snp calling . ngs read mapping can also suffer from sequencing error ( erroneous base calling ) and misaligned reads . the following section focuses on programs tailored for snp discovery and emphasizes some of the precautions and considerations to minimize erroneous snp calling . in theory , a snp is identified when a nucleotide from an accession read differs from the reference genome at the same nucleotide position . in the absence of a reference genome , this is achieved by comparing reads from different genotypes using de novo assembly strategies . read assembly files generated by mapping programs are used to perform snp calling . in practice , various empirical and statistical criteria are used to call snps , such as a minimum and maximum number of reads considering the read depth , the quality score and the consensus base ratio for examples . thresholds for these criteria are adjusted based on the read length and the genome coverage achieved by the ngs data . in assemblies generated allowing single nucleotide variants and insertions / deletions ( indels ) , a list of snp and indel coordinates is generated and the read mapping results can be visualized using graphical user interface programs such as tablet ( figure 1 ) , snp - vista , or savant ( refer to table 4 for download information ) . tablet has a user - friendly interface and is widely used because it supports a wide array of commonly used file formats such as sam , bam , soap , ace , fastq , and fasta generated by different read assemblers such as bowtie , bwa , soap , maq , and seqman ngen . it displays contig overview , coverage information , read names and it allows searching for specific coordinates on scaffolds . samtools is popular because of its various modules for file conversion ( sam to bam and vice - versa ) , mapping statistics , variant calling , and assembly visualization . recently , soapsnp has gained popularity because of its tight integration with soap aligner and other soap modules which are constantly upgraded and provide a one stop shop for the sequencing analysis continuum . variant calling algorithms such as samtools and snver can be used as stand - alone programs or incorporated into pipelines for snp calling . some of the main features of the current commonly used software are listed in table 2 ( refer to table 4 for download information ) . snp discovery is more robust when multiple and divergent genotypes are used simultaneously , creating the necessary basis to capture the genetic variability of a species . large parts of plant genomes consist of repetitive elements which can cause spurious snp calling by erroneous read mapping to paralogous repeat element sequences . in polyploid genomes such as cotton ( allotetraploid ) , homoeologous sequences can cause similar misalignment . improved read assembly and filtering of snps become even more important factors for accurate snp calling in these cases because they can mitigate the effects of errors caused by paralogs and homoeologs . read assembly algorithms such as bowtie and soap as well as variant calling / genotyping softwares such as gatk are rapidly evolving to accommodate an ever increasing number of reads , increased read length , nucleotide quality values , and mate - pair information of pe reads . assembly programs such as novoalign ( http://www.novocraft.com/main/index.php ) and stampy , although memory and time intensive , are highly sensitive for simultaneous mapping of short reads from multiple individuals . snp calls can be significantly improved using filtering criteria that are specific to the genome characteristics and the dataset . for instance , projects aimed at resequencing can compare different datasets from the same genotype and thus eliminate data with large discrepancies . this strategy identifies the most common sources of error and is applied in the 1000 genome project . reduced representation libraries ( rrls ) , that is , sequencing an enriched subset of a genome by eliminating a proportion of its repetitive fractions , reduce the probability of misalignments to repeats and thus potential downstream erroneous snp calling . filtering criteria that can improve snp accuracy include ( i ) a minimum read depth ( often 3 per genotype ) , ( ii ) > 90% nucleotides within a genotype having identical call at a given position ( ~<10% sequencing error ) , ( iii ) a read depth mean of the sequence depth over the entire mapping assembly , ( iv ) the elimination of ribosomal dna and other repetitive elements in the 50 nt flanking any snp call , and ( v ) masking of homopolymer snps with a given base string length ( often 2 ) . additionally , in polyploid species , separate assembly of homoeologs using stringent mapping parameters is often essential for genome - wide snp identification to avoid spurious snp calls caused by erroneous homoeologous read mapping . prior to any snp applications , the discovered snps must be validated to identify the true snps and get an idea of the percentage of potentially false snps resulting from an snp discovery exercise . the need for validation arises because a proportion of the discovered snps could have been wrongly called for various reasons including those outlined above . snp validation can be accomplished using a variety of material such as a biparental segregating population or a diverse panel of genotypes . usually a small subset of the snps is used for validation through assays such as the illumina goldengate , kbiosciences competitive allelespecific - pcr snp genotyping system ( kaspar ) ( http://www.lgcgenomics.com/ ) or the high resolution melting ( hrm ) curve analysis . validation can serve as an iterative and informative process to modify and optimize the snp filtering criteria to improve snp calling . for example , a subset of 144 snps from a total of 2,113,120 snps were validated using the goldengate assay on 160 accessions in apple . another example is illustrated in figure 2 where a kaspar assay was performed on 92 genotypes from a segregating population illustrating the validation of a single with the continuously competitive pricing of ngs , genotyping - by - sequencing ( gbs ) is becoming a viable snp validation method . either biparental segregating populations or a collection of diverse genotypes can be sequenced at a reasonable cost using indexing , that is , combining multiple independently tagged genotypes in a single ngs run to obtain genome - wide or reduced representation genome sequences at a lower coverage but potentially validating a much larger number of snps than the methods described above . sequencing of segregating populations or diverse genotypes may also lead to the discovery of additional snps . the two major factors affecting the snp validation rate are sequencing and read mapping errors as discussed above . ngs platforms have different levels of sequencing accuracies , and this may be the most important factor determining the variation in the validation , from 88.2% for solid followed by illumina at 85.4% and roche 454 at 71% . the snp validation rates can be improved using rrl for snp discovery and choosing snps within the nonrepetitive sequences including predicted single copy genes and single copy repeat junctions shown to have high validation rates . snp applications include phylogenic analysis , marker - assisted selection , genetic mapping of quantitative trait loci ( qtl ) , bulked segregant analysis , genome selection , and genome - wide association studies ( gwas ) . the number of snps and individuals to screen are of primary importance in choosing an snp genotyping assay , though cost of the assay and/or equipment and the level of accuracy are also important considerations . illumina goldengate is a commonly used genotyping assay because of its flexibility in interrogating 96 to 3,072 snp loci simultaneously ( http://www.illumina.com/ ) . hrm analysis is suitable for a few to an intermediate number of snps and can be performed within a typical laboratory setting . kaspar and snpline genotyping systems ( http://www.lgcgenomics.com/ ) can be used for genotyping a few to thousands of snps in a laboratory setting . the snpline system is available in snplite or snpline xl versions to allow flexibility in sample number and snp assays . the iplex gold technology developed by sequenom ( http://www.sequenom.com/ ) is based on the massarray system which uses primer extension chemistry and matrix - assisted laser desorption / ionisation - time of flight ( maldi - tof ) mass spectrometry for genotyping . the iplex gold system has gained acceptance due to its high precision and cost - effective implementation . high throughput chip - based genotyping assays such as the affymetrix genechip arrays ( http://www.affymetrix.com/estore/ ) and the illumina beadchips ( http://www.illumina.com/ ) are capable of validating up to a million snps per reaction across an entire genome . detailed analyses of snp genotyping assays and their features are reviewed in tsuchihashi and dracopoli , sobrino and carracedo , giancola et al . , kim and misra , gupta et al . , and ragoussis . a list of the most commonly used genotyping assays describing the assay type , technology , throughput , multiplexing ability , and relative scalability can be found in table 3 . array - based technologies such as infinium and goldengate substantially improved snp genotyping efficiency , but they are species - specific , expensive to design and require specific equipment and chemistry . pcr and primer extension technologies like kaspar and taqman ( http://www.lifetechnologies.com/global/en/home.html ) are limited by their low snp throughput but can be useful to assay a large number of genotypes with few snps . ngs technologies have become viable for genotyping studies and may offer advantages over other genotyping methods in cost and efficiency . there have been a number of approaches developed that use complexity reduction strategies to lower the cost and simplify the discovery of snp markers using ngs , rna - seq , complexity reduction of polymorphic sequences ( crops ) , restriction - site - associated dna sequencing ( rad - seq ) , and gbs . of these methodologies gbs holds the greatest promise to serve the widest base of plant researchers because of its ability to allow simultaneous marker discovery and genotyping with low cost and a simple molecular biology workflow . briefly , gbs involves digesting the genome of each individual in a population to be studied with a restriction enzyme . one unique and one common adapter are ligated to the fragments and a pcr is carried out which is biased towards amplifying smaller dna fragments . the amplicons are not fragmented so only the ends of the pcr products are sequenced . the unique adapter acts as an i d tag so sequencing reads can be associated with an individual . the choice of enzyme has an effect on the number of markers identified and the amount of sequence coverage required . the more frequent the restriction recognition site , the higher the number of fragments and therefore more potential markers . use of more frequent cutters may necessitate greater amounts of sequencing depending on the application . recently demonstrated the use of two restriction enzymes to perform gbs in bread wheat , a hexaploid genome . gbs has the potential to be a truly revolutionary technology in the arena of plant genomics . it brings high density genotyping to the vast majority of plant species that , until now , have had almost no investment in genomics resources . with little capital investment requirement and an affordable per sample cost , all plant researchers now have powerful genomic and genetic methodologies available to them . uses of gbs include applications in marker discovery , phylogenetics , bulked segregant analysis , qtl mapping in biparental lines , gwas , and genome selection . gbs can also be applied to fine mapping in candidate gene discovery and be used to generate high - density snp genetic maps to assist in de novo genome assembly . we predict tremendous advances in functional genomics and plant breeding from the implementation of gbs because it is truly a democratizing application for ngs in nonmodel plant systems . ngs and snp genotyping technologies have made snps the most widely used marker for genetic studies in plant species such as arabidopsis and rice . snps can help to decipher breeding pedigree , to identify genomic divergence of species to elucidate speciation and evolution , and to associate genomic variations to phenotypic traits . the ease of snp development , reasonable genotyping costs , and the sheer number of snps present within a collection of individuals allow an assortment of applications that can have a tremendous impact on basic and applied research in plant species . a genetic map refers to the arrangement of traits , genes , and markers relative to each other as measured by their recombination frequency . genetic maps are essential tools in molecular breeding for plant genetic improvement as they enable gene localization , map - based cloning , and the identification of qtl . snps have greatly facilitated the production of much higher density maps than previous marker systems . snps discovered using rna - seq and expressed sequence tags ( ests ) have the added advantage of being gene specific . their high abundance and rapidly improving genotyping technologies make snps an ideal marker type for generating new genetic maps as well as saturating existing maps created with other markers . most snps are biallelic thereby having a lower polymorphism information content ( pic ) value as compared to most other marker types which are often multiallelic . the limited information associated with their biallelic nature is greatly compensated by their high frequency , and a map of 700900 snps has been found to be equivalent to a map of 300400 simple sequence repeat ( ssr ) markers . snp - based linkage maps have been constructed in many economically important species such as rice , cotton and brassica . the identification of candidate genes for flowering time in brassica and maize are practical examples of gene discovery through snp - based genetic maps . association mapping ( am ) panels provide a better resolution , consider numerous alleles , and may provide faster marker - trait association than biparental populations . am , often referred to as linkage disequilibrium ( ld ) mapping , relies on the nonrandom association between markers and traits . high marker saturation is required to detect marker - trait association , hence the need for densely saturated maps . in general , gwass require 10,000100,000 markers applied to a collection of genotypes representing a broad genetic basis . in the past few years , ngs technologies have led to the discovery of thousands , even millions of snps , and novel application platforms have made it possible to produce genome - wide haplotypes of large numbers of genotypes , making snps the ideal marker for gwass . so far , 951 gwass have been reported in humans ( http://www.bing.com/search?q=www.genome.gov%2fgwastudies%2f&src=ie9tr ) . in plants , such a study was first reported in arabidopsis for flowering time and pathogen - resistance genes . a gwas performed in rice using ~3.6 million snps identified genomic regions associated with 14 agronomic traits . the genetic structure of northern leaf blight , southern leaf blight , and leaf architecture was studied using ~1.6 million snps in maize [ 133135 ] . snp - based gwas was also performed on species such as barley for which a reference genome sequence is not available . soto - cerda and cloutier have reviewed the concepts , benefits , and limitations of am in plants . however , the biological inferences from results of these two marker types may be misinterpreted due to homoplasy , a phenomenon in which similarity in traits or markers occurs due to reasons other than ancestry , such as convergent evolution , evolutionary reversal , gene duplication , and horizontal gene transfer . the advantage of snps over microsatellites and mitochondrial dna resides in the fact that snps represent single base nucleotide substitutions and , as such , they are less affected by homoplasy because their origin can be explained by mutation models . snps have been employed to quantify genetic variation , for individual identification , to determine parentage relatedness and population structure . seed shattering ( or loss thereof ) has been associated with an snp through a gwas aimed at unraveling the evolution of rice that led to its domestication . snps have also been used to study the evolution of genes such as wag-2 in wheat . algorithms such as neighbor - joining and maximum likelihood implemented in the phylip and mega software are commonly used to generate phylogenetic trees . the main advantage of snps is unquestionably their large numbers . as with all marker systems the researcher must be aware of ascertainment biases that exist in the panel of snps being used . these biases exist because snps are often developed from examining a small group of individuals and selecting the markers that maximize the amount of polymorphism that can be detected in the population used . this results in a collection of markers that sample only a fraction of the diversity that exists in the species but that are nevertheless used to infer relatedness and determine genetic distance for whole populations . ideally , a set of snp markers randomly distributed throughout the genome would be developed for each population studied . gbs moves us closer to this goal by incorporating simultaneous discovery of snps and genotyping of individuals . with this approach genome sample bias snp discovery incontestably made a quantum leap forward with the advent of ngs technologies and large numbers of snps are now available from several genomes including large and complex ones ( see section 4 ) . unlike model systems such as humans and arabidopsis , snps from crop plants remain limited for the time being , but broad access to reasonable cost ngs promises to rapidly increase the production of reference genome sequences as well as snp discovery . many issues remain to be addressed , such as the ascertainment bias of popular biparental populations and the low validation rate of some array - based genotyping platforms . the area of epigenetic regulation of various genome components can be better understood as accurate and deeper sequencing is achieved . rna and chip - sequencing projects , similar to rna - seq in the nonmodel plant sweet cherry to identify snps and haplotypes , can be undertaken to study functional genomics . a great deal of knowledge that is still elusive about the noncoding and repetitive elements can be determined with the next wave of modern and efficient sequencing technologies . the first ( sanger ) and the second ( next ) generation sequencing technologies have enabled researchers to characterize dna sequence variation , sequence entire genomes , quantify transcript abundance , and understand mechanisms such as alternative splicing and epigenetic regulation . numerous plant genomes are now sequenced at various levels of completion and many more are underway . the ngs technologies have made snp discovery affordable even in complex genomes and the technologies themselves have improved tremendously in the past decade . improvements in tgs promise synergies with ngs technologies to further assist our understanding of plant genetics and genomics . ngs has revolutionized genomics - related research , and it is our belief that the ngs - enabled discoveries will continue in the next decade .

the decreasing cost along with rapid progress in next - generation sequencing and related bioinformatics computing resources has facilitated large - scale discovery of snps in various model and nonmodel plant species . large numbers and genome - wide availability of snps make them the marker of choice in partially or completely sequenced genomes . although excellent reviews have been published on next - generation sequencing , its associated bioinformatics challenges , and the applications of snps in genetic studies , a comprehensive review connecting these three intertwined research areas is needed . this paper touches upon various aspects of snp discovery , highlighting key points in availability and selection of appropriate sequencing platforms , bioinformatics pipelines , snp filtering criteria , and applications of snps in genetic analyses . the use of next - generation sequencing methodologies in many non - model crops leading to discovery and implementation of snps in various genetic studies is discussed . development and improvement of bioinformatics software that are open source and freely available have accelerated the snp discovery while reducing the associated cost . key considerations for snp filtering and associated pipelines are discussed in specific topics . a list of commonly used software and their sources is compiled for easy access and reference .

1. Introduction 2. History and Evolution of Sequencing 3. Computing Resources for Sequence Assembly 4. SNP Discovery 5. SNP Genotyping 6. Applications of SNPS 7. Future Perspectives

single nucleotide polymorphisms ( snps ) are currently the marker of choice due to their large numbers in virtually all populations of individuals . the applications of snp markers have clearly been demonstrated in human genomics where complete sequencing of the human genome led to the discovery of several million snps and technologies to analyze large sets of snps ( up to 1 million ) have been developed . snps have been applied in areas as diverse as human forensics and diagnostics , aquaculture , marker assisted - breeding of dairy cattle , crop improvement , conservation , and resource management in fisheries . until recently large scale snp discovery in plants genetic applications such as linkage mapping , population structure , association studies , map - based cloning , marker - assisted plant breeding , and functional genomics continue to be enabled by access to large collections of snps . with the ever increasing throughput of next - generation sequencing ( ngs ) , de novo and reference - based snp discovery and application are now feasible for numerous plant species . this book chapter focuses on the a priori discovery of snps through ngs , bioinformatics tools and resources , and the various downstream applications of snps . the chemical modification followed by cleavage at specific sites method also published in 1977 quickly became the less favored of the two methods because of its technical complexities , use of hazardous chemicals , and inherent difficulty in scale - up . this section will focus on the three main ngs platforms as well as the two main third - generation sequencing ( tgs ) platforms , their throughput and relative cost . it is an sbs method where single fragments of dna are hybridized to a capture bead array and the beads are emulsified with regents necessary to pcr amplifying the individually bound template . the individual beads are deposited into a picotiter plate along with dna polymerase , primers , and the enzymes necessary to create fluorescence through the consumption of inorganic phosphate produced during sequencing . the molecules are sequenced by flooding the flow cell with a new class of cleavable fluorescent nucleotides and the reagents necessary for dna polymerization . unlike 454 sequencing , all four bases are present for the polymerization step and only a single molecule is incorporated per cycle . the solid system was jointly developed by the harvard medical school and the howard hughes medical institute . the library preparation in solid is very similar to roche/454 in which clonal bead populations are prepared in microreactors containing dna template , beads , primers , and pcr components . when the oligonucleotide matches the template sequence , it is ligated onto the primer and the unincorporated nucleotides are washed away . the advantages offered by tgs technology are ( i ) lower cost , ( ii ) high throughput , ( iii ) faster turnaround , and ( iv ) longer reads [ 19 , 29 ] . the tgs can broadly be classified into three different categories : ( i ) sbs where individual nucleotides are observed as they incorporate ( pacific biosciences single molecule real time ( smart ) , heliscope true single molecule sequencing ( tsms ) , and life technologies / starlight and ion torrent ) , ( ii ) nanopore sequencing where single nucleotides are detected as they pass through a nanopore ( oxford / nanopore ) , and ( iii ) direct imaging of individual molecules ( ibm ) . among the tgs technologies , pacific biosciences smart and heliscope tsms have been used in characterizing bacterial genomes and in human - disease - related studies ; however , tgs has yet to be capitalized upon in plant genomes . the pacific biosciences reads have high error rates which limit their direct use in snp discovery . hybrid assemblies incorporating short ( illumina , solid ) , medium ( 454/roche ) , and long reads ( pac - bio ) have the potential to yield better quality reference genomes and , as such , would provide an improved tool for snp discovery . the choice of a sequencing strategy must take into account the research goals , ability to store and analyze data , the ongoing changes in performance parameters , and the cost of ngs / tgs platforms . some key considerations include cost per raw base , cost per consensus base , raw and consensus accuracy of bases , read length , cost per read , and availability of pe or single end reads . the pre- and postprocessing protocols such as library construction and pipeline development and implementation for data analysis are also important . genome - wide analyses of rna sequences and their qualitative and quantitative measurements provide insights into the complex nature of regulatory networks . rna sequencing has been performed on a number of plant species including arabidopsis , soybean , rice , and maize for transcript profiling and detection of splice variants . rna sequencing has been used in de novo assemblies followed by snp discovery performed in nonmodel plants such as eucalyptus grandis , brassica napus , and medicago sativa . as such , chip - seq does not aid snp discovery , but the availability of snp data along with chip - seq allows the study of allele - specific states of chromatin organization . chip - seq can be performed on serial analysis of gene expression ( sage ) tags or pe using sanger , 454 , and illumina platforms [ 55 , 56 ] . the dna , rna , and chip - seq data is analysed using a reference sequence if available or , in the absence of such reference , it requires de novo assembly , all of which is performed using specialized software , algorithms , pipelines , and hardware . the next - generation platforms generate a considerable amount of data and the impact of this with respect to data storage and processing time can be overlooked when designing an experiment . bioinformatics research is constantly developing new software and algorithms , data storage approaches , and even new computer architectures to better meet the computation requirements for projects incorporating ngs . recommends linux - based programs because they are often free , not specific to any sequencing platform , and less computing power hungry and , as a consequence , tend to perform faster . linux - based software such as bowtie , bwa , and soap2/3 have been used widely for the analysis of ngs data . , and treangen and salzberg . characteristics of the most common ngs software and their attributes are listed in table 1 , and their download information can be found in table 4 . in selecting software for ngs data analysis one must consider , among other things , the sequencing platform , the availability of a reference genome , the computing and storage resources necessary , and the bioinformatics expertise available . for example , in aligning 2 13,326,195 paired - end reads ( 76 bp ) from the cancer genome atlas project ( srr018643 ) , shrimp took 1,065 hrs with a peak memory footprint of 12 gigabytes to achieve the mapping of 81% of the reads to the human genome reference whereas bowtie used 2.9 gigabytes of memory , a run time of 2.2 hrs but only achieved a 67% mapping rate . it should be noted that a higher percentage of mapped reads is not a strict measure of quality because it may be indicative of a higher level of misaligned reads or reads aligned against repetitive elements , features that are not desirable . the genomes of arabidopsis thaliana , rice , and maize were generated using a bac - by - bac approach while poplar , grape , and sorghum genomic sequences were obtained through wgs . a list of current plant genome sequencing projects , their sequencing strategies , and status from standard draft to finished can be found in the review by feuillet et al . software programs such as mira , soapdenovo , abyss , and velvet have been used for de novo assembly . the assembly generated by soapdenovo can be used for snp discovery using soapsnp as implemented for the apple genome . the most common application of ngs is snp discovery , whose downstream usefulness in linkage map construction , genetic diversity analyses , association mapping , and marker - assisted selection has been demonstrated in several species . ngs - derived snps have been reported in humans , drosophila , wheat [ 81 , 82 ] , eggplant , rice [ 8486 ] , arabidopsis [ 87 , 88 ] , barley [ 14 , 89 ] , sorghum , cotton , common beans , soybean , potato , flax , aegilops tauschii , alfalfa , oat , and maize to name a few . although snp discovery in complex genomes without a reference genome such as wheat [ 81 , 82 ] , barley [ 14 , 89 ] , oat , and beans can be achieved through ngs , several challenges remain in other nonmodel but economically important crops . the following section focuses on programs tailored for snp discovery and emphasizes some of the precautions and considerations to minimize erroneous snp calling . in theory , a snp is identified when a nucleotide from an accession read differs from the reference genome at the same nucleotide position . in practice , various empirical and statistical criteria are used to call snps , such as a minimum and maximum number of reads considering the read depth , the quality score and the consensus base ratio for examples . in assemblies generated allowing single nucleotide variants and insertions / deletions ( indels ) , a list of snp and indel coordinates is generated and the read mapping results can be visualized using graphical user interface programs such as tablet ( figure 1 ) , snp - vista , or savant ( refer to table 4 for download information ) . tablet has a user - friendly interface and is widely used because it supports a wide array of commonly used file formats such as sam , bam , soap , ace , fastq , and fasta generated by different read assemblers such as bowtie , bwa , soap , maq , and seqman ngen . samtools is popular because of its various modules for file conversion ( sam to bam and vice - versa ) , mapping statistics , variant calling , and assembly visualization . variant calling algorithms such as samtools and snver can be used as stand - alone programs or incorporated into pipelines for snp calling . some of the main features of the current commonly used software are listed in table 2 ( refer to table 4 for download information ) . improved read assembly and filtering of snps become even more important factors for accurate snp calling in these cases because they can mitigate the effects of errors caused by paralogs and homoeologs . read assembly algorithms such as bowtie and soap as well as variant calling / genotyping softwares such as gatk are rapidly evolving to accommodate an ever increasing number of reads , increased read length , nucleotide quality values , and mate - pair information of pe reads . snp calls can be significantly improved using filtering criteria that are specific to the genome characteristics and the dataset . filtering criteria that can improve snp accuracy include ( i ) a minimum read depth ( often 3 per genotype ) , ( ii ) > 90% nucleotides within a genotype having identical call at a given position ( ~<10% sequencing error ) , ( iii ) a read depth mean of the sequence depth over the entire mapping assembly , ( iv ) the elimination of ribosomal dna and other repetitive elements in the 50 nt flanking any snp call , and ( v ) masking of homopolymer snps with a given base string length ( often 2 ) . additionally , in polyploid species , separate assembly of homoeologs using stringent mapping parameters is often essential for genome - wide snp identification to avoid spurious snp calls caused by erroneous homoeologous read mapping . prior to any snp applications , the discovered snps must be validated to identify the true snps and get an idea of the percentage of potentially false snps resulting from an snp discovery exercise . validation can serve as an iterative and informative process to modify and optimize the snp filtering criteria to improve snp calling . either biparental segregating populations or a collection of diverse genotypes can be sequenced at a reasonable cost using indexing , that is , combining multiple independently tagged genotypes in a single ngs run to obtain genome - wide or reduced representation genome sequences at a lower coverage but potentially validating a much larger number of snps than the methods described above . sequencing of segregating populations or diverse genotypes may also lead to the discovery of additional snps . the two major factors affecting the snp validation rate are sequencing and read mapping errors as discussed above . ngs platforms have different levels of sequencing accuracies , and this may be the most important factor determining the variation in the validation , from 88.2% for solid followed by illumina at 85.4% and roche 454 at 71% . the snp validation rates can be improved using rrl for snp discovery and choosing snps within the nonrepetitive sequences including predicted single copy genes and single copy repeat junctions shown to have high validation rates . snp applications include phylogenic analysis , marker - assisted selection , genetic mapping of quantitative trait loci ( qtl ) , bulked segregant analysis , genome selection , and genome - wide association studies ( gwas ) . the number of snps and individuals to screen are of primary importance in choosing an snp genotyping assay , though cost of the assay and/or equipment and the level of accuracy are also important considerations . illumina goldengate is a commonly used genotyping assay because of its flexibility in interrogating 96 to 3,072 snp loci simultaneously ( http://www.illumina.com/ ) . hrm analysis is suitable for a few to an intermediate number of snps and can be performed within a typical laboratory setting . kaspar and snpline genotyping systems ( http://www.lgcgenomics.com/ ) can be used for genotyping a few to thousands of snps in a laboratory setting . detailed analyses of snp genotyping assays and their features are reviewed in tsuchihashi and dracopoli , sobrino and carracedo , giancola et al . , and ragoussis . a list of the most commonly used genotyping assays describing the assay type , technology , throughput , multiplexing ability , and relative scalability can be found in table 3 . there have been a number of approaches developed that use complexity reduction strategies to lower the cost and simplify the discovery of snp markers using ngs , rna - seq , complexity reduction of polymorphic sequences ( crops ) , restriction - site - associated dna sequencing ( rad - seq ) , and gbs . of these methodologies gbs holds the greatest promise to serve the widest base of plant researchers because of its ability to allow simultaneous marker discovery and genotyping with low cost and a simple molecular biology workflow . the choice of enzyme has an effect on the number of markers identified and the amount of sequence coverage required . recently demonstrated the use of two restriction enzymes to perform gbs in bread wheat , a hexaploid genome . it brings high density genotyping to the vast majority of plant species that , until now , have had almost no investment in genomics resources . uses of gbs include applications in marker discovery , phylogenetics , bulked segregant analysis , qtl mapping in biparental lines , gwas , and genome selection . we predict tremendous advances in functional genomics and plant breeding from the implementation of gbs because it is truly a democratizing application for ngs in nonmodel plant systems . ngs and snp genotyping technologies have made snps the most widely used marker for genetic studies in plant species such as arabidopsis and rice . snps can help to decipher breeding pedigree , to identify genomic divergence of species to elucidate speciation and evolution , and to associate genomic variations to phenotypic traits . the ease of snp development , reasonable genotyping costs , and the sheer number of snps present within a collection of individuals allow an assortment of applications that can have a tremendous impact on basic and applied research in plant species . a genetic map refers to the arrangement of traits , genes , and markers relative to each other as measured by their recombination frequency . genetic maps are essential tools in molecular breeding for plant genetic improvement as they enable gene localization , map - based cloning , and the identification of qtl . the limited information associated with their biallelic nature is greatly compensated by their high frequency , and a map of 700900 snps has been found to be equivalent to a map of 300400 simple sequence repeat ( ssr ) markers . snp - based linkage maps have been constructed in many economically important species such as rice , cotton and brassica . association mapping ( am ) panels provide a better resolution , consider numerous alleles , and may provide faster marker - trait association than biparental populations . in the past few years , ngs technologies have led to the discovery of thousands , even millions of snps , and novel application platforms have made it possible to produce genome - wide haplotypes of large numbers of genotypes , making snps the ideal marker for gwass . so far , 951 gwass have been reported in humans ( http://www.bing.com/search?q=www.genome.gov%2fgwastudies%2f&src=ie9tr ) . the genetic structure of northern leaf blight , southern leaf blight , and leaf architecture was studied using ~1.6 million snps in maize [ 133135 ] . soto - cerda and cloutier have reviewed the concepts , benefits , and limitations of am in plants . however , the biological inferences from results of these two marker types may be misinterpreted due to homoplasy , a phenomenon in which similarity in traits or markers occurs due to reasons other than ancestry , such as convergent evolution , evolutionary reversal , gene duplication , and horizontal gene transfer . the advantage of snps over microsatellites and mitochondrial dna resides in the fact that snps represent single base nucleotide substitutions and , as such , they are less affected by homoplasy because their origin can be explained by mutation models . snps have been employed to quantify genetic variation , for individual identification , to determine parentage relatedness and population structure . the main advantage of snps is unquestionably their large numbers . ideally , a set of snp markers randomly distributed throughout the genome would be developed for each population studied . gbs moves us closer to this goal by incorporating simultaneous discovery of snps and genotyping of individuals . with this approach genome sample bias snp discovery incontestably made a quantum leap forward with the advent of ngs technologies and large numbers of snps are now available from several genomes including large and complex ones ( see section 4 ) . unlike model systems such as humans and arabidopsis , snps from crop plants remain limited for the time being , but broad access to reasonable cost ngs promises to rapidly increase the production of reference genome sequences as well as snp discovery . rna and chip - sequencing projects , similar to rna - seq in the nonmodel plant sweet cherry to identify snps and haplotypes , can be undertaken to study functional genomics . the first ( sanger ) and the second ( next ) generation sequencing technologies have enabled researchers to characterize dna sequence variation , sequence entire genomes , quantify transcript abundance , and understand mechanisms such as alternative splicing and epigenetic regulation . the ngs technologies have made snp discovery affordable even in complex genomes and the technologies themselves have improved tremendously in the past decade . ngs has revolutionized genomics - related research , and it is our belief that the ngs - enabled discoveries will continue in the next decade .

in older adults , performance of many habitual daily activities and tasks requires moving and controlling the center of gravity ( defined as the vertical projection of the center of mass ) to various positions within the base of support . however , effective displacement of the center of gravity during either self - initiated voluntary movements or externally triggered perturbations requires the capacity to remain within the limits of stability , which is described as the maximum distance a person can intentionally displace his or her center of gravity from a midline vertical position in any direction without losing balance and stepping or grasping for support.14 thus , a constricted limits of stability boundary or uncontrolled center of gravity movement can impair functional balance of older adults and constrain safe performance of their daily activities such as reaching , bending , walking , gait initiation , and moving from sit - to - stand.5 current literature suggests that aging is associated with diminished limits of stability capacity69 due to multiple factors , including reduced ankle muscle strength10 and poor cutaneous mechanoreceptor function in the soles of the feet , thus exposing older adults to more instability in functional daily activities and a higher risk of falling.11,12 therefore , from the perspectives of injury prevention and/or clinical rehabilitation of physical function , developing exercise intervention strategies that maximize the center of gravity sway envelope ( ie , the area an individual can move his or her center of gravity with control within the base of support)4 and subsequently expand the limits of stability boundary becomes functionally important for improving balance and mobility and , critically , for reducing the risk of falling among the elderly , which is a significant public health problem worldwide.13,14 because of its functional significance and implications for risk of falling , limits of stability should be integrated into exercise balance training programs that target fall prevention in older adults . however , among the many types of group- and evidence - based exercise programs1518 designed to prevent falls , few have a specific , tailored focus on integrating training elements that would maximize limits of stability . given that constrained limits of stability can lead to an increased propensity to fall in older adults,1,19 fall prevention programs that explicitly integrate therapeutic balance training of limits of stability are needed . taking a therapeutic approach , li et al20,21 developed the tai ji quan : moving for better balance ( tjqmbb ) program that specifically includes an element of training on limits of stability . integrating tai ji yin - yang theory22 and theories of motor control and clinical practice,1,2,23 tjqmbb uses an innovative strategy of embracing a complementary interplay between stability and instability ( dualities of yin - yang ) during training to achieve balance equilibrium.20 more specifically , training focuses on eliciting both reactive and anticipatory responses to self - induced , deliberately controlled and coordinated tai ji quan movements aimed at stimulating postural adjustments to progressively increase the sway envelope . the primary purpose of this study , therefore , was to determine whether a year - long training intervention with tjqmbb , given its specific focus on training limits of sway , could enhance limits of stability among community - dwelling older adults . it was hypothesized that tjqmbb would enhance indicators of limits of stability . in light of the functional significance of the limits of stability , a secondary purpose was to examine concomitant changes between limits of stability and physical performance resulting from the intervention . the study design involved a single - group pre- and post - intervention with repeated measures , conducted between may 2012 and january 2014 . participants in this study were a subset of community - dwelling older adults recruited for a large community - based dissemination project through promotions ( newsletters , flyers , sign - up sheets ) at local senior centers in lane county , or , usa . the research protocol was approved by the institutional review board of oregon research institute , and an informed consent was obtained from all participants . those who met the following criteria were eligible for participation : ( 1 ) 65 years of age or over ; ( 2 ) physically mobile ( objectively assessed by the ability to walk at least one block with or without an assistive device ) ; ( 3 ) showing no severe cognitive deficits , as defined by the mini - mental state evaluation ( 23);24 and ( 4 ) having medical clearance from a health care provider . individuals who responded to the promotions were subsequently contacted and scheduled for an in - office visit where the details of the study were explained . following a consent process , baseline data were collected at a research laboratory prior to the beginning of the tjqmbb program with follow - ups at 24 and 48 weeks . trained research staff collected demographic and study outcome data . the tjqmbb program , with the conceptual basis and technical detail fully described elsewhere,20 is a balance training modality based on applications of tai ji quan with a specific focus on training limits of stability and sensory integration . eligible participants participated in a full 60-minute tjqmbb class session taught by a trained instructor twice per week for 48 consecutive weeks . in terms of the training protocol , each session began with tai ji quan - based movement warm - up exercises ( 510 minutes ) , followed by teaching and practicing the core components ( described in the following section ) ( 4045 minutes ) , and ending with a simple set of breathing exercises ( 35 minutes ) . the training protocol involves a core set of adapted yang style22 tai ji quan - based forms with built - in practice variations and a subset of mini therapeutic movements aimed at stimulating and integrating musculoskeletal and sensory systems via performance of self - initiated tai ji quan movements . exercise sessions were structured from an easy - to - difficult learning - and - practice progression with an emphasis on within - session practice integration of forms , variations of the forms , and mini movements . both seated and standing exercises were implemented , with seated exercises emphasizing weight shift , trunk rotation , transitioning from sit - to - stand and stand - to - sit , and standing exercises focusing on the features described in the following section . during each session , participants practiced choreographed tai ji quan forms / movements ( eg , part wild horse s mane ) while engaging in controlled circular ankle sways ( ie , lateral , anterior posterior , medial lateral together ) that moved the center of gravity progressively toward and near the edge of the base of support with planted feet ; trunk - driven rotational ( laterally and posteriorly ) weight shift ; plantar / dorsiflexion during transitioning between moves ; reactive and proactive ( anticipatory ) postural adjustments in response to self - induced perturbed actions on either fixed support or change - in - support;25 and active and coordinated eye head hand movements . throughout the training and practice , emphasis was placed on ( 1 ) an interplay between stability ( static movements ) and instability ( self - induced movement perturbations ) in performing each form / move ; ( 2 ) functional ankle - based training by mimicking reaching , tilting , stepping , and turning tasks ; and ( 3 ) a variable approach that stressed practice variations in movement speeds , range of joint motion , movement directions and patterns , and base of foot support . the primary outcome was limits of stability , which consisted of three indicators : ( 1 ) endpoint excursion ; ( 2 ) movement velocity ; and ( 3 ) directional control , assessed by computerized dynamic posturography ( balance master system ; neurocom , clackamas , or , usa ) . endpoint excursion assesses the limits of self - initiated movements as participants shift or lean their center of gravity toward the theoretical limit ( 100% ) in eight prespecified target directions without loss of balance ( scores expressed as a percentage ; % los ) . movement velocity assesses the average speed of the center of gravity movement from the center to the eight targets ( scores expressed as degrees / second ) . directional control provides a measure of movement accuracy by comparing the amount of movement toward the target to the amount of extraneous movement ( scores expressed as % of 100 ) . reliability of limits of stability has been established26 or shown in previous studies.21,27 in this study , 24-week test retest reliability ( correlation ) ranged from 0.520.64 ( 0.57 for endpoint excursion , 0.52 for movement velocity , and 0.64 for directional control ) . secondary measures included physical performance measures of the : ( 1 ) timed up and go test28 and ( 2 ) 50-foot speed walk , adopted from the physical performance test.29 timed up and go measured the time ( in seconds ) taken to rise from a chair , walk 10 feet ( 3 m ) , return , and sit down , with a lower time indicating better mobility . the 50-foot speed walk measured the time ( in seconds ) it took participants to walk as quickly as possible 25 feet out and 25 feet back . the 6-month test retest reliability was 0.82 for timed up and go and 0.91 for the 50-foot walk test , respectively . intervention outcomes were analyzed on a sample size of 145 participants who provided at least two data points ( baseline , 24-week , and/or 48-week follow - up ) . descriptive statistics ( mean , standard deviation ) were performed to describe the study sample characteristics . coefficient of variation was calculated for each variable across time as the ratio of standard deviation to the mean , multiplied by 100 . minimal detectable change ( mdc ) scores were calculated for each of the limits of stability indicators to determine whether the change score was at or above the minimal level of detectible change . mdc is based on the standard error of measurement and calculated at a 95% confidence interval ( mdc95 ) using the formula of : standard error of measurementn1.96,(1 ) where the standard error of measurement is derived from the square root of the mean square error term in the repeated measures analysis of variance and n is the number of measurements used . intervention effect on the primary and secondary outcomes was analyzed via latent growth curve analysis . in the latent curve analysis , the primary interest was on the following estimates : ( 1 ) slope means on each of three limits of stability indicators and ( 2 ) a slope - to - slope correlation between each of the limits of stability indicators and the two physical performance measures . given the single - group intervention design , change at 48 weeks from baseline was estimated for the indicators of endpoint excursion and directional control using the pooled estimates from prior studies.21,27 assuming an alpha level of 0.05 with 80% power to detect 7% los points difference in endpoint excursion and 8% of 100 points in directional control with an anticipated dropout rate of 30% for the 48-week intervention , a minimal sample size of 70 participants for the tjqmbb was planned . all descriptive analyses including mdc scores were performed using spss version 17 ( spss inc . , chicago , il , usa ) and microsoft office excel software for windows ( microsoft corporation , redmond , wa , usa ) . changes in the latent means of the limits of stability indicators and correlations of changes between limits of stability and physical performance measures were performed using mplus software ( v 6 ; muthn & muthn , los angeles , ca , usa).30 the study design involved a single - group pre- and post - intervention with repeated measures , conducted between may 2012 and january 2014 . participants in this study were a subset of community - dwelling older adults recruited for a large community - based dissemination project through promotions ( newsletters , flyers , sign - up sheets ) at local senior centers in lane county , or , usa . the research protocol was approved by the institutional review board of oregon research institute , and an informed consent was obtained from all participants . those who met the following criteria were eligible for participation : ( 1 ) 65 years of age or over ; ( 2 ) physically mobile ( objectively assessed by the ability to walk at least one block with or without an assistive device ) ; ( 3 ) showing no severe cognitive deficits , as defined by the mini - mental state evaluation ( 23);24 and ( 4 ) having medical clearance from a health care provider . individuals who responded to the promotions were subsequently contacted and scheduled for an in - office visit where the details of the study were explained . following a consent process , baseline data were collected at a research laboratory prior to the beginning of the tjqmbb program with follow - ups at 24 and 48 weeks . trained research staff collected demographic and study outcome data . the tjqmbb program , with the conceptual basis and technical detail fully described elsewhere,20 is a balance training modality based on applications of tai ji quan with a specific focus on training limits of stability and sensory integration . eligible participants participated in a full 60-minute tjqmbb class session taught by a trained instructor twice per week for 48 consecutive weeks . in terms of the training protocol , each session began with tai ji quan - based movement warm - up exercises ( 510 minutes ) , followed by teaching and practicing the core components ( described in the following section ) ( 4045 minutes ) , and ending with a simple set of breathing exercises ( 35 minutes ) . the training protocol involves a core set of adapted yang style22 tai ji quan - based forms with built - in practice variations and a subset of mini therapeutic movements aimed at stimulating and integrating musculoskeletal and sensory systems via performance of self - initiated tai ji quan movements . exercise sessions were structured from an easy - to - difficult learning - and - practice progression with an emphasis on within - session practice integration of forms , variations of the forms , and mini movements . both seated and standing exercises were implemented , with seated exercises emphasizing weight shift , trunk rotation , transitioning from sit - to - stand and stand - to - sit , and standing exercises focusing on the features described in the following section . during each session , participants practiced choreographed tai ji quan forms / movements ( eg , part wild horse s mane ) while engaging in controlled circular ankle sways ( ie , lateral , anterior posterior , medial lateral together ) that moved the center of gravity progressively toward and near the edge of the base of support with planted feet ; trunk - driven rotational ( laterally and posteriorly ) weight shift ; plantar / dorsiflexion during transitioning between moves ; reactive and proactive ( anticipatory ) postural adjustments in response to self - induced perturbed actions on either fixed support or change - in - support;25 and active and coordinated eye head hand movements . throughout the training and practice , emphasis was placed on ( 1 ) an interplay between stability ( static movements ) and instability ( self - induced movement perturbations ) in performing each form / move ; ( 2 ) functional ankle - based training by mimicking reaching , tilting , stepping , and turning tasks ; and ( 3 ) a variable approach that stressed practice variations in movement speeds , range of joint motion , movement directions and patterns , and base of foot support . the primary outcome was limits of stability , which consisted of three indicators : ( 1 ) endpoint excursion ; ( 2 ) movement velocity ; and ( 3 ) directional control , assessed by computerized dynamic posturography ( balance master system ; neurocom , clackamas , or , usa ) . endpoint excursion assesses the limits of self - initiated movements as participants shift or lean their center of gravity toward the theoretical limit ( 100% ) in eight prespecified target directions without loss of balance ( scores expressed as a percentage ; % los ) . movement velocity assesses the average speed of the center of gravity movement from the center to the eight targets ( scores expressed as degrees / second ) . directional control provides a measure of movement accuracy by comparing the amount of movement toward the target to the amount of extraneous movement ( scores expressed as % of 100 ) . reliability of limits of stability has been established26 or shown in previous studies.21,27 in this study , 24-week test retest reliability ( correlation ) ranged from 0.520.64 ( 0.57 for endpoint excursion , 0.52 for movement velocity , and 0.64 for directional control ) . secondary measures included physical performance measures of the : ( 1 ) timed up and go test28 and ( 2 ) 50-foot speed walk , adopted from the physical performance test.29 timed up and go measured the time ( in seconds ) taken to rise from a chair , walk 10 feet ( 3 m ) , return , and sit down , with a lower time indicating better mobility . the 50-foot speed walk measured the time ( in seconds ) it took participants to walk as quickly as possible 25 feet out and 25 feet back . the 6-month test retest reliability was 0.82 for timed up and go and 0.91 for the 50-foot walk test , respectively . intervention outcomes were analyzed on a sample size of 145 participants who provided at least two data points ( baseline , 24-week , and/or 48-week follow - up ) . descriptive statistics ( mean , standard deviation ) were performed to describe the study sample characteristics . coefficient of variation was calculated for each variable across time as the ratio of standard deviation to the mean , multiplied by 100 . minimal detectable change ( mdc ) scores were calculated for each of the limits of stability indicators to determine whether the change score was at or above the minimal level of detectible change . mdc is based on the standard error of measurement and calculated at a 95% confidence interval ( mdc95 ) using the formula of : standard error of measurementn1.96,(1 ) where the standard error of measurement is derived from the square root of the mean square error term in the repeated measures analysis of variance and n is the number of measurements used . intervention effect on the primary and secondary outcomes was analyzed via latent growth curve analysis . in the latent curve analysis , the primary interest was on the following estimates : ( 1 ) slope means on each of three limits of stability indicators and ( 2 ) a slope - to - slope correlation between each of the limits of stability indicators and the two physical performance measures . given the single - group intervention design , change at 48 weeks from baseline was estimated for the indicators of endpoint excursion and directional control using the pooled estimates from prior studies.21,27 assuming an alpha level of 0.05 with 80% power to detect 7% los points difference in endpoint excursion and 8% of 100 points in directional control with an anticipated dropout rate of 30% for the 48-week intervention , a minimal sample size of 70 participants for the tjqmbb was planned . all descriptive analyses including mdc scores were performed using spss version 17 ( spss inc . , chicago , il , usa ) and microsoft office excel software for windows ( microsoft corporation , redmond , wa , usa ) . changes in the latent means of the limits of stability indicators and correlations of changes between limits of stability and physical performance measures were performed using mplus software ( v 6 ; muthn & muthn , los angeles , ca , usa).30 the major reasons for being determined ineligible were age ( n=13 ) , time conflict ( n=3 ) , or significant change in health ( n=2 ) . in addition , six eligible participants ( 4% ) were removed because they attended only one session and provided no follow - up data . the mean age at enrollment was approximately 75 years , 79% of the participants were women , and approximately 92% were caucasian . in all , 124 participants ( 85% ) reported having two or more chronic conditions , 70 ( 48% ) were taking two or more prescribed medications , and 73 ( 50% ) reported having fallen at least once in the previous 6 months , with about 25% of those having fallen two or more times . a total of 98 participants ( 68% ) reported having some level of fear of falling . of the total 145 participants , 21 ( 14% ) dropped out of the study during the 48-week intervention period , due primarily to reasons such as deterioration in medical conditions ( n=14 ) , change in health status ( n=5 ) , or relocation ( n=2 ) . there were , however , no significant differences in baseline demographics and functional characteristics between the participants who completed the intervention and those who dropped out of the study ( completers : n=124 versus dropouts : n=21 ) . the average number of class sessions attended per participant for the intervention ( a total of 96 sessions ) was 66 sessions ( range : 596 sessions ; median : 73 sessions ) for the whole sample ( n=145 , including dropouts [ n=21 ] ) compared to 73 sessions ( range : 2896 sessions ; median : 78 sessions ) for those who completed the 48-week intervention ( n=124 ) . the percentage attendance for the intervention was 69% ( median : 76% ) for the whole sample ( n=145 ) and 76% ( median : 81% ) for the completers ( n=124 ) . table 2 provides descriptive statistics ( mean , standard deviation ) for the three primary and two secondary outcome variables across the three measurement time points . the observed means for each of the three limits of stability show a linear improvement over time . change from baseline values were 17.21% los ( 32% ) for endpoint execution , 1.71 degrees / second ( 68% ) for movement velocity , and 12.41% of 100 ( 19% ) for directional control compared to mdc95 values , which were 16.88% los for endpoint excursion , 1.63 degrees / second for movement velocity , and 12.14% of 100 for directional control , respectively . results of the latent growth analysis are presented in table 3 , which further indicates that there was a significant rate of change ( as reflected in latent linear slope means ) in the indicators of endpoint excursion , movement velocity , and directional control over the 48-week intervention period . the analyses also show a significant small - to - moderate negative correlation ( indicating improvement ) between rate of change in each of the limits of stability indicators and the two physical performance measures over time ( table 3 ) . there were no significant adverse events attributable to the intervention observed during the intervention period . three falls were reported during warm - up sessions , but the participants sustained no injuries and continued the classes . the mean age at enrollment was approximately 75 years , 79% of the participants were women , and approximately 92% were caucasian . in all , 124 participants ( 85% ) reported having two or more chronic conditions , 70 ( 48% ) were taking two or more prescribed medications , and 73 ( 50% ) reported having fallen at least once in the previous 6 months , with about 25% of those having fallen two or more times . a total of 98 participants ( 68% ) reported having some level of fear of falling . of the total 145 participants , 21 ( 14% ) dropped out of the study during the 48-week intervention period , due primarily to reasons such as deterioration in medical conditions ( n=14 ) , change in health status ( n=5 ) , or relocation ( n=2 ) . there were , however , no significant differences in baseline demographics and functional characteristics between the participants who completed the intervention and those who dropped out of the study ( completers : n=124 versus dropouts : n=21 ) . the average number of class sessions attended per participant for the intervention ( a total of 96 sessions ) was 66 sessions ( range : 596 sessions ; median : 73 sessions ) for the whole sample ( n=145 , including dropouts [ n=21 ] ) compared to 73 sessions ( range : 2896 sessions ; median : 78 sessions ) for those who completed the 48-week intervention ( n=124 ) . the percentage attendance for the intervention was 69% ( median : 76% ) for the whole sample ( n=145 ) and 76% ( median : 81% ) for the completers ( n=124 ) . table 2 provides descriptive statistics ( mean , standard deviation ) for the three primary and two secondary outcome variables across the three measurement time points . the observed means for each of the three limits of stability show a linear improvement over time . change from baseline values were 17.21% los ( 32% ) for endpoint execution , 1.71 degrees / second ( 68% ) for movement velocity , and 12.41% of 100 ( 19% ) for directional control compared to mdc95 values , which were 16.88% los for endpoint excursion , 1.63 degrees / second for movement velocity , and 12.14% of 100 for directional control , respectively . results of the latent growth analysis are presented in table 3 , which further indicates that there was a significant rate of change ( as reflected in latent linear slope means ) in the indicators of endpoint excursion , movement velocity , and directional control over the 48-week intervention period . the analyses also show a significant small - to - moderate negative correlation ( indicating improvement ) between rate of change in each of the limits of stability indicators and the two physical performance measures over time ( table 3 ) . there were no significant adverse events attributable to the intervention observed during the intervention period . three falls were reported during warm - up sessions , but the participants sustained no injuries and continued the classes . this is the very first study to evaluate the effect of a specifically tailored tai ji quan training program on improving limits of stability , an important prerequisite for performing many of the functional activities of daily living in older adults . the results showed that 48 weeks of tjqmbb training significantly improved older adult limits of stability as reflected in increases in the indicators of movement excursion , velocity , and control , and that increased limits of stability were concomitantly associated with improved physical performance . thus , with no significant adverse events during the study period , it appears that the intervention is an appropriate one for this at - risk population . while cross - sectional studies that showed that older adult practitioners of tai ji quan tended to exhibit better stability limits compared to nonpractitioners31,32 provided early nonintervention data on the potential of tai ji quan to enhance limits of stability , more recent intervention studies from the author s group that integrated limits of sway training demonstrated the benefit of this focused tai ji quan program for improving limits of stability in older adults and patients with parkinson s disease.21,27 in contrast , other tai ji quan - based studies , ranging from 4 to 12 months of intervention,33,34 have not shown significant improvements in postural stability measures in older adults . the tjqmbb program differs from the programs in these other studies in a clinically important way in that this approach transforms traditional martial arts - based training into a therapeutically oriented training paradigm that specifically emphasizes the practice of deliberate , voluntarily controlled tai ji quan postural actions . these actions include manipulation of the center of gravity movements , variations in the base of support , and adaptation training with reactive and proactive actions to self - induced perturbations , with the purpose of increasing the sway envelope and thereby expanding the limits of stability , with the ultimate goal of reducing the risk of falling . although there are currently no guidelines for gauging clinically meaningful change in the measures of limits of stability , this 48-week , twice - per - week training schedule intervention appears to have provided a substantive change over time in the measures used in this study : 32% on endpoint excursion , 68% on movement velocity , and 19% on directional control . improvements in these limits of stability indicators support the potential for the tjqmbb program to address problems of improper weight shifts that are observed in elderly people residing in long - term care.35 consistent with findings from a cross - sectional study,5 the results demonstrate that improved measures on limits of stability through this tai ji quan training in older adults was sufficiently sensitive to elicit change in clinical measures of balance and mobility , suggesting that training with tjqmbb offers the potential to improve functional activities and activities of daily living , including sit - to - stand , walking , turning , and stand - to - sit tasks . the author s intervention approach linking martial - arts - based tai ji quan to clinical and functional training of balance in older adults offers some practical implications . from an applied perspective , the training approach used in this study can be readily introduced in various settings . for example , as an effective balance training modality , tjqmbb can be readily adopted in diverse communities for the purpose of preventing falls36,37 and in health care settings to assist patients with balance disorders and fall risk problems.21 uniquely different from all other existing tai ji quan - based programs , tjqmbb also has a specific , tailored clinical focus in that training incorporates therapeutic approaches such as closed and open kinetic chain movements;38 motor control mechanisms through manipulation of movement magnitude , speed , direction , and base of support ; and training of functional activities that take into account interactions between individuals , tasks at hand , and environmental constraints imposed on the tasks.23 these specific training features thus make the program amenable and applicable to clinical training and rehabilitation that address neuromuscular and movement disorders in patients with balance and gait problems and who are at high risk of falls . the mechanism by which the training protocol improves the limits of stability indicators in this study was not explored and therefore remains unclear . limits of stability is known to be influenced by a number of physiological and biomechanical factors,4,23 including lower extremity strength , range of ankle motion , and cutaneous mechanoreceptors in the feet,2 acting either alone or in combination . through integration of tai ji quan and physical therapy , the protocol in this study puts great emphasis on controlled weight shifting and ankle movement sway , alternation between a narrow stance and a wide stance to continually change the base of support , rotational trunk - driven movements , and dorsiflexion and plantar flexion during movement transitioning . these therapeutically tailored training features may have made an impact on important sensorimotor elements that contribute to enhanced limits of stability . therefore , it is plausible that improvement in the limits of stability performance may be attributed to unmeasured improvements in flexion / extension ankle torques , the range of ankle motion , movement recovery strategies , or sensory integration features that are inherent in tjqmbb training . some of these mechanisms of action may be revealed by evaluating the kinematics of body movement or conducting kinetic analysis of these ankle swing - based static and dynamic movement exercises , thus offering a better understanding of the movement strategies and forces contributing to the intervention - induced change in limits of stability . its design does not include a control condition , making it difficult to draw a firm conclusion regarding the true intervention effect of tjqmbb on limits of stability . therefore , the results should be corroborated by future clinical randomized controlled trials . given the importance of plantarflexor muscle strength in limits of stability,10 another limitation is the lack of a measure of ankle muscle strength , which precludes the opportunity to examine the role of ankle strength in the observed improved limits of stability outcomes . finally , the study used theoretical limits of stability values derived from computerized dynamic posturography that correspond to the maximum range in which center of gravity can be moved safely without changing the base of support.26 however , the capacity of stability limits may vary depending on the task performed;23 therefore , consideration of performance - based or functional limits of stability resembling real - world situations may be needed in future assessments.39 the tjqmbb program , a transformed protocol of traditional tai ji quan training approaches , was found to improve limits of stability in older adults , and the improved limits of stability were associated with improved functional performance . within the limitations of the current study , the combined results provide promising preliminary data that suggest the potential utility of tjqmbb for increasing limits of stability in older adults , thereby enhancing functional performance of habitual daily activities .

backgroundlimits of stability , defined as the ability to maintain the center of gravity within the boundary of the base of support , is critically important for older adults in performing their activities of daily living . however , few exercise programs specifically tailored to enhance limits of stability exist . the primary purpose of this study was to determine whether a therapeutically designed intervention , tai ji quan : moving for better balance ( tjqmbb ) , could improve limits of stability in older adults . a secondary purpose was to examine concomitant change in limits of stability and physical performance as a result of the intervention.methodsa single - group design was used in which 145 community - dwelling older adults ( average age : 75 years ) were enrolled in tjqmbb classes , participating twice weekly for 48 weeks . primary outcome measures were three indicators of limits of stability ( los ) ( endpoint excursion , movement velocity , and directional control ) , with secondary measures of physical performance being timed up and go and 50-foot speed walk ( in seconds ) , which were assessed at baseline , 24 weeks , and 48 weeks . changes in the repeated measures of outcome variables were analyzed via latent curve analysis.resultsat 48 weeks , a significant rate of change ( improvement ) over time was observed in the three limits of stability indicators ( endpoint execution : 8.30% los , p<0.001 ; movement velocity : 0.86 degrees / second , p<0.001 ; directional control : 6.79% of 100 , p<0.001 ) ; all reached a threshold of real change as judged by the minimal detectable change values . improvements in the three limits of stability measures were concomitantly correlated with improved ( reduced times ) performance scores in the timed up and go ( 0.30 , 0.45 , and 0.55 , respectively ) and 50-foot walk ( 0.33 , 0.49 , and 0.41 , respectively).conclusionin this single - group study , community - dwelling older adults trained through tjqmbb significantly improved their limits of stability , providing preliminary support for the use of tjqmbb as a therapeutic modality for enhancing functional activities in older adults .

Introduction Methods Study design Participants and procedures Intervention Outcome measures Data analysis Results Baseline characteristics Intervention attrition and adherence Change in primary and secondary outcomes Correlations in change between primary and secondary outcomes Intervention safety Discussion Conclusion

in older adults , performance of many habitual daily activities and tasks requires moving and controlling the center of gravity ( defined as the vertical projection of the center of mass ) to various positions within the base of support . however , effective displacement of the center of gravity during either self - initiated voluntary movements or externally triggered perturbations requires the capacity to remain within the limits of stability , which is described as the maximum distance a person can intentionally displace his or her center of gravity from a midline vertical position in any direction without losing balance and stepping or grasping for support.14 thus , a constricted limits of stability boundary or uncontrolled center of gravity movement can impair functional balance of older adults and constrain safe performance of their daily activities such as reaching , bending , walking , gait initiation , and moving from sit - to - stand.5 current literature suggests that aging is associated with diminished limits of stability capacity69 due to multiple factors , including reduced ankle muscle strength10 and poor cutaneous mechanoreceptor function in the soles of the feet , thus exposing older adults to more instability in functional daily activities and a higher risk of falling.11,12 therefore , from the perspectives of injury prevention and/or clinical rehabilitation of physical function , developing exercise intervention strategies that maximize the center of gravity sway envelope ( ie , the area an individual can move his or her center of gravity with control within the base of support)4 and subsequently expand the limits of stability boundary becomes functionally important for improving balance and mobility and , critically , for reducing the risk of falling among the elderly , which is a significant public health problem worldwide.13,14 because of its functional significance and implications for risk of falling , limits of stability should be integrated into exercise balance training programs that target fall prevention in older adults . given that constrained limits of stability can lead to an increased propensity to fall in older adults,1,19 fall prevention programs that explicitly integrate therapeutic balance training of limits of stability are needed . taking a therapeutic approach , li et al20,21 developed the tai ji quan : moving for better balance ( tjqmbb ) program that specifically includes an element of training on limits of stability . the primary purpose of this study , therefore , was to determine whether a year - long training intervention with tjqmbb , given its specific focus on training limits of sway , could enhance limits of stability among community - dwelling older adults . in light of the functional significance of the limits of stability , a secondary purpose was to examine concomitant changes between limits of stability and physical performance resulting from the intervention . participants in this study were a subset of community - dwelling older adults recruited for a large community - based dissemination project through promotions ( newsletters , flyers , sign - up sheets ) at local senior centers in lane county , or , usa . the tjqmbb program , with the conceptual basis and technical detail fully described elsewhere,20 is a balance training modality based on applications of tai ji quan with a specific focus on training limits of stability and sensory integration . in terms of the training protocol , each session began with tai ji quan - based movement warm - up exercises ( 510 minutes ) , followed by teaching and practicing the core components ( described in the following section ) ( 4045 minutes ) , and ending with a simple set of breathing exercises ( 35 minutes ) . during each session , participants practiced choreographed tai ji quan forms / movements ( eg , part wild horse s mane ) while engaging in controlled circular ankle sways ( ie , lateral , anterior posterior , medial lateral together ) that moved the center of gravity progressively toward and near the edge of the base of support with planted feet ; trunk - driven rotational ( laterally and posteriorly ) weight shift ; plantar / dorsiflexion during transitioning between moves ; reactive and proactive ( anticipatory ) postural adjustments in response to self - induced perturbed actions on either fixed support or change - in - support;25 and active and coordinated eye head hand movements . throughout the training and practice , emphasis was placed on ( 1 ) an interplay between stability ( static movements ) and instability ( self - induced movement perturbations ) in performing each form / move ; ( 2 ) functional ankle - based training by mimicking reaching , tilting , stepping , and turning tasks ; and ( 3 ) a variable approach that stressed practice variations in movement speeds , range of joint motion , movement directions and patterns , and base of foot support . the primary outcome was limits of stability , which consisted of three indicators : ( 1 ) endpoint excursion ; ( 2 ) movement velocity ; and ( 3 ) directional control , assessed by computerized dynamic posturography ( balance master system ; neurocom , clackamas , or , usa ) . endpoint excursion assesses the limits of self - initiated movements as participants shift or lean their center of gravity toward the theoretical limit ( 100% ) in eight prespecified target directions without loss of balance ( scores expressed as a percentage ; % los ) . movement velocity assesses the average speed of the center of gravity movement from the center to the eight targets ( scores expressed as degrees / second ) . reliability of limits of stability has been established26 or shown in previous studies.21,27 in this study , 24-week test retest reliability ( correlation ) ranged from 0.520.64 ( 0.57 for endpoint excursion , 0.52 for movement velocity , and 0.64 for directional control ) . secondary measures included physical performance measures of the : ( 1 ) timed up and go test28 and ( 2 ) 50-foot speed walk , adopted from the physical performance test.29 timed up and go measured the time ( in seconds ) taken to rise from a chair , walk 10 feet ( 3 m ) , return , and sit down , with a lower time indicating better mobility . the 50-foot speed walk measured the time ( in seconds ) it took participants to walk as quickly as possible 25 feet out and 25 feet back . the 6-month test retest reliability was 0.82 for timed up and go and 0.91 for the 50-foot walk test , respectively . minimal detectable change ( mdc ) scores were calculated for each of the limits of stability indicators to determine whether the change score was at or above the minimal level of detectible change . in the latent curve analysis , the primary interest was on the following estimates : ( 1 ) slope means on each of three limits of stability indicators and ( 2 ) a slope - to - slope correlation between each of the limits of stability indicators and the two physical performance measures . given the single - group intervention design , change at 48 weeks from baseline was estimated for the indicators of endpoint excursion and directional control using the pooled estimates from prior studies.21,27 assuming an alpha level of 0.05 with 80% power to detect 7% los points difference in endpoint excursion and 8% of 100 points in directional control with an anticipated dropout rate of 30% for the 48-week intervention , a minimal sample size of 70 participants for the tjqmbb was planned . changes in the latent means of the limits of stability indicators and correlations of changes between limits of stability and physical performance measures were performed using mplus software ( v 6 ; muthn & muthn , los angeles , ca , usa).30 the study design involved a single - group pre- and post - intervention with repeated measures , conducted between may 2012 and january 2014 . participants in this study were a subset of community - dwelling older adults recruited for a large community - based dissemination project through promotions ( newsletters , flyers , sign - up sheets ) at local senior centers in lane county , or , usa . the tjqmbb program , with the conceptual basis and technical detail fully described elsewhere,20 is a balance training modality based on applications of tai ji quan with a specific focus on training limits of stability and sensory integration . in terms of the training protocol , each session began with tai ji quan - based movement warm - up exercises ( 510 minutes ) , followed by teaching and practicing the core components ( described in the following section ) ( 4045 minutes ) , and ending with a simple set of breathing exercises ( 35 minutes ) . during each session , participants practiced choreographed tai ji quan forms / movements ( eg , part wild horse s mane ) while engaging in controlled circular ankle sways ( ie , lateral , anterior posterior , medial lateral together ) that moved the center of gravity progressively toward and near the edge of the base of support with planted feet ; trunk - driven rotational ( laterally and posteriorly ) weight shift ; plantar / dorsiflexion during transitioning between moves ; reactive and proactive ( anticipatory ) postural adjustments in response to self - induced perturbed actions on either fixed support or change - in - support;25 and active and coordinated eye head hand movements . throughout the training and practice , emphasis was placed on ( 1 ) an interplay between stability ( static movements ) and instability ( self - induced movement perturbations ) in performing each form / move ; ( 2 ) functional ankle - based training by mimicking reaching , tilting , stepping , and turning tasks ; and ( 3 ) a variable approach that stressed practice variations in movement speeds , range of joint motion , movement directions and patterns , and base of foot support . the primary outcome was limits of stability , which consisted of three indicators : ( 1 ) endpoint excursion ; ( 2 ) movement velocity ; and ( 3 ) directional control , assessed by computerized dynamic posturography ( balance master system ; neurocom , clackamas , or , usa ) . endpoint excursion assesses the limits of self - initiated movements as participants shift or lean their center of gravity toward the theoretical limit ( 100% ) in eight prespecified target directions without loss of balance ( scores expressed as a percentage ; % los ) . movement velocity assesses the average speed of the center of gravity movement from the center to the eight targets ( scores expressed as degrees / second ) . reliability of limits of stability has been established26 or shown in previous studies.21,27 in this study , 24-week test retest reliability ( correlation ) ranged from 0.520.64 ( 0.57 for endpoint excursion , 0.52 for movement velocity , and 0.64 for directional control ) . secondary measures included physical performance measures of the : ( 1 ) timed up and go test28 and ( 2 ) 50-foot speed walk , adopted from the physical performance test.29 timed up and go measured the time ( in seconds ) taken to rise from a chair , walk 10 feet ( 3 m ) , return , and sit down , with a lower time indicating better mobility . the 50-foot speed walk measured the time ( in seconds ) it took participants to walk as quickly as possible 25 feet out and 25 feet back . the 6-month test retest reliability was 0.82 for timed up and go and 0.91 for the 50-foot walk test , respectively . minimal detectable change ( mdc ) scores were calculated for each of the limits of stability indicators to determine whether the change score was at or above the minimal level of detectible change . in the latent curve analysis , the primary interest was on the following estimates : ( 1 ) slope means on each of three limits of stability indicators and ( 2 ) a slope - to - slope correlation between each of the limits of stability indicators and the two physical performance measures . given the single - group intervention design , change at 48 weeks from baseline was estimated for the indicators of endpoint excursion and directional control using the pooled estimates from prior studies.21,27 assuming an alpha level of 0.05 with 80% power to detect 7% los points difference in endpoint excursion and 8% of 100 points in directional control with an anticipated dropout rate of 30% for the 48-week intervention , a minimal sample size of 70 participants for the tjqmbb was planned . changes in the latent means of the limits of stability indicators and correlations of changes between limits of stability and physical performance measures were performed using mplus software ( v 6 ; muthn & muthn , los angeles , ca , usa).30 the major reasons for being determined ineligible were age ( n=13 ) , time conflict ( n=3 ) , or significant change in health ( n=2 ) . the mean age at enrollment was approximately 75 years , 79% of the participants were women , and approximately 92% were caucasian . the observed means for each of the three limits of stability show a linear improvement over time . change from baseline values were 17.21% los ( 32% ) for endpoint execution , 1.71 degrees / second ( 68% ) for movement velocity , and 12.41% of 100 ( 19% ) for directional control compared to mdc95 values , which were 16.88% los for endpoint excursion , 1.63 degrees / second for movement velocity , and 12.14% of 100 for directional control , respectively . results of the latent growth analysis are presented in table 3 , which further indicates that there was a significant rate of change ( as reflected in latent linear slope means ) in the indicators of endpoint excursion , movement velocity , and directional control over the 48-week intervention period . the analyses also show a significant small - to - moderate negative correlation ( indicating improvement ) between rate of change in each of the limits of stability indicators and the two physical performance measures over time ( table 3 ) . the observed means for each of the three limits of stability show a linear improvement over time . change from baseline values were 17.21% los ( 32% ) for endpoint execution , 1.71 degrees / second ( 68% ) for movement velocity , and 12.41% of 100 ( 19% ) for directional control compared to mdc95 values , which were 16.88% los for endpoint excursion , 1.63 degrees / second for movement velocity , and 12.14% of 100 for directional control , respectively . results of the latent growth analysis are presented in table 3 , which further indicates that there was a significant rate of change ( as reflected in latent linear slope means ) in the indicators of endpoint excursion , movement velocity , and directional control over the 48-week intervention period . the analyses also show a significant small - to - moderate negative correlation ( indicating improvement ) between rate of change in each of the limits of stability indicators and the two physical performance measures over time ( table 3 ) . this is the very first study to evaluate the effect of a specifically tailored tai ji quan training program on improving limits of stability , an important prerequisite for performing many of the functional activities of daily living in older adults . the results showed that 48 weeks of tjqmbb training significantly improved older adult limits of stability as reflected in increases in the indicators of movement excursion , velocity , and control , and that increased limits of stability were concomitantly associated with improved physical performance . while cross - sectional studies that showed that older adult practitioners of tai ji quan tended to exhibit better stability limits compared to nonpractitioners31,32 provided early nonintervention data on the potential of tai ji quan to enhance limits of stability , more recent intervention studies from the author s group that integrated limits of sway training demonstrated the benefit of this focused tai ji quan program for improving limits of stability in older adults and patients with parkinson s disease.21,27 in contrast , other tai ji quan - based studies , ranging from 4 to 12 months of intervention,33,34 have not shown significant improvements in postural stability measures in older adults . these actions include manipulation of the center of gravity movements , variations in the base of support , and adaptation training with reactive and proactive actions to self - induced perturbations , with the purpose of increasing the sway envelope and thereby expanding the limits of stability , with the ultimate goal of reducing the risk of falling . although there are currently no guidelines for gauging clinically meaningful change in the measures of limits of stability , this 48-week , twice - per - week training schedule intervention appears to have provided a substantive change over time in the measures used in this study : 32% on endpoint excursion , 68% on movement velocity , and 19% on directional control . improvements in these limits of stability indicators support the potential for the tjqmbb program to address problems of improper weight shifts that are observed in elderly people residing in long - term care.35 consistent with findings from a cross - sectional study,5 the results demonstrate that improved measures on limits of stability through this tai ji quan training in older adults was sufficiently sensitive to elicit change in clinical measures of balance and mobility , suggesting that training with tjqmbb offers the potential to improve functional activities and activities of daily living , including sit - to - stand , walking , turning , and stand - to - sit tasks . for example , as an effective balance training modality , tjqmbb can be readily adopted in diverse communities for the purpose of preventing falls36,37 and in health care settings to assist patients with balance disorders and fall risk problems.21 uniquely different from all other existing tai ji quan - based programs , tjqmbb also has a specific , tailored clinical focus in that training incorporates therapeutic approaches such as closed and open kinetic chain movements;38 motor control mechanisms through manipulation of movement magnitude , speed , direction , and base of support ; and training of functional activities that take into account interactions between individuals , tasks at hand , and environmental constraints imposed on the tasks.23 these specific training features thus make the program amenable and applicable to clinical training and rehabilitation that address neuromuscular and movement disorders in patients with balance and gait problems and who are at high risk of falls . the mechanism by which the training protocol improves the limits of stability indicators in this study was not explored and therefore remains unclear . through integration of tai ji quan and physical therapy , the protocol in this study puts great emphasis on controlled weight shifting and ankle movement sway , alternation between a narrow stance and a wide stance to continually change the base of support , rotational trunk - driven movements , and dorsiflexion and plantar flexion during movement transitioning . therefore , it is plausible that improvement in the limits of stability performance may be attributed to unmeasured improvements in flexion / extension ankle torques , the range of ankle motion , movement recovery strategies , or sensory integration features that are inherent in tjqmbb training . some of these mechanisms of action may be revealed by evaluating the kinematics of body movement or conducting kinetic analysis of these ankle swing - based static and dynamic movement exercises , thus offering a better understanding of the movement strategies and forces contributing to the intervention - induced change in limits of stability . given the importance of plantarflexor muscle strength in limits of stability,10 another limitation is the lack of a measure of ankle muscle strength , which precludes the opportunity to examine the role of ankle strength in the observed improved limits of stability outcomes . finally , the study used theoretical limits of stability values derived from computerized dynamic posturography that correspond to the maximum range in which center of gravity can be moved safely without changing the base of support.26 however , the capacity of stability limits may vary depending on the task performed;23 therefore , consideration of performance - based or functional limits of stability resembling real - world situations may be needed in future assessments.39 the tjqmbb program , a transformed protocol of traditional tai ji quan training approaches , was found to improve limits of stability in older adults , and the improved limits of stability were associated with improved functional performance . within the limitations of the current study , the combined results provide promising preliminary data that suggest the potential utility of tjqmbb for increasing limits of stability in older adults , thereby enhancing functional performance of habitual daily activities .

in a previous study , we established that 11(r)-hete was a significant cox-2-derived aa metabolite in epithelial cells.(1 ) however , the consequences of this finding were not clear at that time . a number of toxic substances including arsenite,(2 ) dioxin,(3 ) benzo[a]pyrene diol epoxide,(4 ) and cigarette smoke(5 ) upregulate cox-2 expression , which in turn regulates numerous intracellular biochemical pathways . this occurs primarily through the biosynthesis of cox-2-derived aa metabolites , which can exert cell - specific effects on inflammation , cell growth , and proliferation . for example cox-2-derived prostaglandin ( pg ) e2 increases tumor proliferation through multiple mechanisms including activation of plasma membrane g - protein - coupled receptors and the nuclear peroxisome proliferator - activated receptor ( ppar).(6 ) steady - state levels of pge2 are maintained by pge - synthase - mediated biosynthesis from cox-2-derived pgh2 and catabolism by 15-pgdh - mediated inactivation to 15-oxo - pge2 ( figure 1 ) . the 15-oxo - pge2 is then converted to 13,14-dihydro-15-oxo - pge2 by 15-oxoprostaglandin-(13 ) reductase.(9 ) loss of 15-pgdh expression is associated with tumor formation in bladder , breast , colon , intestine , kidney , lung , pancreas , stomach , and skin cancer . thus , upregulation of cox-2(6 ) and downregulation of 15-pgdh(10 ) provides a switch toward endogenous mediators that are significant contributors to cancer progression.(15 ) formation and action of cox-2-derived eicosanoids in epithelial cell models . aa is released from membrane phospholipids by calcium - dependent cytosolic phospholipase a2 ( cpla2 ) . the released aa undergoes cox-2-mediated metabolism to pgs or forms the lipid hydroperoxides , 15(s)-hydroperoxyeicosatetraenoic acid ( hpete ) , 15(r)-hpete and 11(r)-hpete , which are reduced to the corresponding hetes . both 15-oxo - pgd2 and 15-oxo - pge2 are converted to 13,14-dihydro-5-oxo - pg metabolites . intact pgd2 secreted by the epithelial cells can undergo albumin - mediated dehydration to 15d - pgj2 . pge2 secreted from the epithelial cells by the abcc4 transporter is proproliferative for tumor cells . reuptake of pge2 by oatp2a1 into the epithelial cells leads to further 15-pgdh - mediated inactivation . in contrast to pge2 and pgd2 , 15(s)-hete and 11(r)-hete are activated by 15-pgdh - mediated oxidation to 15-oxo - ete and 11-oxo - ete , respectively . . secreted 15- and 11-oxo - ete that escape further metabolism can then inhibit endothelial cell proliferation . therefore , downregulation of 15-pgdh and oatp2a1 would result in increased pge2-mediated tumor and endothelial cell proliferation . pgd2 , another cox-2-derived metabolite , is also metabolized by 15-pgdh to form 15-oxo - pgd2 , which is then converted to the corresponding inactive 13,14-dihydro derivative ( figure 1).(16 ) alternatively , pgd2 undergoes albumin - mediated dehydration to give pgj2 , followed by a further dehydration to give 15-deoxy--prostaglandin j2 ( 15d - pgj2 ) ( figure 2).(17 ) previous studies have shown that 15d - pgj2 is a ppar agonist,(18 ) which inhibits huvec proliferation in culture.(19 ) in addition , 15d - pgj2 can induce caspase - mediated endothelial cell apoptosis(20 ) and inhibit the nuclear factor b ( nfb ) pathway . it can also increase levels of p53 in huvecs , activate p53 phosphorylation , and induce p21.(23 ) chemical structures of cox-2-derived eicosanoids . studies of purified cox enzymes have shown that 11(r)-hete , 15(s)-hydroxy-5,8,11,13-(z , z , z , e)-eicosatetraenoic acid ( 15(s)-hete ) , and 15(r)-hete are the major hetes that are formed.(24 ) the hetes arise from reduction of the corresponding hydroperoxyeicosatetraenoic acids ( hpetes ) primarily through the peroxidase ( pox ) activity of coxs ( figure 1).(25 ) 11(r)-hete was a significant eicosanoid secreted by aa - treated rat intestinal epithelial cells that stably express cox-2 ( ries cells ) , but it was rapidly metabolized.(1 ) 15(s)-hete was formed in lower abundance and metabolized to 15-oxo - ete , as expected from its 15(s)-configuration and the substrate specificity of 15-pgdh . 15-oxo - ete was also found to inhibit endothelial cell proliferation , although at concentrations higher than 15d - pgj2.(26 ) however , there is little evidence that 15d - pgj2 can be formed in vivo at concentrations commensurate with an endogenous antiproliferative role.(27 ) in our earlier study,(1 ) the metabolic fate of 11(r)-hete secreted by the ries cells was not established . 11-oxo - ete has now been synthesized , and a lc - selected reaction monitoring ( srm)/ms method for its analysis has been developed . this has made it possible to determine whether 11-oxo - ete is secreted by human epithelial cell lines with upregulated cox-2 expression and to identify the dehydrogenase responsible . aa ( peroxide - free ) , 11(r , s)-hete , 15(r , s)-hete , [ h8]-15(s)-hete , [ c20]-15-oxo - ete , 15-oxo - ete , pge2 , [ h4]-pge2 , 13,14-dihydro-15-keto - pge2 , [ h4]-13,14-dihydro-15-keto - pge2 , 15d - pgj2 , cay10397 , protease inhibitor cocktail and recombinant human 15-pgdh were purchased from cayman ( ann arbor , mi ) . the dess martin reagent [ 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1h)-one ] , 2,3,4,5,6-pentafluorobenzyl ( pfb ) bromide , trizma - hcl , lipoxidase from glycine max ( soybean ) , sodium borohydride , and nad were purchased from sigma - aldrich ( st . louis , mo ) . fetal bovine serum ( fbs ) was from gemini bioproducts ( west sacramento , ca ) . f12k medium , dmem medium , medium 200 ( m200 ) , d - glucose , l - glutamine , low - serum growth supplement ( lsgs ) kit , penicillin , and streptomycin were supplied by invitrogen ( carlsbad , ca ) . lc ms grade water , hexane , methanol , isopropanol , acetonitrile and dichloromethane were obtained from fisher scientific ( pittsburgh , pa ) . human colorectal adenocarcinoma lovo cells ( atcc , manassas , va ) were cultured in f12k medium supplemented with 10% fbs , 2 mm l - glutamine , 100,000 units / l penicillin and 100 mg / l streptomycin . louis , mo ) were grown in dmem supplemented with 10% fbs , 2 mm l - glutamine , 110 mg / l sodium pyruvate , 100,000 units / l penicillin and 100 mg / l streptomycin . for lipidomics analysis , the culture medium was replaced with serum - free f12k or dmem medium before the treatment . huvecs were obtained from invitrogen ( carlsbad , ca ) and cultured on collagen i - coated tissue culture dishes in medium 200 supplemented with lsgs kit . cell proliferation assays were performed using huvecs from passage 4 . a triple stage quadrupole ( tsq quantum ) mass spectrometer ( thermo electron , san jose , ca ) targeted chiral lc - ecapci / srm / ms analysis was conducted using pfb derivatives of 7 lipids and 4 heavy isotope analogue internal standards . for the lipidomics profile , the instrument was operated in the negative ion mode , and unit resolution was maintained for both precursor and fragment ions . operating conditions for the tsq quantum were as follows : vaporizer temperature at 450 c ; heated capillary temperature at 250 c with the corona discharge needle set at 30 a ; nitrogen as sheath ( 25 psi ) and auxiliary ( 5 arbitrary units ) gas . collision - induced dissociation ( cid ) was performed using argon as the collision gas at 2.7 mtorr in the rf - only quadrupole . the following srm transitions were used : 11-oxo - ete - pfb , m / z 317 165 ( collision energy ( ce ) , 25 ev ) ; 15-oxo - ete - pfb , m / z 317 113 ( ce , 18 ev ) ; [ c20]-15-oxo - ete - pfb , m / z 337 120 ( ce , 18 ev ) ; 11(r)-hete - pfb , m / z 319 167 ( ce , 16 ev ) ; [ h8]-15(s)-hete - pfb , m / z 327 226 ( ce , 13 ev ) ; pge2-pfb , m / z 351 271 ( ce , 18 ev ) ; [ h4]-pge2-pfb , m / z 355 275 ( ce , 18 ev ) ; 13,14-dihydro-15-keto - pge2-pfb , m / z 351 235 ( ce , 22 ev ) ; [ h4]-13,14-dihydro-15-keto - pge2-pfb , m / z 355 239 ( ce , 22 ev ) . for gsh adduct analysis , the mass spectrometer was operated in the positive ion mode with an electrospray ionization ( esi ) source . the operating conditions were as follows : spray voltage at 4 kv ; capillary temperature at 350 c ; nitrogen as sheath ( 35 psi ) and auxiliary ( 13 arbitrary units ) gas . cid was performed using argon as the collision gas at 2.7 mtorr in the rf - only quadrupole . the following srm transition ( m / z 626 497 ) was monitored for 11-oxo - ete - gsh ( ce , 18 ev ) . a chiralpak ad - h column ( 250 4.6 mm inner diameter , 5 m ; daicel ) was employed for normal phase separation ( flow rate 1 ml / min ) of pfb derivatives of eicosanoids . gradient 1 was used for separating pfb - derivatives of hetes and pge2 , whereas gradient 2 was used for pfb derivatives of oxo - etes . for gradient 1 , solvent a was hexane , and solvent b was methanol / isopropanol ( 1:1 ; v / v ) . gradient 1 was as follows : 2% b at 0 min , 2% b at 3 min , 3.6% b at 11 min , 8% b at 15 min , 8% b at 27 min , 50% b at 30 min , 50% at 35 min , and 2% b at 37 min . solvent a was hexane , and solvent b was isopropanol / hexane ( 6:4 ; v / v ) . gradient 2 was as follows : 2% b at 0 min , 2% b at 14.5 min , 12% b at 15 min , 23% b at 19 min , 90% b at 19.5 min , 90% b at 23.5 min , and 2% b at 24 min . a chiralpak ad - rh column ( 150 4.6 mm inner diameter , 5 m ; daicel ) was used for reversed phase ( isocratic method 1 , flow rate 0.5 ml / min ) separation of the underivatized 11-oxo - ete . the mobile phase for isocratic separations was methanol / water / formic acid ( 95:5:0.1 ; v / v ) . chemically synthesized 11-oxo - ete was purified by normal - phase ( isocratic method 2 ) preparative lc ( ultrasphere 250 10 mm , inner diameter , 5 m ; beckman ) using waters alliance 2690 hplc system by monitoring the uv absorbance at 236 nm . the mobile phase for the isocratic method 2 ( flow rate 2.5 ml / min ) was hexane / isopropanol / acetic acid ( 98.5:1.5:0.1 ; v / v ) . gsh adducts were separated by reversed phase using gradient 3 on waters alliance 2690 hplc system . the separation employed a phenomenex synergi hydro - rp column ( 150 4.6 mm inner diameters , 5 m ) . solvent a was 0.1% aqueous formic acid , and solvent b was methanol / acetonitrile ( 50:50 ; v / v ) . gradient 3 was as follows : 2% b at 0 min , 2% b at 14 min , 30% b at 20 min , 42% b at 21 min , 65% b at 27 min , 80% b at 29 min , 90% b at 33 min , 90% b at 34 min , 2% b at 35 min . eicosanoids were dissolved in 100 l of acetonitrile and then reacted with 100 l of pfb bromide in acetonitrile ( 1:19 ; v / v ) and 100 l of diisopropylethylamine in acetonitrile ( 1:9 ; v / v ) at room temperature for 30 min . the derivatives were evaporated to dryness , dissolved in 100 l of hexane / ethanol ( 95:5 ; v / v ) and analyzed by chiral lc - ecapci / srm / ms . various concentrations of 11(r)-hete ( 0 , 2.3 m , 4.6 m , 6.9 m , 9.2 m and 23 m ) were incubated with 9 nm recombinant human 15-pgdh ( 52.2 ng , 1.8 pmol ) and cofactor nad ( 400 m ) in 50 mm tris - cl ( ph 7.9 ) for 3.5 min at 37 c . each total reaction volume was 200 l . after a 3.5 min incubation , the enzymatic reaction was quenched with 400 l of ice cold methanol and [ c20]-15-oxo - ete ( 8 ng ) added as the internal standard . eicosanoids were extracted with 1.2 ml of dichloromethane / methanol ( 8:1 ; v / v ) . the lower organic layer was then evaporated to dryness under nitrogen and reconstituted in methanol ( 100 l ) . an aliquot ( 25 l ) was separated using the isocratic method 1 and analyzed by lc in separate experiments , the formation of 11-oxo - ete was found to be linear for the first 5 min . eicosanoids were quantified by interpolation from a standard curve prepared with 11-oxo - ete using [ c20]-15-oxo - ete as the internal standard . martin reagent ( 2 mg , 4.7 mol ) was added to a solution of 11(r , s)-hete ( 0.5 mg , 1.6 mol ) in dichloromethane ( 1.0 ml ) and stirred for 2 h at room temperature . ms using gradient 1 as described above , after pfb derivatization until there was no starting material left . the reaction mixture was centrifuged twice at 3,400 rpm ( 10 min ) , and the supernatant was evaporated . the residue was dissolved in the mobile phase ( 800 l ) and purified by isocratic method 2 as described above . high resolution accurate mass measurements were obtained using electrospray ionization on a thermo ltq - ft mass spectrometer at a resolution of 100,000 ( data not shown ) . lovo or hca-7 cells were grown to 90% confluence , washed with 10 ml of phosphate - buffered saline ( pbs ) buffer ( 2 times ) , and then gently scraped in 600 l of lysis buffer containing 0.1 m tris - hcl ( ph 7.9 ) and the protease inhibitor . cell suspension was transferred to 2 ml eppendorf tubes and sonicated for 60 s on ice ( power 5 ) . cell lysate was then incubated with or without the selective 15-pgdh inhibitor ( cay10397 , 50 m ) and its cofactor ( nad , 500 m ) for 10 min at 37 c . the ph was then adjusted to 4 with 10% aqueous acetic acid ( 10 l ) followed by addition of the internal standard mix , [ c20]-15-oxo - ete , [ h8]-15(s)-hete and [ h4]-pge2 ( 50 pg/l , 20 l ) . diethyl ether ( 600 l ) was added , and samples were vortex - mixed and centrifuged ( 15000 rpm 2 min ) . the organic layer was evaporated under nitrogen , and then the eicosanoids were derivatized with pfb bromide as mentioned above . v / v , 100 l ) and analyzed ( 20 l ) by normal phase lc - ecapci / ms . the amounts of eicosanoids were normalized by protein concentrations of each lysate , which were determined by bca assay . lovo or hca-7 cells were grown to 90% confluence in 6-well plates as described above , and then fed fresh serum - free f-12k or dmem medium . cells were then incubated with aa ( 10 m ) for 0 , 5 , 10 , and 30 min , 1 and 2.5 h at 37 c . at each time point , 0.6 ml of medium was taken out , and 20 l of 10% aqueous acetic acid was added to adjust ph to 34 , together with 20 l of internal standards mixture ( 50 pg/l [ c20]-15-oxo - ete , [ h8]-15(s)-hete , [ h4]-pge2 and [ h4]-13,14-dihydro-15-keto - pge2-pfb ) . then diethyl ether ( 1 ml ) was added , and the mixture was vortex - mixed and centrifuged ( 15000 rpm 2 min ) . the upper ether layer was evaporated under nitrogen , and pfb derivatives were synthesized as described above and analyzed by normal phase lc - ecapci / ms . to quantify eicosanoids excreted in the medium , eppendorf tubes containing 0.6 ml of f12k medium were spiked with lipid standards , together with internal standards for [ c20]-15-oxo - ete , [ h8]-15(s)-hete , [ h4]-pge2 and [ h4]-13,14-dihydro-15-keto - pge2-pfb ( 1 ng each ) . to quantify eicosanoids in the cell lysate , lipid standards and internal standards mixture were spiked into 0.2 ml of tris - hcl buffer . lovo cells were grown to 90% confluence and then washed with pbs ( 10 ml ) . cells were gently scraped in 600 l of tris - hcl buffer ( 0.1 m , ph = 7.9 ) , containing protease inhibitor . cell lysates were transferred to 2 ml eppendorf tubes and sonicated for 60 s on ice ( power 5 ) . 11-oxo - ete ( 20 ng in ethanol ) was added to the lysate together with 1 mm gsh . after incubation for 25 min at 37 c , 10 l of 10% acetic acid was added , and the sample was loaded onto an spe column ( oasis hlb , 30 mg ) preconditioned with methanol and then 0.1% formic acid . the column was washed with 1 ml of water and eluted with 250 l of methanol , and then 20 l was analyzed by reversed phase lc brdu incorporation in huvecs was used to assess the effects of 11-oxo - ete on cell proliferation . the brdu assay was performed in a 96-well format using a commercially available colorimetric enzyme - linked immunosorbent assay ( elisa ) kit ( roche ) , and also by immunofluorescence microscopy . equal numbers of huvecs in passage 4 were plated on either collagen - i coated 96-well plates ( 2000 cells / well ) or collagen - i coated 8-chamber tissue culture glass slides ( 10000 cells / chamber ) . eicosanoids were dissolved in dmso , such that the final concentration of dmso in cell medium was always 0.25% or lower . cells were then treated for 24 h with either vehicle ( 0.25% dmso ) , 11-oxo - ete ( 1 nm to 100 m ) , or 15d - pgj2 ( 1 nm to 100 m ) . after 18 h of treatment , brdu ( final concentration , 10 m in 0.25% dmso ) was added to each treatment group for an additional 6 h. for the colorimetric elisa , the manufacturer s protocol was followed to perform the assay . the absorbance at = 370 nm obtained from the assay was transformed to the cell numbers using a standard curve constructed by plating known number of huvecs in triplicate . the ic50 values for eicosanoid inhibition of huvec proliferation were defined as the half maximal inhibitory concentration for endothelial cell proliferation over 24 h when compared with vehicle - treated cells . they were determined from the regression lines of the log inhibitor vs response curves using a least - squares fit . for the immunofluorescence staining , cells were fixed with neutral buffered formalin for 10 min , permeabilized with methanol for 20 min , and then dna was denatured by pressure cooking the slides in 10 mm citric acid buffer , ph 6 for 1 h. cells were then incubated overnight with rat anti - brdu antibody ( 1:1000 , accurate chemical & scientific corp . ) at 4 c , followed by 30 min incubation at 37 c with cy3-conjugated donkey anti - rat secondary antibody ( 1:600 , jackson immuno research ) . cells were counterstained with 4,6-diamidino-2-phenylindole ( dapi , invitrogen ) and visualized using a nikon e600 microscope equipped with differential interference contrast ( nomarski ) optics and photographed ( 200 magnification ) with a fast 1394 qicam ( qimaging ) . positive brdu staining was quantified by image analysis using ivision analysis software ( biovision ) . the percentage of proliferating cells was determined by counting the brdu - positive cells versus the total number of cells in randomly selected microscopic fields ( 10/replicate ) for each treatment group . all experiments were conducted in triplicate , unless otherwise indicated . statistical significance ( p value 0.05 ) was determined using a two - tailed unpaired t test employing graphpad prism software ( v 5.01 ) . various concentrations of 11(r)-hete were incubated with recombinant human 15-pgdh and nad at 37 c . a michaelis menten kinetic analysis of 11-oxo - ete formation provided the vmax ( 296 nmol / min / mg of protein ) , km ( 3.42 m ) , and kcat ( 8.6 min ) values for oxidation of 11(r)-hete ( figure 3 ) and the vmax ( 403.8 nmol / min / mg of protein , km ( 1.65 m ) , and kcat ( 8.6 min ) values for oxidation of 15(s)-hete ( supplementary figure 1 in the supporting information ) . therefore , the catalytic efficiency ( kcat / km ) for 15-pgdh - mediated oxidation of 11(r)-hete ( 2513 min mm , figure 3 ) was 35% of that found for 15(s)-hete ( 7091 min mm ; supplementary figure 1 in the supporting information ) . kinetic plot of the formation of 11-oxo - ete by 15-pgdh . various concentrations of 11(r)-hete ( 023 m ) were incubated with 9 nm recombinant 15-pgdh ( 52.2 ng , 1.8 pmol ) and cofactor nad . determinations for 11-oxo - ete were conducted in triplicate ( means sem ) by stable isotope dilution lc 11-oxo - ete was synthesized chemically by oxidizing racemic 11(r , s)-hete using the dess . a combined total of 10 mg of the racemic 11(r , s)-hete resulted in the isolation of 4.9 mg of pure 11-oxo - ete ( overall yield of 49% ) . high resolution electrospray ionization ms of 11-oxo - ete revealed accurate masses of m / z 319.2263 and m / z 341.2079 for the protonated and sodiated molecules , respectively [ calculated accurate mass for protonated ( c20h31o3 ) and sodiated ( c20h30o3na ) molecules , m / z 319.2273 and m / z 341.2093 , respectively ] . 500 mhz h nmr ( h , cdcl3 ; supplementary figure 2 in the supporting information ) : 7.55 ( dd , j1 = 11.5 hz , j2 = 15.5 , 1h ) , 6.21(d , j = 15.5 hz , 1h ) , 6.156.11 ( m , 1h ) , 5.975.92 ( m , 1h ) , 5.605.58 ( m , 2h ) , 5.415.38 ( m , 2h ) , 3.35 ( d , j = 4.5 hz , 2h ) , 2.832.81 ( m , 2h ) , 2.382.31 ( m , 4h ) , 2.172.12 ( m , 2h ) , 1.741.25 ( m , 8h ) , 0.89 ( t , j = 7.0 hz , 3h ) . analysis of purified 11-oxo - ete as its pfb derivative by lc - ecapci / ms ( figure 4a ) revealed an intense negative ion at m / z 317 corresponding to [ m pfb ] . cid of [ m pfb ] ( m / z 317 ) and ms / ms analysis revealed major product ions at m / z 273 , 219 , 165 , 149 , and 123 . the uv spectrum was consistent with the presence of a conjugated dienone with a uv max at 279 nm and molecular extinction coefficient ( ) of 18,985 m cm . specific product ions observed by cid and ms / ms analysis of [ m - pfb ] ( m / z 317 ) , corresponding to 11-oxo - ete are shown on the relevant chemical structures . ( a ) synthetic 11-oxo - ete - pfb standard ; ( b ) 15-pgdh - derived 11-oxo - ete - pfb from lovo cell lysate to which 100 nm 11(r)-hete had been added . the lovo cell line is known to express both cox-2 and 15-pgdh and only trace amounts of cox-1 ; this was confirmed by western blot analysis ( data not shown ) . cell lysates were incubated with 100 nm 11(r)-hete in the presence of 500 m nad for 10 min . lc ms analysis of pfb derivatives of eicosanoids extracted from the lovo cell lysate showed that there was a single major metabolite , which eluted at 12.8 min ( data not shown ) . the full scan mass spectrum of this metabolite had only one major ion at m / z 317 corresponding to [ m pfb ] ( figure 4b ) . cid and ms / ms analysis revealed the formation of intense product ions at m / z 123 , 149 , 165 , 219 , and 273 ( figure 4b ) . the product ion spectrum was identical with that obtained from authentic synthetic 11-oxo - ete - pfb ( figure 4a ) , which confirmed the identity of 11-oxo - ete from lovo cells . the product ion at m / z 165 in lovo cell - derived and synthetic 11-oxo - ete - pfb corresponded to cleavage between c-9 and c-10 , and m / z 123 was formed from cleavage between c-11 and c-12 ( figure 4 ) . lysates from lovo cells were extracted for the eicosanoids , which were then analyzed ( after pfb derivatization ) by lc - ecapci / srm / ms . a representative chromatogram ( figure 5 ) reveals the separation of 11-oxo - ete ( retention time 12.8 min ) and 15-oxo - ete ( retention time 12.0 min ) that were formed in the lovo cell lysate . additional eicosanoids that were observed ( figure 5 ) include 13,14-dihydro-15-keto - pge2 ( retention time , 33.6 min ) , 11(r)-hete ( retention time , 10.0 min ) , and pge2 ( retention time , 29.4 min ) . lovo cells were lysed ; eicosanoids were extracted , derivatized with pfb bromide , and analyzed by lc - ecapci / srm / ms . lovo cell lysates were pretreated with 50 m cay10397 to inhibit 15-pgdh to be able to detect the 11- , 15-hetes and pge2 . representative chromatograms are shown for ( top to bottom ) ( a ) 11(r)-hete - pfb ( m / z 319 167 ) , ( b ) [ h8]-15(s)-hete - pfb internal standard ( m / z 327 226 ) , ( c ) 11-oxo - ete - pfb ( m / z 317 165 ) and 15-oxo - ete - pfb ( m / z 317 165 ) , ( d ) [ c20]-15-oxo - ete - pfb internal standard ( m / z 337 120 ) , ( e ) pge2-pfb ( m / z 351 271 ) , ( f ) [ h4]-pge2-pfb ( m / z 355 275 ) , ( g ) 13,14-dihydro-15-oxo - pge2-pfb ( m / z 351 235 ) , ( h ) [ h4]-13,14-dihydro-15-oxo - pge2-pfb ( m / z 355 239 ) . in contrast to lovo cells , the hca-7 cell line is known to express cox-2 and only trace amounts of cox-1 and 15-pgdh . lovo ( figure 6a ) or hca-7 ( figure 6b ) cell lysates were incubated with 500 mm nad , with or without the 15-pgdh inhibitor , cay10397 ( 50 m ) , for 10 min . inhibition of 15-pgdh significantly abolished the formation of endogenous 11-oxo - ete ( figure 6a ) by 92% , along with the diminished formation of 15-oxo - ete as well as 13,14-dihydro-15-oxo - pge2 ( figure 6a ) in the lovo cell lysate . however , since the oxo - etes and 13,14-dihydro-15-oxo - pge2 were undetectable in hca-7 cell lysate , their precursors were quantified instead , and cay10397 had no effect on their levels ( figure 6b ) . ( a ) lovo or ( b ) hca-7 cell lysate ( 1 10 cells / treatment group ) was incubated with or without cay10397 ( 50 m to inhibit 15-pgdh ) . eicosanoids were extracted from the lysates , and their levels were determined by chiral lc - ecapci / srm / ms . time - course analyses for the amount of different eicosanoids secreted by lovo and hca-7 cells following aa incubation for 02.5 h are shown in figure 7a and figure 7b , respectively . 11-oxo - ete was secreted into the cell medium by lovo cells along with 15-oxo - ete and 13,14-dihydro-15-oxo - pge2 ( figure 7a ) . interestingly , both 11-oxo - ete and 15-oxo - ete reached maximum concentrations 10 min after the addition of aa to the cells . this was followed by a decline of both eicosanoids to steady - state levels after 2.5 h ( figure 7a ) in lovo cells . in contrast , 13,14-dihydro-15-oxo - pge2 levels did not peak until 1 h after the addition of aa ( figure 7a ) and remained constant throughout the remainder of the incubation period . levels of the eicosanoids ( 11-oxo - ete , 15-oxo - ete or 13,14-dihydro-15-oxo - pge2 ) resulting from 15-pgdh - mediated metabolism were below detection limits in the medium of hca-7 cells incubated with aa and , hence , are not displayed in figure 7b . pge2 was secreted in the medium from hca-7 cells incubated with aa ( figure 7b ) demonstrating that the cox-2-mediated aa metabolism occurred . this ruled out the possibility that an artifact was responsible for lack of detection of 11-oxo - ete , 15-oxo - ete or 13,14-dihydro-15-oxo - pge2 in the hca-7 cell media . ( a ) lovo or ( b ) hca-7 cells were incubated with 10 m aa for 06 h. the different eicosanoids secreted into the medium at various time points were extracted , and their levels were determined by stable isotope dilution lc - ecapci / srm / ms analysis of their pfb derivatives . determinations were conducted in triplicate ( means sem ) , and the quantitation was performed using the standard curves generated for these eicosanoids . biosynthesis of 11-oeg was monitored in the cell lysate after the addition of 100 nm 11-oxo - ete and 1 mm gsh to the lovo cell lysate . oeg ( retention time , 32.2 min , data not shown ) was observed as an intense peak in the lc cid and ms / ms analysis revealed the formation of intense product ions at m / z 497 , 319 , 308 , and 179 ( figure 8) . 11-oeg was synthesized by reacting 200 nm 11-oxo - ete and 1 mm gsh in lovo cell lysate for 25 min at 37 c . specific product ions observed by cid of [ mh ] ( m / z 626 ) are shown with their relevant chemical structures in the ms / ms analysis of 11-oeg . proliferation by huvecs was assessed by quantifying the brdu incorporation into the cells actively synthesizing new dna . the brdu assay was performed using a quantitative colorimetric 96-well format elisa as well as by immunofluorescence microscopy for observing the morphological effects of eicosanoids in huvecs . for the brdu elisa , huvecs ( 2000 cells / well ) were treated with different doses ( 0100 m ) of 11-oxo - ete and 15d - pgj2 for a period of 24 h. the cell numbers corresponding to each treatment dose were computed and used to calculate cell proliferation ( % ) as compared to the vehicle - treated controls . 11-oxo - ete ( ic50 = 2.1 m ) was equipotent with 15d - pgj2 ( ic50 = 2.3 m ) , a known potent inhibitor of endothelial cell proliferation ( figure 9a ) . for the immunofluorescence analysis , huvecs ( 8000 cells / chamber ) were treated with either 2 m 11-oxo - ete or 2 m 15d - pgj2 for 24 h. brdu incorporation was assessed by counting the brdu - positive cells as compared to the total number of cells counted in randomly selected microscopic fields ( 10 fields / treatment ) . the photomicrographs clearly show that treatment with 11-oxo - ete as well as 15d - pgj2 remarkably reduced the total number of brdu - positive cells ( stained red ) as compared to vehicle - treated controls ( figure 9b ) . moreover , there was a distinct change in the morphological appearance of the dapi - stained ( stained blue ) cells in the eicosanoid - treated slides that failed to incorporate brdu ( figure 9b ) . ( a ) for brdu elisa , huvecs ( 2000 cells / well ) were treated with various doses of 11-oxo - ete and 15d - pgj2 for a period of 24 h. cell proliferation ( means sem ) was assessed by measuring absorbance at 370 nm and converting it to cell numbers using a standard curve and thereby used to construct the ic50 plots . ( b ) for immunofluorescence , huvecs ( 8000/chamber ) were treated with 2 m 15d - pgj2 or 2 m 11-oxo - ete for 24 h and then stained for brdu and counterstained with dapi ( cells stained blue ) . photomicrographs were taken at 200 magnification , and brdu - positive ( stained purple ) cells were counted in randomly selected microscopic fields ( 10/replicate ) as compared to the total number of cells in these fields . all experiments were conducted in triplicate . 15-pgdh catalyzes nad - mediated oxidation of the 15(s)-hydroxyl moiety of pgs and other eicosanoids ( figure 1 ) . our previous studies had established that 15-pgdh is also responsible for metabolizing the 15-lipoxygenase - derived 15(s)-hete in macrophages and monocytes to 15-oxo - ete.(26 ) surprisingly , we have now found that 11(r)-hete is also metabolized by 15-pgdh to a novel eicosanoid , that was identified as 11-oxo - ete ( figure 2 ) . the catalytic activity of human 15-pgdh for oxidation of 11(r)-hete ( figure 2 ) was approximately one - third of that observed for 15(s)-hete ( supplementary figure 1 in the supporting information ) . these results were very surprising in view of the lack of a 15(s)-hydroxyl group on 11(r)-hete as well as the incorrect 11(r)-stereochemistry . the structure of 11-oxo - ete metabolite was established by chemically synthesizing authentic 11-oxo - ete from racemic 11-hete . 11-oxo - ete was formed in lovo cell lysates ( figure 6a ) and secreted from intact cells ( figure 7a ) ; whereas 11-oxo - ete was not formed by hca-7 cell lysates ( figure 6b ) or secreted from intact cells ( figure 7b ) . since lovo cells express cox-2 as well as 15-pgdh , whereas hca-7 cells express cox-2 but not 15-pgdh , these data convincingly prove that 11-oxo - ete is an endogenous product formed enzymatically by a combination of cox-2 and 15-pgdh . 11-oxo - ete is isomeric with 15-oxo - ete ( figure 2 ) and also has very similar lc properties . therefore , a reversed phase lc - srm / ms method was developed to separate the underivatized oxo - etes , and normal phase was implemented to separate their pfb derivatives . endogenous eicosanoids formed by lovo and hca-7 epithelial cells and cell lysates as well as those formed after the addition of aa were analyzed as pfb derivatives by chiral lc - ecapci / ms . representative chromatograms of the eicosanoids produced endogenously in the lovo cell lysates are shown in figure 5 . inhibition of 15-pgdh enzyme by cay10397 significantly diminished the formation of endogenous 11-oxo - ete in lovo cell lysates ( figure 6a ) . in addition , the formation of 15-oxo - ete as well as 13,14-dihydro-15-oxo - pge2 , the other two 15-pgdh - dependent metabolites , was also significantly reduced in lovo cell lysates treated with cay10397 ( figure 6a ) . in contrast , the three 15-pgdh products , namely , 11- and 15-oxo - ete and 13,14-dihydro-15-oxo - pge2 , were undetectable in hca-7 cells as these cells do not express any 15-pgdh . furthermore , treatment with cay10397 had no effect on levels of 11(r)-hete , 15(s)-hete or pge2 ( figure 6b ) . intact lovo cells also secreted 11-oxo - ete and 15-oxo - ete as well as 13,14-dihydro-15-oxo - pge2 . however , the 11-oxo - ete was cleared very rapidly ( figure 7a ) , suggesting that further metabolism was occurring . subsequently , it was observed that 11-oxo - ete underwent gsh conjugation to form 11-oeg , similar to the formation of 15-oeg in ries cells ( figure 8).(1 ) gsh conjugation was predominantly catalyzed by the gst enzymes ; however , it can also occur in a nonenzymatic manner . the relative amount of 11-oeg formed from these different pathways was not explored in detail in the present studies . we realized that , despite its being a linear molecule , 11-oxo - ete has structural similarities to the cyclopentenone eicosanoid 15d - pgj2 due to the presence of an 11-oxo moiety and a -diene ( figure 2 ) . this suggested that 11-oxo - ete might be a more effective inhibitor of huvec proliferation than 15-oxo - ete(26 ) with a potency similar to that observed for 15d - pgj2.(19 ) in fact , 11-oxo - ete was six times more potent than 15-oxo - ete ( data not shown ) and equipotent with 15d - pgj2 at inhibition of huvec proliferation ( figure 9 ) . response studies revealed that its ic50 was 2.1 m compared to an ic50 value of 2.3 m for 15d - pgj2 ( figure 9a ) . moreover , immunofluorescence microscopy revealed that 11-oxo - ete not only inhibited brdu incorporation into the huvecs but also caused a dramatic change in the shape and morphology of these cells ( figure 9b ) . although the total number of cells counted in the eicosanoid - treated groups was quite similar to that in the vehicle - treated group , the brdu - negative cells were significantly distorted ( figure 9b ) . typically , if the cells are undergoing death by apoptosis , they would appear more compact and round . however , 11-oxo - treated cells were elongated and stretched ( figure 9b ) , which could be indicative of extensive cytoskeletal remodeling , cell cycle arrest and/or differentiation . interestingly , 11-oxo - ete formation could not be detected in huvec lysate that was incubated with 11(r)-hete ( data not shown ) . furthermore , immunoblot analysis of huvec cell lysate failed to detect cox-2 protein ( data not shown ) . taken together , these data suggest a paracrine role for 11-oxo - ete on endothelial cell proliferation . as noted above , inhibition of 15-pgdh resulted in significant decreases in 11-oxo - ete formation in lovo cells ( figure 6 ) . 15-pgdh is downregulated in numerous cancer types , which would cause a decrease in 11-oxo - ete biosynthesis by preventing the in vivo conversion of 11(r)-hete to 11-oxo - ete ( figure 1 ) . this could be particularly devastating as cox-2 becomes upregulated during tumorigenesis , which would result in elevated pge2 biosynthesis along with decreased 15-pgdh - mediated inactivation to 15-oxo - pge2 ( figure 1 ) . ultimately , this would result in an increase in pge2-mediated proproliferative activity without the counter effect of antiproliferative oxo - etes . increased pge2 activity can also arise through downregulation of the influx pg transporter : the organic anion transporter polypeptide ( oatp ) 2a1 ( figure 1 ) . pge2 and 11-oxo - ete can also arise from the metabolism of aa by constitutively expressed cox-1 . therefore , downregulation of 15-pgdh would also prevent the inactivation of cox-1-derived proproliferative pge2 as well as the biosynthesis of antiproliferative 11-oxo - ete . these metabolic changes could also contribute to the eicosanoid - mediated effects that occur during tumorigenesis . there is some evidence that 11-oxo - ete can activate nuclear ppar.(34 ) ppar is a ligand - dependent transcription factor responsible for the regulation of a number of cellular events ranging from lipid metabolism to apoptosis.(35 ) 15d - pgj2 is a ppar agonist , which might account for its ability to inhibit endothelial cell proliferation . however , this effect is only observed with pharmacological amounts of 15d - pgj2 rather than endogenous concentrations.(27 ) besides ppar agonistic activity , 15d - pgj2 is also known to be an inhibitor of nfb signaling , a pathway critical to cell proliferation as well as tumorigenesis . in view of the ability of cox-2/15-pgdh to rapidly metabolize aa to nm amounts of oxo - etes , together with the similar structural features of the oxo - etes and 15d - pgj2 ( figure 2 ) , it will be important to determine which of these activities are shared by both classes of eicosanoids . it is noteworthy that docosahexaenoic acid and docosapentaenoic acid can be metabolized by the sequential action of cox-2 and dehydrogenases into oxoeicosanoids that modulate the antioxidant response.(38 ) similarly 5-lipoxygenase - derived 5(s)-hete is metabolized by 5-hydroxyeicosanoid dehydrogenase into the chemoattractant 5-oxo - ete.(39 ) therefore , , our studies have revealed that inhibition of 15-pgdh prevents the formation of endogenous antiproliferative eicosanoid , 11-oxo - ete . therefore , 15-pgdh has two quite distinct properties : it can either inactivate pgs or activate hetes to oxo - etes that exert a paracrine effect on endothelial cells ( figure 1 ) . 11-oxo - ete , a member of the oxo - ete family , was observed previously as an endogenously derived lipid in human atherosclerotic plaques.(42 ) however , the biosynthesis of 11-oxo - ete and its biological activity were not evaluated in that study . furthermore , there does not appear to be any subsequent report of its formation either in vitro or in vivo . we have now shown that in fact 11-oxo - ete is derived from cox-2/15-pgdh - mediated aa metabolism and that it inhibits endothelial cell proliferation with an ic50 that is very similar to that of 15d - pgj2 .

previously , we established that 11(r)-hydroxy-5,8,12,14-(z , z , e , z)-eicosatetraenoic acid ( hete ) was a significant cyclooxygenase ( cox)-2-derived arachidonic acid ( aa ) metabolite in epithelial cells . stable isotope dilution chiral liquid chromatography ( lc)-electron capture atmospheric pressure chemical ionization ( ecapci)/mass spectrometry ( ms ) was used to quantify cox-2-derived eicosanoids in the human colorectal adenocarcinoma ( lovo ) epithelial cell line , which expresses both cox-2 and 15-hydroxyprostaglandin dehydrogenase ( 15-pgdh ) . 11(r)-hete secretion reached peak concentrations within minutes after aa addition before rapidly diminishing , suggesting further metabolism had occurred . surprisingly , recombinant 15-pgdh , which is normally specific for oxidation of eicosanoid 15(s)-hydroxyl groups , was found to convert 11(r)-hete to 11-oxo-5,8,12,14-(z , z , e , z)-eicosatetraenoic acid ( ete ) . furthermore , lovo cell lysates converted 11(r)-hete to 11-oxo - ete and inhibition of 15-pgdh with 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy - benzeneacetic acid ( cay10397 ) ( 50 m ) significantly suppressed endogenous 11-oxo - ete production with a corresponding increase in 11(r)-hete . these data confirmed cox-2 and 15-pgdh as enzymes responsible for 11-oxo - ete biosynthesis . finally , addition of aa to the lovo cells resulted in rapid secretion of 11-oxo - ete into the media , reaching peak levels within 20 min of starting the incubation . this was followed by a sharp decrease in 11-oxo - ete levels . glutathione ( gsh ) s - transferase ( gst ) was found to metabolize 11-oxo - ete to the 11-oxo - ete - gsh ( oeg)-adduct in lovo cells , as confirmed by lc ms / ms analysis . bromodeoxyuridine ( brdu)-based cell proliferation assays in human umbilical vein endothelial cells ( huvecs ) revealed that the half - maximal inhibitory concentration ( ic50 ) of 11-oxo - ete for inhibition of huvec proliferation was 2.1 m . these results show that 11-oxo - ete is a novel cox-2/15-pgdh - derived eicosanoid , which inhibits endothelial cell proliferation with a potency that is similar to that observed for 15d - pgj2 .

Introduction Experimental Procedures Results Discussion

in a previous study , we established that 11(r)-hete was a significant cox-2-derived aa metabolite in epithelial cells. (15 ) formation and action of cox-2-derived eicosanoids in epithelial cell models . secreted 15- and 11-oxo - ete that escape further metabolism can then inhibit endothelial cell proliferation . pgd2 , another cox-2-derived metabolite , is also metabolized by 15-pgdh to form 15-oxo - pgd2 , which is then converted to the corresponding inactive 13,14-dihydro derivative ( figure 1). (16 ) alternatively , pgd2 undergoes albumin - mediated dehydration to give pgj2 , followed by a further dehydration to give 15-deoxy--prostaglandin j2 ( 15d - pgj2 ) ( figure 2). (17 ) previous studies have shown that 15d - pgj2 is a ppar agonist,(18 ) which inhibits huvec proliferation in culture. (19 ) in addition , 15d - pgj2 can induce caspase - mediated endothelial cell apoptosis(20 ) and inhibit the nuclear factor b ( nfb ) pathway . (25 ) 11(r)-hete was a significant eicosanoid secreted by aa - treated rat intestinal epithelial cells that stably express cox-2 ( ries cells ) , but it was rapidly metabolized. 15-oxo - ete was also found to inhibit endothelial cell proliferation , although at concentrations higher than 15d - pgj2. this has made it possible to determine whether 11-oxo - ete is secreted by human epithelial cell lines with upregulated cox-2 expression and to identify the dehydrogenase responsible . aa ( peroxide - free ) , 11(r , s)-hete , 15(r , s)-hete , [ h8]-15(s)-hete , [ c20]-15-oxo - ete , 15-oxo - ete , pge2 , [ h4]-pge2 , 13,14-dihydro-15-keto - pge2 , [ h4]-13,14-dihydro-15-keto - pge2 , 15d - pgj2 , cay10397 , protease inhibitor cocktail and recombinant human 15-pgdh were purchased from cayman ( ann arbor , mi ) . human colorectal adenocarcinoma lovo cells ( atcc , manassas , va ) were cultured in f12k medium supplemented with 10% fbs , 2 mm l - glutamine , 100,000 units / l penicillin and 100 mg / l streptomycin . the following srm transition ( m / z 626 497 ) was monitored for 11-oxo - ete - gsh ( ce , 18 ev ) . a chiralpak ad - rh column ( 150 4.6 mm inner diameter , 5 m ; daicel ) was used for reversed phase ( isocratic method 1 , flow rate 0.5 ml / min ) separation of the underivatized 11-oxo - ete . an aliquot ( 25 l ) was separated using the isocratic method 1 and analyzed by lc in separate experiments , the formation of 11-oxo - ete was found to be linear for the first 5 min . the ph was then adjusted to 4 with 10% aqueous acetic acid ( 10 l ) followed by addition of the internal standard mix , [ c20]-15-oxo - ete , [ h8]-15(s)-hete and [ h4]-pge2 ( 50 pg/l , 20 l ) . 11-oxo - ete ( 20 ng in ethanol ) was added to the lysate together with 1 mm gsh . the column was washed with 1 ml of water and eluted with 250 l of methanol , and then 20 l was analyzed by reversed phase lc brdu incorporation in huvecs was used to assess the effects of 11-oxo - ete on cell proliferation . cells were then treated for 24 h with either vehicle ( 0.25% dmso ) , 11-oxo - ete ( 1 nm to 100 m ) , or 15d - pgj2 ( 1 nm to 100 m ) . the ic50 values for eicosanoid inhibition of huvec proliferation were defined as the half maximal inhibitory concentration for endothelial cell proliferation over 24 h when compared with vehicle - treated cells . a michaelis menten kinetic analysis of 11-oxo - ete formation provided the vmax ( 296 nmol / min / mg of protein ) , km ( 3.42 m ) , and kcat ( 8.6 min ) values for oxidation of 11(r)-hete ( figure 3 ) and the vmax ( 403.8 nmol / min / mg of protein , km ( 1.65 m ) , and kcat ( 8.6 min ) values for oxidation of 15(s)-hete ( supplementary figure 1 in the supporting information ) . determinations for 11-oxo - ete were conducted in triplicate ( means sem ) by stable isotope dilution lc 11-oxo - ete was synthesized chemically by oxidizing racemic 11(r , s)-hete using the dess . a combined total of 10 mg of the racemic 11(r , s)-hete resulted in the isolation of 4.9 mg of pure 11-oxo - ete ( overall yield of 49% ) . analysis of purified 11-oxo - ete as its pfb derivative by lc - ecapci / ms ( figure 4a ) revealed an intense negative ion at m / z 317 corresponding to [ m pfb ] . cid of [ m pfb ] ( m / z 317 ) and ms / ms analysis revealed major product ions at m / z 273 , 219 , 165 , 149 , and 123 . specific product ions observed by cid and ms / ms analysis of [ m - pfb ] ( m / z 317 ) , corresponding to 11-oxo - ete are shown on the relevant chemical structures . ( a ) synthetic 11-oxo - ete - pfb standard ; ( b ) 15-pgdh - derived 11-oxo - ete - pfb from lovo cell lysate to which 100 nm 11(r)-hete had been added . the lovo cell line is known to express both cox-2 and 15-pgdh and only trace amounts of cox-1 ; this was confirmed by western blot analysis ( data not shown ) . lc ms analysis of pfb derivatives of eicosanoids extracted from the lovo cell lysate showed that there was a single major metabolite , which eluted at 12.8 min ( data not shown ) . the product ion spectrum was identical with that obtained from authentic synthetic 11-oxo - ete - pfb ( figure 4a ) , which confirmed the identity of 11-oxo - ete from lovo cells . the product ion at m / z 165 in lovo cell - derived and synthetic 11-oxo - ete - pfb corresponded to cleavage between c-9 and c-10 , and m / z 123 was formed from cleavage between c-11 and c-12 ( figure 4 ) . lysates from lovo cells were extracted for the eicosanoids , which were then analyzed ( after pfb derivatization ) by lc - ecapci / srm / ms . a representative chromatogram ( figure 5 ) reveals the separation of 11-oxo - ete ( retention time 12.8 min ) and 15-oxo - ete ( retention time 12.0 min ) that were formed in the lovo cell lysate . representative chromatograms are shown for ( top to bottom ) ( a ) 11(r)-hete - pfb ( m / z 319 167 ) , ( b ) [ h8]-15(s)-hete - pfb internal standard ( m / z 327 226 ) , ( c ) 11-oxo - ete - pfb ( m / z 317 165 ) and 15-oxo - ete - pfb ( m / z 317 165 ) , ( d ) [ c20]-15-oxo - ete - pfb internal standard ( m / z 337 120 ) , ( e ) pge2-pfb ( m / z 351 271 ) , ( f ) [ h4]-pge2-pfb ( m / z 355 275 ) , ( g ) 13,14-dihydro-15-oxo - pge2-pfb ( m / z 351 235 ) , ( h ) [ h4]-13,14-dihydro-15-oxo - pge2-pfb ( m / z 355 239 ) . in contrast to lovo cells , the hca-7 cell line is known to express cox-2 and only trace amounts of cox-1 and 15-pgdh . lovo ( figure 6a ) or hca-7 ( figure 6b ) cell lysates were incubated with 500 mm nad , with or without the 15-pgdh inhibitor , cay10397 ( 50 m ) , for 10 min . inhibition of 15-pgdh significantly abolished the formation of endogenous 11-oxo - ete ( figure 6a ) by 92% , along with the diminished formation of 15-oxo - ete as well as 13,14-dihydro-15-oxo - pge2 ( figure 6a ) in the lovo cell lysate . ( a ) lovo or ( b ) hca-7 cell lysate ( 1 10 cells / treatment group ) was incubated with or without cay10397 ( 50 m to inhibit 15-pgdh ) . 11-oxo - ete was secreted into the cell medium by lovo cells along with 15-oxo - ete and 13,14-dihydro-15-oxo - pge2 ( figure 7a ) . interestingly , both 11-oxo - ete and 15-oxo - ete reached maximum concentrations 10 min after the addition of aa to the cells . this was followed by a decline of both eicosanoids to steady - state levels after 2.5 h ( figure 7a ) in lovo cells . this ruled out the possibility that an artifact was responsible for lack of detection of 11-oxo - ete , 15-oxo - ete or 13,14-dihydro-15-oxo - pge2 in the hca-7 cell media . ( a ) lovo or ( b ) hca-7 cells were incubated with 10 m aa for 06 h. the different eicosanoids secreted into the medium at various time points were extracted , and their levels were determined by stable isotope dilution lc - ecapci / srm / ms analysis of their pfb derivatives . biosynthesis of 11-oeg was monitored in the cell lysate after the addition of 100 nm 11-oxo - ete and 1 mm gsh to the lovo cell lysate . oeg ( retention time , 32.2 min , data not shown ) was observed as an intense peak in the lc cid and ms / ms analysis revealed the formation of intense product ions at m / z 497 , 319 , 308 , and 179 ( figure 8) . 11-oeg was synthesized by reacting 200 nm 11-oxo - ete and 1 mm gsh in lovo cell lysate for 25 min at 37 c . specific product ions observed by cid of [ mh ] ( m / z 626 ) are shown with their relevant chemical structures in the ms / ms analysis of 11-oeg . for the brdu elisa , huvecs ( 2000 cells / well ) were treated with different doses ( 0100 m ) of 11-oxo - ete and 15d - pgj2 for a period of 24 h. the cell numbers corresponding to each treatment dose were computed and used to calculate cell proliferation ( % ) as compared to the vehicle - treated controls . 11-oxo - ete ( ic50 = 2.1 m ) was equipotent with 15d - pgj2 ( ic50 = 2.3 m ) , a known potent inhibitor of endothelial cell proliferation ( figure 9a ) . for the immunofluorescence analysis , huvecs ( 8000 cells / chamber ) were treated with either 2 m 11-oxo - ete or 2 m 15d - pgj2 for 24 h. brdu incorporation was assessed by counting the brdu - positive cells as compared to the total number of cells counted in randomly selected microscopic fields ( 10 fields / treatment ) . the photomicrographs clearly show that treatment with 11-oxo - ete as well as 15d - pgj2 remarkably reduced the total number of brdu - positive cells ( stained red ) as compared to vehicle - treated controls ( figure 9b ) . ( a ) for brdu elisa , huvecs ( 2000 cells / well ) were treated with various doses of 11-oxo - ete and 15d - pgj2 for a period of 24 h. cell proliferation ( means sem ) was assessed by measuring absorbance at 370 nm and converting it to cell numbers using a standard curve and thereby used to construct the ic50 plots . ( b ) for immunofluorescence , huvecs ( 8000/chamber ) were treated with 2 m 15d - pgj2 or 2 m 11-oxo - ete for 24 h and then stained for brdu and counterstained with dapi ( cells stained blue ) . our previous studies had established that 15-pgdh is also responsible for metabolizing the 15-lipoxygenase - derived 15(s)-hete in macrophages and monocytes to 15-oxo - ete. (26 ) surprisingly , we have now found that 11(r)-hete is also metabolized by 15-pgdh to a novel eicosanoid , that was identified as 11-oxo - ete ( figure 2 ) . the catalytic activity of human 15-pgdh for oxidation of 11(r)-hete ( figure 2 ) was approximately one - third of that observed for 15(s)-hete ( supplementary figure 1 in the supporting information ) . 11-oxo - ete was formed in lovo cell lysates ( figure 6a ) and secreted from intact cells ( figure 7a ) ; whereas 11-oxo - ete was not formed by hca-7 cell lysates ( figure 6b ) or secreted from intact cells ( figure 7b ) . since lovo cells express cox-2 as well as 15-pgdh , whereas hca-7 cells express cox-2 but not 15-pgdh , these data convincingly prove that 11-oxo - ete is an endogenous product formed enzymatically by a combination of cox-2 and 15-pgdh . 11-oxo - ete is isomeric with 15-oxo - ete ( figure 2 ) and also has very similar lc properties . endogenous eicosanoids formed by lovo and hca-7 epithelial cells and cell lysates as well as those formed after the addition of aa were analyzed as pfb derivatives by chiral lc - ecapci / ms . representative chromatograms of the eicosanoids produced endogenously in the lovo cell lysates are shown in figure 5 . inhibition of 15-pgdh enzyme by cay10397 significantly diminished the formation of endogenous 11-oxo - ete in lovo cell lysates ( figure 6a ) . in addition , the formation of 15-oxo - ete as well as 13,14-dihydro-15-oxo - pge2 , the other two 15-pgdh - dependent metabolites , was also significantly reduced in lovo cell lysates treated with cay10397 ( figure 6a ) . intact lovo cells also secreted 11-oxo - ete and 15-oxo - ete as well as 13,14-dihydro-15-oxo - pge2 . however , the 11-oxo - ete was cleared very rapidly ( figure 7a ) , suggesting that further metabolism was occurring . subsequently , it was observed that 11-oxo - ete underwent gsh conjugation to form 11-oeg , similar to the formation of 15-oeg in ries cells ( figure 8). we realized that , despite its being a linear molecule , 11-oxo - ete has structural similarities to the cyclopentenone eicosanoid 15d - pgj2 due to the presence of an 11-oxo moiety and a -diene ( figure 2 ) . this suggested that 11-oxo - ete might be a more effective inhibitor of huvec proliferation than 15-oxo - ete(26 ) with a potency similar to that observed for 15d - pgj2. (19 ) in fact , 11-oxo - ete was six times more potent than 15-oxo - ete ( data not shown ) and equipotent with 15d - pgj2 at inhibition of huvec proliferation ( figure 9 ) . response studies revealed that its ic50 was 2.1 m compared to an ic50 value of 2.3 m for 15d - pgj2 ( figure 9a ) . moreover , immunofluorescence microscopy revealed that 11-oxo - ete not only inhibited brdu incorporation into the huvecs but also caused a dramatic change in the shape and morphology of these cells ( figure 9b ) . taken together , these data suggest a paracrine role for 11-oxo - ete on endothelial cell proliferation . as noted above , inhibition of 15-pgdh resulted in significant decreases in 11-oxo - ete formation in lovo cells ( figure 6 ) . 15-pgdh is downregulated in numerous cancer types , which would cause a decrease in 11-oxo - ete biosynthesis by preventing the in vivo conversion of 11(r)-hete to 11-oxo - ete ( figure 1 ) . pge2 and 11-oxo - ete can also arise from the metabolism of aa by constitutively expressed cox-1 . (35 ) 15d - pgj2 is a ppar agonist , which might account for its ability to inhibit endothelial cell proliferation . (27 ) besides ppar agonistic activity , 15d - pgj2 is also known to be an inhibitor of nfb signaling , a pathway critical to cell proliferation as well as tumorigenesis . (39 ) therefore , , our studies have revealed that inhibition of 15-pgdh prevents the formation of endogenous antiproliferative eicosanoid , 11-oxo - ete . 11-oxo - ete , a member of the oxo - ete family , was observed previously as an endogenously derived lipid in human atherosclerotic plaques. (42 ) however , the biosynthesis of 11-oxo - ete and its biological activity were not evaluated in that study . we have now shown that in fact 11-oxo - ete is derived from cox-2/15-pgdh - mediated aa metabolism and that it inhibits endothelial cell proliferation with an ic50 that is very similar to that of 15d - pgj2 .

the number of new cases of lung cancer in the eu will increase by 27% between 2008 and 2025 , in part due to population ageing , and in part due to long - term trends in tobacco use . surgery remains the mainstay of treatment , with curative intent for non - small - cell lung cancer ( nsclc ) patients who are medically fit , with lobectomy as the treatment of first choice . length of stay ( los ) in hospital after surgery impacts on cost and hospital performance [ 35 ] . there is little definitive information on los following lung cancer resection ; yet , the rate of lung cancer resection ( as a proportion of nsclc cases ) is increasing in several european countries [ 68 ] . internationally , the few available studies suggest that there is much variation in the reported median los after nsclc resection [ 5 , 912 ] . postoperative complications are common after lung resection and can result in prolonged hospitalization and early readmission [ 9 , 13 ] . we conducted a population - based analysis of time trends in los and predictors of prolonged los following resection for nsclc in ireland . we further investigated the factors predicting emergency readmission within 28 days of discharge after the index procedure ( ip ) . the primary data sources for this study were the national cancer registry ( ncr ) and the hospital in - patient enquiry ( hipe ) database in ireland [ 14 , 15 ] . hipe is a computer - based information system that records data on discharges from all acute public hospitals and a few private hospitals . lung cancer patients ( icd - o2 : c34 ) newly diagnosed between 2002 and 2008 were identified from the ncr . individuals who had another primary cancer prior to the lung cancer ( other than non - melanoma skin ) were excluded . the dataset was then limited to those who had lung cancer resection according to ncr records ( icd9-cm codes 32.2x , 32.3 , 32.4 , 32.5 , 32.6 , 32.9 , 34.4x ) . using probabilistic matching techniques , these patients were linked to hipe episodes ( fig . 1 ) . coverage of private hospitals by hipe is very incomplete and we limited our analysis to patients treated in public hospitals . cases with tumour morphology codes of m8039 - 8046 ( small cell carcinoma ) were then excluded . the date of surgical resection ( ip ) recorded by the ncr was matched to the corresponding hipe episode . the final analysis dataset included 1284 patients with nsclc who underwent a resection ( fig . los was calculated as the number of days between the admission date for the ip and the discharge date , or death ( if the patient died in hospital ) ( i.e. this was the total of preoperative and postoperative los ) . duration of discharge was calculated as the time from discharge following the ip to the first emergency readmission to a public hospital ( if any ) . emergency when the patient required immediate care and treatment as a result of a severe , life - threatening or potentially disabling condition . in the uk , 28-day readmission rate is a key hospital performance indicator [ http://www.nchod.nhs.uk ] ; therefore , we based our analysis on emergency readmissions where the duration of discharge was < 29 days . a range of patient , tumour and health - service - related variables were abstracted from the ncr and hipe databases and investigated for associations with los and emergency readmission . details on age at diagnosis , gender and marital status were available from the ncr . socioeconomic status was measured in terms of the level of deprivation of each patient 's local area of residence at diagnosis using a score created from 2002 census variables . the score was derived for each of the 3409 electoral districts in ireland ; the districts were categorized into five levels of deprivation . in our dataset , the number of patients resident within a quintile 5 district ( most deprived ) was double that of any other quintile in our study . we therefore pooled quintiles 14 ( less deprived ) for comparison with quintile 5 ; this is also consistent with what was done in a large us study of los and lung resections among medicare recipients . each case was classified according to smoking status at diagnosis , which was derived by the ncr from hospital charts , and defined as : ( i ) never smoked , ( ii ) ex - smoker did not smoke more than once a month for the past year and ( iii ) cases were classified by summary stage of disease . where information on distant metastasis ( mx ) was not recorded , these cases were treated as m0 . a comorbidity score for each patient , based on the charlson index , was derived from all diagnoses recorded in hipe for the ip episode ; the lung cancer diagnosis was disregarded in this calculation . resection procedures were categorized as : lobectomy , pneumonectomy , segmental / wedge resections and other procedures. patients were classified as private or public ( this refers to whether the patient saw the surgeon as a private or public patient ) . the patient 's destination at discharge after ip was classified as : home , other healthcare facility ( i.e. another acute / step - down hospital , nursing home or hospice ) , or death in hospital after ip . resections for nsclc were performed in six public hospitals during 20028 . lower volume depending on whether they were operated on in a hospital above , or below , the median resection count / hospital / year respectively ( 40/hospital / year ) . median los , and ranges ( upper and lower decile of los ) were computed at each level of the sociodemographic , clinical and health - service - related variables . wallis equality - of - populations rank test and cuzick 's test for trend . in the absence of any published definition of prolonged los in europe , the cut - point defining the upper quartile of patients ( > 20 days ) was selected as a practical threshold for prolonged los ; this also happened to be 1 week longer than the median los ( 13 days ) . as the outcome was common , instead of using logistic regression to estimate odds ratios , we modelled prolonged los using poisson regression with a log link and robust variance to estimate risk ratios and associated 95% confidence intervals . three types of variables were considered for inclusion in the model : sociodemographic ( age , gender , marital status , deprivation , smoking status and public / private status ) ; clinical ( ip type , stage and comorbidity ) and health - service related ( hospital volume , destination at discharge ) . a backward stepwise variable selection approach for modelling was used , removing variables in order of least significance until all retained variables achieved p < 0.1 in likelihood ratio tests ( lrts ) . as a sensitivity analysis , we also modelled los as a continuous variable using linear regression after a log transformation to normalize the distribution of los . poisson regression with a log link and robust variance was also used to estimate risk ratios for factors predicting emergency readmissions within 28 days after discharge . patients who had not died in hospital were classified as : ( a ) emergency readmission within 28 days of discharge , or ( b ) not readmitted as an emergency within 28 days of discharge . the primary reason for the emergency readmission was derived from the hipe diagnostic codes for that admission . median los , and ranges ( upper and lower decile of los ) were computed at each level of the sociodemographic , clinical and health - service - related variables . wallis equality - of - populations rank test and cuzick 's test for trend . in the absence of any published definition of prolonged los in europe , the cut - point defining the upper quartile of patients ( > 20 days ) was selected as a practical threshold for prolonged los ; this also happened to be 1 week longer than the median los ( 13 days ) . as the outcome was common , instead of using logistic regression to estimate odds ratios , we modelled prolonged los using poisson regression with a log link and robust variance to estimate risk ratios and associated 95% confidence intervals . three types of variables were considered for inclusion in the model : sociodemographic ( age , gender , marital status , deprivation , smoking status and public / private status ) ; clinical ( ip type , stage and comorbidity ) and health - service related ( hospital volume , destination at discharge ) . a backward stepwise variable selection approach for modelling was used , removing variables in order of least significance until all retained variables achieved p < 0.1 in likelihood ratio tests ( lrts ) . as a sensitivity analysis , we also modelled los as a continuous variable using linear regression after a log transformation to normalize the distribution of los . poisson regression with a log link and robust variance was also used to estimate risk ratios for factors predicting emergency readmissions within 28 days after discharge . patients who had not died in hospital were classified as : ( a ) emergency readmission within 28 days of discharge , or ( b ) not readmitted as an emergency within 28 days of discharge . the primary reason for the emergency readmission was derived from the hipe diagnostic codes for that admission . of 12 139 incident cases of invasive lung cancer diagnosed in ireland between 2002 and 2008 , 1284 had nsclc and underwent lung resection in public hospitals ( fig . 1 ) . of these , 72% underwent lobectomy , 17% underwent pneumonectomy , 4% had segmental / wedge resection and 7% had another excision procedure . there was a significant increase in lung cancer resections performed per annum ( expressed as a proportion of nsclc cases ) between 2002 and 2008 ( 142/1415 ( 10% ) in 2002 , rising to 228/1613 ( 14% ) in 2008 ; p - trend = 0.000012 ) . the median total los was 13 days ( inter - decile range ( idr ) : 735 days , mean = 18 days and range : 1343 days ) . the median los decreased significantly from 15 days in 2002 to 12 days in 2008 ( p - trend = 0.037 ) ( table 1 ) . the median preoperative los for the period 20028 was 1 day ( idr : 06 days ) . this decreased significantly over time , from 3 days [ idr : 116 ] in 2002 , to 1 day [ idr : 03 ] in 2008 ( p - trend = 0.0000000000021 ) . table 1:patients with nsclc undergoing resection , 20028 : numbers ( n ) , percentages ( % ) and median and idr los and p - valuesvariablescategoriesn%median losidrp - valuetotal1284100%13[735]age at diagnosis<55 year20416%10[525]0.0000000175564 year42333%12[730]6574 year47937%13[737]>75 year17814%15[750]genderfemale54142%12[732]0.22male74358%13[736]marital statusmarried81063%12[731]0.019other47437%13[740]deprivation1 least23018%12[635]0.016216613%12[527]314411%12.5[730]418715%12[628]5 most48037%13[739]unknown776%13[731]smoking statusnever smoker12410%12.5[634]0.67ex - smoker43434%12[733]current smoker60447%13[738]unknown12210%10[223]health insurancepublic88469%13[736]0.017private ( held private health insurance)40031%12[631]stagestage i / ii85467%13[736]0.023stage iii+30924%13[733]unstaged1219%10[422]comorbidity093173%12[632]0.000000026126421%13[836]2 + 897%18[859]procedurelobectomy90971%12[735]0.14pneumonectomy23418%13[733]segmental / wedge excision494%13[642]other excision927%15[437]discharge statusto home97076%12[729]0.00000059to care ( other acute hospital / nursing home / hospice)23018%12[245]died in hospital post - ip847%21.5[1091]hospital volumehigher volume : 40/year66151%12[631]0.000076lower volume : < 40/year62349%13[737]year of incidence200214211%15[836]0.037200317414%12[734]200415312%13[832]200518715%12[434]200617614%13[729]200722417%13[733]200822818%12[640]p - value : kruskal wallis test for binary and categorical variables and test for trend with ordinal categorical variables.small area ( electoral district ) based quintile of deprivation.patient had private health insurance or settled own account ( private ) , or did not have health insurance ( public ) at discharge.the patients were classified as high volume or low volume depending on whether they were operated in a hospital above or below the median resection count / hospital / year respectively ( 40/hospital / year).idr : inter - decile range ( 10th90th centile ) . patients with nsclc undergoing resection , 20028 : numbers ( n ) , percentages ( % ) and median and idr los and p - values p - value : kruskal wallis test for binary and categorical variables and test for trend with ordinal categorical variables . patient had private health insurance or settled own account ( private ) , or did not have health insurance ( public ) at discharge . high volume or low volume depending on whether they were operated in a hospital above or below the median resection count / hospital / year respectively ( 40/hospital / year ) . the characteristics of the 1284 cases , together with median and idr los , are shown in table 1 . table 2 presents crude and adjusted risk ratios ( rr ) for factors that were significantly associated with prolonged los in multivariate analyses ( greater than the upper quartile of los , i.e. > 20 days ) . in the adjusted analysis , the risk of prolonged los was significantly higher in patients who were older than 75 years ; lived in the most deprived areas ; had two or more comorbid conditions ; and had undergone surgery in a lower - volume hospital . patients who died in hospital subsequent to the ip were also significantly more likely to have prolonged los . although the percentage of patients who had prolonged los varied slightly according to the procedure received ( lobectomy 25% , pneumonectomy 23% , segmental 31% and other excision 31% ) , this was not statistically significant in adjusted analysis . similarly , stage of disease was not significantly associated with prolonged los after adjusting for other variables ( stage i / ii 25% , stage iii+ 25% and unstaged 16% ) . in the sensitivity analysis , using linear regression , the same variables predicted los as predicted prolonged los . table 2:factors significantly associated with prolonged los in patients having resection for nsclc , 20028 : number ( n ) of total ( n ) ( % ) who had prolonged los ( > 20 days ) , univariate and adjusted rr , with 95% ci and lrtsprolonged los ( > 20 days)univariateadjustedlrtnn%rr95% cirr95% cip - valuetotal312128424%age < 55 year3720418%110.064 5564 year8542320%1.11[0.78 , 1.57]1.03[0.73 , 1.45 ] 6574 year12847927%1.47[1.06 , 2.04]1.32[0.95 , 1.82 ] > 75 year6217835%1.92[1.35 , 2.74]1.55[1.08 , 2.23]sex female13254124%1 male18074324%0.99[0.82 , 1.21]deprivation less deprived ( q14)15872722%110.060 most deprived ( q5)13848029%1.32[1.09 , 1.61]1.30[1.07 , 1.58 ] unknown167721%0.96[0.61 , 1.51]1.03[0.66 , 1.62]comorbidity none20693122%110.073 17026427%1.20[0.95 , 1.51]1.11[0.88 , 1.40 ] 2 + 368940%1.83[1.38 , 2.42]1.60[1.20 , 2.13]hospital volume higher : 40/year14166121%110.086 lower : < 40/year17162327%1.29[1.03 , 1.61]1.24[0.99 , 1.56]discharge status alive at discharge266120022%110.000074 died in hospital post ip468455%2.47[1.98 , 3.08]2.03[1.60 , 2.57]mutually adjusted for age , deprivation , comorbidity , hospital volume and discharge status ( and year of incidence , not shown).rr : risk ratio ; lrt : likelihood ratio test for exclusion of that variable from multivariable model . factors significantly associated with prolonged los in patients having resection for nsclc , 20028 : number ( n ) of total ( n ) ( % ) who had prolonged los ( > 20 days ) , univariate and adjusted rr , with 95% ci and lrts mutually adjusted for age , deprivation , comorbidity , hospital volume and discharge status ( and year of incidence , not shown ) . rr : risk ratio ; lrt : likelihood ratio test for exclusion of that variable from multivariable model . of 1200 patients who were discharged alive after surgery ( none of whom died within 28 days of discharge ) , 119 ( 10% ) table 3 presents crude and adjusted risk ratios for factors significantly associated with risk of emergency readmission . in the adjusted analysis , risk of readmission was increased in patients from the most deprived areas , with two or more comorbid conditions , and with stage iii+ disease ( table 3 ) . patients > 75 years ( 14% of whom were readmitted , compared with 7 , 10 and 9% in the < 55 year , 5564 year and 6574 year age groups , respectively ) , and those who underwent segmental procedures ( 17% readmitted , compared with 9% for lobectomy , 12% for pneumonectomy and 9% for other procedures ) were also more prone to emergency readmission in univariate analyses ; however , age and procedure type were not significantly associated with readmission after adjustment for the factors in the model . the main reasons for emergency readmission were : pulmonary complications ( 29% ) , cardiovascular / vascular events ( 21% ) and infections ( 20% ) ( table 4 ) . table 3:factors significantly associated with emergency readmission in patients having resection for nsclc , 20028 : number ( n ) of total ( n ) ( % ) who were readmitted within 28 days ) , univariate and adjusted rr with 95% ci and lrtreadmitted within 28 dayscrudeadjustedlrtnn%rr[95% ci]rr[95% ci]p - valuetotal119120010%age < 55 year152017%1 5564 year4240610%1.39[0.79 , 2.44 ] 6574 year414419%1.25[0.71 , 2.20 ] > 75 year2115214%1.85[0.99 , 3.47]sex female5052110%1 male6967910%1.06[0.75 , 1.50]deprivation less deprived ( q14)576858%110.0095 most deprived ( q5)5944113%1.61[1.14 , 2.27]1.56[1.11 , 2.20 ] unknown3744%0.49[0.16 , 1.52]0.48[0.16 , 1.45]comorbidity none748798%110.011 13024412%1.46[0.98 , 2.18]1.43[0.96 , 2.12 ] 2 + 157719%2.31[1.40 , 3.83]2.38[1.43 , 3.96]stage stage i / ii728019%110.039 stage iii+3928114%1.54[1.07 , 2.23]1.62[1.13 , 2.34 ] unstaged81187%0.75[0.37 , 1.53]0.83[0.41 , 1.69]mutually adjusted for deprivation , comorbidity and stage.rr : risk ratio ; lrt : p - value of likelihood ratio test for exclusion of that variable in multivariable model . table 4:primary reason for first emergency readmission within 28 days of ipreadmission causedescriptionn ( % ) subtotal number ( % ) pulmonary complicationatelectasis11 ( 9%)pneumonia8 ( 7%)pulmonary air leak6 ( 5%)pleural effusion7 ( 6%)empyema2 ( 2%)34 ( 29%)cardiovascular / vasculardysrhythmia18 ( 15%)myocardial infarction4 ( 3%)cerebrovascular event3 ( 3%)25 ( 21%)infectionwound infection6 ( 5%)fever14 ( 12%)urinary tract infection3 ( 3%)23 ( 20%)othersrenal insufficiency2 ( 2%)others35 ( 29%)37 ( 31%)total119 ( 100% ) factors significantly associated with emergency readmission in patients having resection for nsclc , 20028 : number ( n ) of total ( n ) ( % ) who were readmitted within 28 days ) , univariate and adjusted rr with 95% ci and lrt mutually adjusted for deprivation , comorbidity and stage . rr : risk ratio ; lrt : p - value of likelihood ratio test for exclusion of that variable in multivariable model . our dataset of > 1200 lung cancer resections conducted in patients incident during 20028 is relatively large by european standards and provides for the first time , as far as we are aware , detailed information on factors predicting prolonged los , and emergency readmission from a european population - based series . to our knowledge , apart from one study in japan , it is the only population - based study to investigate los in patients of all ages ; the other large population - based studies of los from the usa were restricted to patients aged 65 and older [ 4 , 5 ] . only 24 ( < 2% ) cases recorded by the ncr as having a resection in a public hospital had no corresponding hipe record . failure to find a match can occur for several reasons including : typographical errors in fields used for matching , missing data on either system or no mention of cancer on the hipe record , in which case the record would not be made available to ncr . the small numbers of missing episodes were distributed across hospitals and years . compared with other cancers , surgery with curative intent for nsclc is relatively uncommon , typically being undertaken in a minority of patients ( e.g. 1011% in england and 1619% in norway ) . we did not have access to data on lung function and derived a comorbidity score instead to provide some measure of likely health status . in our study , the median los ( 13 days ) was considerably greater than that observed in canada ( 6 days , based on 360 patients ) and spain ( 7 days , n = 727 ) . however , these data derived from case series from single centres may not be directly comparable to other hospitals or countries . several large population - based studies have presented median los after lung cancer resection : us thoracic surgery database audit ( 6 days ) , us medicare database audit ( 10 days ) , us seer subset of medicare recipients ( 6 days ) , japan ( national audit of lobectomy , mean 13.7 days ) , and a uk multicentre cohort study ( 9 days for pneumonectomy , n = 312 , 2005 ) . there is no single obvious reason why ireland should have such prolonged los for lung resection compared with other countries . in published papers , it is not always entirely clear how los has been computed and whether the authors included ( as we have done ) preoperative los . when we discounted preoperative los , postoperative los in ireland ( median = 12 days , data not shown ) was still much greater than that of any other country with published information . los in ireland following colorectal cancer surgery was also relatively high compared with international norms ; this suggests that system - level or hospital - level practices account for the findings to some degree . the largest studies on los were undertaken within the us healthcare system where every cost item ( including los ) is rigorously controlled by health insurers , which probably explains why the los in these studies is markedly lower than that of ireland . the ( postoperative mean ) estimate of los in ireland was not much greater than that of japan . note that unlike in the usa , immediate postoperative care and subsequent nursing care tend to be combined within the same hospital episode , which is why los is much greater in japan than in the usa . excess days los in irish hospitals may be devoted to subsequent nursing care rather than immediate postoperative care , as happens in japan , but we are unable to determine this from the data available . the dutch national medical registration system has proposed that all hospitals should aspire to achieve an average los for each specialty , equal to that of a benchmark model hospital for that specialty . shorter hospital stay as a result of improved discharge efficiency could reduce cost per patient and increase patient throughput . the observed associations between older age and more comorbidities and increased risk of prolonged los in our study are probably unsurprising . however , they do suggest that los following lung resection is not easily reduced , short of restricting surgery to the youngest and healthiest patients . our finding of reduced risk of prolonged los in higher - volume hospitals is consistent with a large national audit in japan , but not with a large national audit of the us medicare population ( > 65 years ) in the usa . apart from the much smaller number of hospitals performing lung cancer surgery in ireland , the number of nsclc resections / hospital carried out within irish hospitals was within the same order of magnitude as that of the usa and japan . although the excess los for patients in lower - volume hospitals in our study was modest ( only 2 days on average ) , this has the potential to impact significantly on hospital budgets in ireland ( and elsewhere ) ; this should be a particular concern for service providers in light of the increasing incidence of lung cancer projected in many populations and the increasing resort to surgical interventions [ 68 ] . one potential implication of our findings is that further centralization of surgical expertise towards higher - volume hospitals in a small county like ireland could provide a way to constrain los and achieve better economy of scale . we also observed for the first time as far as we are aware that patients resident in the most deprived areas were more likely to have a prolonged los . another study reported that patients with longer los following lung cancer surgery have higher mortality rates 2.5 years post - surgery . this suggests that the association between deprivation and los explains , at least in part , the poorer survival found in socioeconomically disadvantaged lung cancer patients . in terms of potential reasons why more deprived patients might have longer los , it is possible that they are not as prepared for discharge as less deprived patients , for reasons other than clinical factors . for example , in some instances , los could simply depend on access to transport on the proposed day of discharge ( e.g. money for a taxi or availability of a car ) ; such access may be more limited in patients resident in the most deprived areas . also , some characteristics of the most deprived patients were different from other patients . for example , those resident in the most deprived areas were less likely to be married than those resident in other areas ( 41 vs 35% ) . clinicians may be less likely to discharge a patient quickly if they lack a spouse who can provide support and care at home . fewer patients in the most deprived areas were treated privately by the managing consultant ( 22 vs 36% ) , which effectively means that they did not have private health insurance . we have shown , for colorectal cancer , that private patients have shorter los post - surgery , most likely due to pressure from the insurer ( or the patient ) to constrain costs . finally , current smoking was more common among patients resident in the most deprived areas ( 51 vs 45% ) . smokers often have significant co - existing conditions ( which may not have been fully captured by our measure of comorbidity ) and may have poorer general health status , or be in poorer physical condition , thus resulting in longer los . moreover , it is possible that they have poorer lung function , which has been previously reported to be associated with los [ 3 , 10 ] . the main reasons for emergency readmission within 28 days of discharge pulmonary complications , cardio / cerebrovascular events and infection were similar to those described in an audit of a us specialist centre . in common with research in the usa , we found that multiple comorbidities and advanced stage predisposed to readmission , and these associations are unsurprising . to our knowledge , the differentials between the most and least deprived for various characteristics were detailed above . smoking , which was more common in the deprived category , may have been a factor driving readmissions , as was lack of social support in the unmarried . it is also possible that patients in the most deprived areas were inherently more prone to adverse events due to greater comorbidity , and perhaps were slower to recognize and act upon the development of an adverse event before it became serious enough for emergency readmission , but we are not aware of any data to support this hypothesis . similar to the seer subset study then , any efforts to reduce los in ireland ( or elsewhere ) may not necessarily result in increased readmissions . our dataset of > 1200 lung cancer resections conducted in patients incident during 20028 is relatively large by european standards and provides for the first time , as far as we are aware , detailed information on factors predicting prolonged los , and emergency readmission from a european population - based series . to our knowledge , apart from one study in japan , it is the only population - based study to investigate los in patients of all ages ; the other large population - based studies of los from the usa were restricted to patients aged 65 and older [ 4 , 5 ] . only 24 ( < 2% ) cases recorded by the ncr as having a resection in a public hospital had no corresponding hipe record . failure to find a match can occur for several reasons including : typographical errors in fields used for matching , missing data on either system or no mention of cancer on the hipe record , in which case the record would not be made available to ncr . the small numbers of missing episodes were distributed across hospitals and years . compared with other cancers , surgery with curative intent for nsclc is relatively uncommon , typically being undertaken in a minority of patients ( e.g. 1011% in england and 1619% in norway ) . we did not have access to data on lung function and derived a comorbidity score instead to provide some measure of likely health status . in our study , the median los ( 13 days ) was considerably greater than that observed in canada ( 6 days , based on 360 patients ) and spain ( 7 days , n = 727 ) . however , these data derived from case series from single centres may not be directly comparable to other hospitals or countries . several large population - based studies have presented median los after lung cancer resection : us thoracic surgery database audit ( 6 days ) , us medicare database audit ( 10 days ) , us seer subset of medicare recipients ( 6 days ) , japan ( national audit of lobectomy , mean 13.7 days ) , and a uk multicentre cohort study ( 9 days for pneumonectomy , n = 312 , 2005 ) . there is no single obvious reason why ireland should have such prolonged los for lung resection compared with other countries . in published papers , it is not always entirely clear how los has been computed and whether the authors included ( as we have done ) preoperative los . when we discounted preoperative los , postoperative los in ireland ( median = 12 days , data not shown ) was still much greater than that of any other country with published information . los in ireland following colorectal cancer surgery was also relatively high compared with international norms ; this suggests that system - level or hospital - level practices account for the findings to some degree . the largest studies on los were undertaken within the us healthcare system where every cost item ( including los ) is rigorously controlled by health insurers , which probably explains why the los in these studies is markedly lower than that of ireland . the ( postoperative mean ) estimate of los in ireland was not much greater than that of japan . note that unlike in the usa , immediate postoperative care and subsequent nursing care tend to be combined within the same hospital episode , which is why los is much greater in japan than in the usa . excess days los in irish hospitals may be devoted to subsequent nursing care rather than immediate postoperative care , as happens in japan , but we are unable to determine this from the data available . the dutch national medical registration system has proposed that all hospitals should aspire to achieve an average los for each specialty , equal to that of a benchmark model hospital for that specialty . shorter hospital stay as a result of improved discharge efficiency could reduce cost per patient and increase patient throughput . the observed associations between older age and more comorbidities and increased risk of prolonged los in our study are probably unsurprising . however , they do suggest that los following lung resection is not easily reduced , short of restricting surgery to the youngest and healthiest patients . our finding of reduced risk of prolonged los in higher - volume hospitals is consistent with a large national audit in japan , but not with a large national audit of the us medicare population ( > 65 years ) in the usa . apart from the much smaller number of hospitals performing lung cancer surgery in ireland , the number of nsclc resections / hospital carried out within irish hospitals was within the same order of magnitude as that of the usa and japan . although the excess los for patients in lower - volume hospitals in our study was modest ( only 2 days on average ) , this has the potential to impact significantly on hospital budgets in ireland ( and elsewhere ) ; this should be a particular concern for service providers in light of the increasing incidence of lung cancer projected in many populations and the increasing resort to surgical interventions [ 68 ] . one potential implication of our findings is that further centralization of surgical expertise towards higher - volume hospitals in a small county like ireland could provide a way to constrain los and achieve better economy of scale . we also observed for the first time as far as we are aware that patients resident in the most deprived areas were more likely to have a prolonged los . another study reported that patients with longer los following lung cancer surgery have higher mortality rates 2.5 years post - surgery . this suggests that the association between deprivation and los explains , at least in part , the poorer survival found in socioeconomically disadvantaged lung cancer patients . in terms of potential reasons why more deprived patients might have longer los , it is possible that they are not as prepared for discharge as less deprived patients , for reasons other than clinical factors . for example , in some instances , los could simply depend on access to transport on the proposed day of discharge ( e.g. money for a taxi or availability of a car ) ; such access may be more limited in patients resident in the most deprived areas . also , some characteristics of the most deprived patients were different from other patients . for example , those resident in the most deprived areas were less likely to be married than those resident in other areas ( 41 vs 35% ) . clinicians may be less likely to discharge a patient quickly if they lack a spouse who can provide support and care at home . fewer patients in the most deprived areas were treated privately by the managing consultant ( 22 vs 36% ) , which effectively means that they did not have private health insurance . we have shown , for colorectal cancer , that private patients have shorter los post - surgery , most likely due to pressure from the insurer ( or the patient ) to constrain costs . finally , current smoking was more common among patients resident in the most deprived areas ( 51 vs 45% ) . smokers often have significant co - existing conditions ( which may not have been fully captured by our measure of comorbidity ) and may have poorer general health status , or be in poorer physical condition , thus resulting in longer los . moreover , it is possible that they have poorer lung function , which has been previously reported to be associated with los [ 3 , 10 ] . the main reasons for emergency readmission within 28 days of discharge pulmonary complications , cardio / cerebrovascular events and infection were similar to those described in an audit of a us specialist centre . in common with research in the usa , we found that multiple comorbidities and advanced stage predisposed to readmission , and these associations are unsurprising . to our knowledge , the differentials between the most and least deprived for various characteristics were detailed above . smoking , which was more common in the deprived category , may have been a factor driving readmissions , as was lack of social support in the unmarried . it is also possible that patients in the most deprived areas were inherently more prone to adverse events due to greater comorbidity , and perhaps were slower to recognize and act upon the development of an adverse event before it became serious enough for emergency readmission , but we are not aware of any data to support this hypothesis . similar to the seer subset study , prolonged los did not predict emergency readmission . by implication then , any efforts to reduce los in ireland ( or elsewhere ) may not necessarily result in increased readmissions . half of the patients undergoing resection for nsclc stay in hospital for > 13 days . deprivation , greater age , comorbidity and treatment at a lower - volume hospital were identified as risk factors for prolonged los . deprivation also predicted emergency readmission with 28 days , as did comorbidity and more advanced stage . since socioeconomic disadvantage is related to poorer survival from lung cancer , in the interests of equity , the reasons for the observed associations between deprivation and los and readmission require elucidation .

objectiveswe conducted a population - based analysis of time trends in length of stay ( los ) , predictors of prolonged los and emergency readmission following resection for non - small - cell lung cancer ( nsclc).methodsincident lung cancers ( icdo2:c34 ) , diagnosed between 2002 and 2008 , were identified from the national cancer registry ( ncr ) of ireland , and linked to hospital in - patient episodes ( hipe ) . for those with nsclc who underwent lung resection , the associated hospital episode was identified . factors predicting longer los ( upper quartile , > 20 days ) , and emergency readmission within 28 days of the index procedure ( ip ) were investigated using poisson regression.resultsa total of 1284 patients underwent resection . eighty - four ( 7% ) subsequently died in hospital and 1200 ( 93% ) were discharged . hundred and nineteen of 1200 ( 10% ) were readmitted as an emergency within 28 days of discharge . median los after the ip was 13 days ( inter - decile range : 735 ) . risk of prolonged los was significantly greater in patients > 75 years , resident in an area of highest deprivation , with 2 + comorbidities , who had undergone surgery in a lower - volume hospital , and died in hospital subsequent to the ip . emergency readmission was significantly more likely in patients who were resident in an area of highest deprivation , with 2 + comorbidities , and had stage iii disease or worse . the main reasons for emergency readmission were : pulmonary complications ( 29% ) , cardio / cerebrovascular events ( 21% ) or infection ( 20%).conclusionshalf of the patients had a los in excess of 13 days , which was longer than any other country with published data . patient and health - service factors were associated with prolonged los , while patient and tumour characteristics were associated with risk of emergency readmission . deprivation was a conspicuous determinant of both los and readmission .

INTRODUCTION METHODS Statistical analysis RESULTS DISCUSSION Strengths and limitations International comparisons in length of stay Factors associated with length of stay Emergency readmissions CONCLUSIONS Funding

surgery remains the mainstay of treatment , with curative intent for non - small - cell lung cancer ( nsclc ) patients who are medically fit , with lobectomy as the treatment of first choice . length of stay ( los ) in hospital after surgery impacts on cost and hospital performance [ 35 ] . we conducted a population - based analysis of time trends in los and predictors of prolonged los following resection for nsclc in ireland . we further investigated the factors predicting emergency readmission within 28 days of discharge after the index procedure ( ip ) . the primary data sources for this study were the national cancer registry ( ncr ) and the hospital in - patient enquiry ( hipe ) database in ireland [ 14 , 15 ] . lung cancer patients ( icd - o2 : c34 ) newly diagnosed between 2002 and 2008 were identified from the ncr . individuals who had another primary cancer prior to the lung cancer ( other than non - melanoma skin ) were excluded . a range of patient , tumour and health - service - related variables were abstracted from the ncr and hipe databases and investigated for associations with los and emergency readmission . median los , and ranges ( upper and lower decile of los ) were computed at each level of the sociodemographic , clinical and health - service - related variables . in the absence of any published definition of prolonged los in europe , the cut - point defining the upper quartile of patients ( > 20 days ) was selected as a practical threshold for prolonged los ; this also happened to be 1 week longer than the median los ( 13 days ) . three types of variables were considered for inclusion in the model : sociodemographic ( age , gender , marital status , deprivation , smoking status and public / private status ) ; clinical ( ip type , stage and comorbidity ) and health - service related ( hospital volume , destination at discharge ) . patients who had not died in hospital were classified as : ( a ) emergency readmission within 28 days of discharge , or ( b ) not readmitted as an emergency within 28 days of discharge . median los , and ranges ( upper and lower decile of los ) were computed at each level of the sociodemographic , clinical and health - service - related variables . in the absence of any published definition of prolonged los in europe , the cut - point defining the upper quartile of patients ( > 20 days ) was selected as a practical threshold for prolonged los ; this also happened to be 1 week longer than the median los ( 13 days ) . three types of variables were considered for inclusion in the model : sociodemographic ( age , gender , marital status , deprivation , smoking status and public / private status ) ; clinical ( ip type , stage and comorbidity ) and health - service related ( hospital volume , destination at discharge ) . patients who had not died in hospital were classified as : ( a ) emergency readmission within 28 days of discharge , or ( b ) not readmitted as an emergency within 28 days of discharge . of 12 139 incident cases of invasive lung cancer diagnosed in ireland between 2002 and 2008 , 1284 had nsclc and underwent lung resection in public hospitals ( fig . there was a significant increase in lung cancer resections performed per annum ( expressed as a proportion of nsclc cases ) between 2002 and 2008 ( 142/1415 ( 10% ) in 2002 , rising to 228/1613 ( 14% ) in 2008 ; p - trend = 0.000012 ) . the median total los was 13 days ( inter - decile range ( idr ) : 735 days , mean = 18 days and range : 1343 days ) . table 1:patients with nsclc undergoing resection , 20028 : numbers ( n ) , percentages ( % ) and median and idr los and p - valuesvariablescategoriesn%median losidrp - valuetotal1284100%13[735]age at diagnosis<55 year20416%10[525]0.0000000175564 year42333%12[730]6574 year47937%13[737]>75 year17814%15[750]genderfemale54142%12[732]0.22male74358%13[736]marital statusmarried81063%12[731]0.019other47437%13[740]deprivation1 least23018%12[635]0.016216613%12[527]314411%12.5[730]418715%12[628]5 most48037%13[739]unknown776%13[731]smoking statusnever smoker12410%12.5[634]0.67ex - smoker43434%12[733]current smoker60447%13[738]unknown12210%10[223]health insurancepublic88469%13[736]0.017private ( held private health insurance)40031%12[631]stagestage i / ii85467%13[736]0.023stage iii+30924%13[733]unstaged1219%10[422]comorbidity093173%12[632]0.000000026126421%13[836]2 + 897%18[859]procedurelobectomy90971%12[735]0.14pneumonectomy23418%13[733]segmental / wedge excision494%13[642]other excision927%15[437]discharge statusto home97076%12[729]0.00000059to care ( other acute hospital / nursing home / hospice)23018%12[245]died in hospital post - ip847%21.5[1091]hospital volumehigher volume : 40/year66151%12[631]0.000076lower volume : < 40/year62349%13[737]year of incidence200214211%15[836]0.037200317414%12[734]200415312%13[832]200518715%12[434]200617614%13[729]200722417%13[733]200822818%12[640]p - value : kruskal wallis test for binary and categorical variables and test for trend with ordinal categorical variables.small area ( electoral district ) based quintile of deprivation.patient had private health insurance or settled own account ( private ) , or did not have health insurance ( public ) at discharge.the patients were classified as high volume or low volume depending on whether they were operated in a hospital above or below the median resection count / hospital / year respectively ( 40/hospital / year).idr : inter - decile range ( 10th90th centile ) . table 2 presents crude and adjusted risk ratios ( rr ) for factors that were significantly associated with prolonged los in multivariate analyses ( greater than the upper quartile of los , i.e. in the adjusted analysis , the risk of prolonged los was significantly higher in patients who were older than 75 years ; lived in the most deprived areas ; had two or more comorbid conditions ; and had undergone surgery in a lower - volume hospital . patients who died in hospital subsequent to the ip were also significantly more likely to have prolonged los . although the percentage of patients who had prolonged los varied slightly according to the procedure received ( lobectomy 25% , pneumonectomy 23% , segmental 31% and other excision 31% ) , this was not statistically significant in adjusted analysis . table 2:factors significantly associated with prolonged los in patients having resection for nsclc , 20028 : number ( n ) of total ( n ) ( % ) who had prolonged los ( > 20 days ) , univariate and adjusted rr , with 95% ci and lrtsprolonged los ( > 20 days)univariateadjustedlrtnn%rr95% cirr95% cip - valuetotal312128424%age < 55 year3720418%110.064 5564 year8542320%1.11[0.78 , 1.57]1.03[0.73 , 1.45 ] 6574 year12847927%1.47[1.06 , 2.04]1.32[0.95 , 1.82 ] > 75 year6217835%1.92[1.35 , 2.74]1.55[1.08 , 2.23]sex female13254124%1 male18074324%0.99[0.82 , 1.21]deprivation less deprived ( q14)15872722%110.060 most deprived ( q5)13848029%1.32[1.09 , 1.61]1.30[1.07 , 1.58 ] unknown167721%0.96[0.61 , 1.51]1.03[0.66 , 1.62]comorbidity none20693122%110.073 17026427%1.20[0.95 , 1.51]1.11[0.88 , 1.40 ] 2 + 368940%1.83[1.38 , 2.42]1.60[1.20 , 2.13]hospital volume higher : 40/year14166121%110.086 lower : < 40/year17162327%1.29[1.03 , 1.61]1.24[0.99 , 1.56]discharge status alive at discharge266120022%110.000074 died in hospital post ip468455%2.47[1.98 , 3.08]2.03[1.60 , 2.57]mutually adjusted for age , deprivation , comorbidity , hospital volume and discharge status ( and year of incidence , not shown).rr : risk ratio ; lrt : likelihood ratio test for exclusion of that variable from multivariable model . factors significantly associated with prolonged los in patients having resection for nsclc , 20028 : number ( n ) of total ( n ) ( % ) who had prolonged los ( > 20 days ) , univariate and adjusted rr , with 95% ci and lrts mutually adjusted for age , deprivation , comorbidity , hospital volume and discharge status ( and year of incidence , not shown ) . of 1200 patients who were discharged alive after surgery ( none of whom died within 28 days of discharge ) , 119 ( 10% ) table 3 presents crude and adjusted risk ratios for factors significantly associated with risk of emergency readmission . in the adjusted analysis , risk of readmission was increased in patients from the most deprived areas , with two or more comorbid conditions , and with stage iii+ disease ( table 3 ) . patients > 75 years ( 14% of whom were readmitted , compared with 7 , 10 and 9% in the < 55 year , 5564 year and 6574 year age groups , respectively ) , and those who underwent segmental procedures ( 17% readmitted , compared with 9% for lobectomy , 12% for pneumonectomy and 9% for other procedures ) were also more prone to emergency readmission in univariate analyses ; however , age and procedure type were not significantly associated with readmission after adjustment for the factors in the model . the main reasons for emergency readmission were : pulmonary complications ( 29% ) , cardiovascular / vascular events ( 21% ) and infections ( 20% ) ( table 4 ) . table 3:factors significantly associated with emergency readmission in patients having resection for nsclc , 20028 : number ( n ) of total ( n ) ( % ) who were readmitted within 28 days ) , univariate and adjusted rr with 95% ci and lrtreadmitted within 28 dayscrudeadjustedlrtnn%rr[95% ci]rr[95% ci]p - valuetotal119120010%age < 55 year152017%1 5564 year4240610%1.39[0.79 , 2.44 ] 6574 year414419%1.25[0.71 , 2.20 ] > 75 year2115214%1.85[0.99 , 3.47]sex female5052110%1 male6967910%1.06[0.75 , 1.50]deprivation less deprived ( q14)576858%110.0095 most deprived ( q5)5944113%1.61[1.14 , 2.27]1.56[1.11 , 2.20 ] unknown3744%0.49[0.16 , 1.52]0.48[0.16 , 1.45]comorbidity none748798%110.011 13024412%1.46[0.98 , 2.18]1.43[0.96 , 2.12 ] 2 + 157719%2.31[1.40 , 3.83]2.38[1.43 , 3.96]stage stage i / ii728019%110.039 stage iii+3928114%1.54[1.07 , 2.23]1.62[1.13 , 2.34 ] unstaged81187%0.75[0.37 , 1.53]0.83[0.41 , 1.69]mutually adjusted for deprivation , comorbidity and stage.rr : risk ratio ; lrt : p - value of likelihood ratio test for exclusion of that variable in multivariable model . table 4:primary reason for first emergency readmission within 28 days of ipreadmission causedescriptionn ( % ) subtotal number ( % ) pulmonary complicationatelectasis11 ( 9%)pneumonia8 ( 7%)pulmonary air leak6 ( 5%)pleural effusion7 ( 6%)empyema2 ( 2%)34 ( 29%)cardiovascular / vasculardysrhythmia18 ( 15%)myocardial infarction4 ( 3%)cerebrovascular event3 ( 3%)25 ( 21%)infectionwound infection6 ( 5%)fever14 ( 12%)urinary tract infection3 ( 3%)23 ( 20%)othersrenal insufficiency2 ( 2%)others35 ( 29%)37 ( 31%)total119 ( 100% ) factors significantly associated with emergency readmission in patients having resection for nsclc , 20028 : number ( n ) of total ( n ) ( % ) who were readmitted within 28 days ) , univariate and adjusted rr with 95% ci and lrt mutually adjusted for deprivation , comorbidity and stage . our dataset of > 1200 lung cancer resections conducted in patients incident during 20028 is relatively large by european standards and provides for the first time , as far as we are aware , detailed information on factors predicting prolonged los , and emergency readmission from a european population - based series . to our knowledge , apart from one study in japan , it is the only population - based study to investigate los in patients of all ages ; the other large population - based studies of los from the usa were restricted to patients aged 65 and older [ 4 , 5 ] . in our study , the median los ( 13 days ) was considerably greater than that observed in canada ( 6 days , based on 360 patients ) and spain ( 7 days , n = 727 ) . several large population - based studies have presented median los after lung cancer resection : us thoracic surgery database audit ( 6 days ) , us medicare database audit ( 10 days ) , us seer subset of medicare recipients ( 6 days ) , japan ( national audit of lobectomy , mean 13.7 days ) , and a uk multicentre cohort study ( 9 days for pneumonectomy , n = 312 , 2005 ) . when we discounted preoperative los , postoperative los in ireland ( median = 12 days , data not shown ) was still much greater than that of any other country with published information . our finding of reduced risk of prolonged los in higher - volume hospitals is consistent with a large national audit in japan , but not with a large national audit of the us medicare population ( > 65 years ) in the usa . apart from the much smaller number of hospitals performing lung cancer surgery in ireland , the number of nsclc resections / hospital carried out within irish hospitals was within the same order of magnitude as that of the usa and japan . although the excess los for patients in lower - volume hospitals in our study was modest ( only 2 days on average ) , this has the potential to impact significantly on hospital budgets in ireland ( and elsewhere ) ; this should be a particular concern for service providers in light of the increasing incidence of lung cancer projected in many populations and the increasing resort to surgical interventions [ 68 ] . the main reasons for emergency readmission within 28 days of discharge pulmonary complications , cardio / cerebrovascular events and infection were similar to those described in an audit of a us specialist centre . our dataset of > 1200 lung cancer resections conducted in patients incident during 20028 is relatively large by european standards and provides for the first time , as far as we are aware , detailed information on factors predicting prolonged los , and emergency readmission from a european population - based series . to our knowledge , apart from one study in japan , it is the only population - based study to investigate los in patients of all ages ; the other large population - based studies of los from the usa were restricted to patients aged 65 and older [ 4 , 5 ] . in our study , the median los ( 13 days ) was considerably greater than that observed in canada ( 6 days , based on 360 patients ) and spain ( 7 days , n = 727 ) . several large population - based studies have presented median los after lung cancer resection : us thoracic surgery database audit ( 6 days ) , us medicare database audit ( 10 days ) , us seer subset of medicare recipients ( 6 days ) , japan ( national audit of lobectomy , mean 13.7 days ) , and a uk multicentre cohort study ( 9 days for pneumonectomy , n = 312 , 2005 ) . when we discounted preoperative los , postoperative los in ireland ( median = 12 days , data not shown ) was still much greater than that of any other country with published information . our finding of reduced risk of prolonged los in higher - volume hospitals is consistent with a large national audit in japan , but not with a large national audit of the us medicare population ( > 65 years ) in the usa . apart from the much smaller number of hospitals performing lung cancer surgery in ireland , the number of nsclc resections / hospital carried out within irish hospitals was within the same order of magnitude as that of the usa and japan . the main reasons for emergency readmission within 28 days of discharge pulmonary complications , cardio / cerebrovascular events and infection were similar to those described in an audit of a us specialist centre . deprivation , greater age , comorbidity and treatment at a lower - volume hospital were identified as risk factors for prolonged los . since socioeconomic disadvantage is related to poorer survival from lung cancer , in the interests of equity , the reasons for the observed associations between deprivation and los and readmission require elucidation .

intracranial hemangiopericytoma ( hpc ) was first reported by begg and garret4 ) , who noted its microscopic characteristics including meningiomas . hpc arises from zimmerman pericytes around capillaries and postcapillary venules ; therefore , these tumors can occur anywhere capillaries are found , especially in the lower extremities , retroperitoneum and pelvis24,45 ) . intracranial hpcs rarely present in the central nervous system , and they account for 2.5% of all meningeal tumors and less than 1% of all cns tumors22,23 ) . these tumors also have malignant features , such as a tendency to recur locally and metastasize distantly . despite the high rate of local recurrence and distant metastasis , we reviewed our experiences with 13 patients with intracranial hpcs to analyze the disease features , treatment strategies and clinical courses . in particular , we focused on the efficacy of resection and adjuvant radiotherapy ( rt ) . a total of 13 patients underwent microsurgical resection for intracranial hpc at least once at our institute from january 1995 to may 2013 . four patients had preoperative digital subtraction angiography ( dsa ) and only one patient underwent tumor embolization using dimethyl sulfoxide ( dmso ) and onyx . a total of 20 operations were performed for the resection of either primary or recurrent intracranial hpcs . after 2002 , all operations were performed by assisted neuronavigation . and intraoperative electrophysiologic monitoring was performed after 2009 . we reviewed the original operation records to determine the estimated blood loss ( ebl ) , macroscopic features and extent of tumor resection . immunohistochemistry was examined in all cases , including ema , s-100 , cd34 and the ki-67 proliferative index . the mean follow - up period was 54.3 months , with a range of 13 to 185 months . information regarding postoperative status was obtained from our outpatient department medical records and phone contacts . statistical analysis was carried out using kaplan - meier method , and the comparison of recurrence free survival rate between different two groups was conducted using log - rank test . the patient group consisted of 12 ( 92.3% ) males and 1 ( 7.7% ) female . patient ages at the time of initial diagnosis ranged from 26 to 73 years , with a mean of 48 years . the most common presenting symptom was headache , and four patients had visual field defects due to a tumor in the occipital lobe . during the study period , we diagnosed 13 intracranial hpcs and 598 meningiomas . the ratio of intracranial hpcs to meningiomas was 1 : 46 , and the proportion of hpcs was only 2.2% . the preferential areas of tumor origin were parasagittal ( 5 patients , 38% ) and falx ( 3 patients , 23% ) . one patient had a homogeneous enhancing pineal gland tumor which turned out to be hpc . distributions of the remaining tumor locations were as follows : two convexity , one sphenoid wing and one cerebellopontine angle . the preoperative contrast - enhanced ct scans of 12 patients ( ct was not available for one patient ) revealed hyperdense and homogeneous or heterogeneous enhancing lesions with no evidence of calcification . radiologic findings of osteolysis of the skull were noted in 2 patients ( 15.4% ) ( fig . enhanced mr images , all patients had well demarcated tumor margins and detailed features of mr images are described in table 1 . preoperative dsas were performed on 4 patients ( 30.8% ) because they had many fluid void signals on the t2-weighted images . each dsa image showed a hypervascular contrast staining mass with an arterial feeder from the middle meningeal artery ( case no . 8) , distal branches of the pericallosal artery and some posterior choroidal arteries ( case no . two patients ( 15.4% ) showed hemorrhage from the hpc on the initial ct scans ( case nos . 1 , 2 ) one day preceding the operation for tumor resection , embolization was performed by dmso and onyx via the left distal mca . post - embolic angiography showed a decrease in the size of the hypervascular contrast staining mass ( fig . 3 ) . an mr image obtained immediately after embolization of the tumor showed no acute infarction or venous congestion . all patients were initially treated with surgical resections and there were no perioperative mortalities . at the initial operation , a grossly total resection ( gtr ) was accomplished in 9 patients ( 69.2% ) , a partial resection ( pr ) in 2 patients ( 15.4% ) and an endoscopic biopsy in 1 patient , while results were not available for one patient . one patient underwent only an endoscopic biopsy because of a deep seated tumor ( pineal lesion , case no . another patient underwent the initial operation at an outside institution , so we could not estimate the extent of the resection . according to the original operation records , ebl ranged from 350 ml to 1200 ml and the mean ebl was 850 ml , for all patients . postoperative external beam radiotherapy ( ebrt ) was delivered in 11 patients ( 84.6% ) regardless of the extent of resection . 9 ) rejected an active treatment after the initial surgery and another patient ( case no . 4 ) was not irradiated postoperatively due to misdiagnosis . however , he received irradiation ( 1500 cgy ) by cyberknife 3 months later . the mean total ebrt dose was 5770 cgy ( range : 5400 - 6000 cgy ) . two patients ( 15.4% ) underwent stereotactic radiosurgery using a cyberknife for treatment of recurrent tumors . his initial pathologic diagnosis was pineal parenchymal neoplasm with intermediate undifferentiation , and he received a total of 2 cycles of ice chemotherapy ( a regimen employing ifosfamide , carboplatin and etoposide ) . but , the tumor progressed during chemotherapy and mental confusion developed due to an obstructed hydrocephalus . six patients ( 46.1% ) had low grade hpcs ( who grade ii ) and 7 patients ( 53.9% ) had anaplastic hpcs ( who grade iii ) , a percentage that is relatively higher than in other studies . interestingly , the pathologic diagnosis of the resected recurrent tumors changed from low grade to anaplastic hpcs in 2 patients ( 15.4% , case nos . 2 , 4 ) . low grade hpcs ( who grade ii ) showed dense cellularity with branched thin walled vessels ( fig . anaplastic hpcs ( who grade iii ) showed cellular pleomorphism with frequent mitosis and necrosis ( fig . all resected tumors had well developed reticulin fibers , and cd34 was positive in 10 patients ( 83.3% ) and negative in 2 patients ( 16.7% ) . the ki-67 proliferative index ranged from 1% to 30% , wi th a mean of 11.3% . the mean ki-67 proliferative index was 2.2% in the low grade hpc group , and 18.3% in the anaplastic hpc groups . six patients ( 46.2% ) developed local recurrences at 104 , 73 , 19 , 4 and 3 ( two patients ) months after initial surgery . of the 6 patients , one underwent five operations , another underwent four operations and the other two underwent two operations . the two remaining patients underwent only the initial operation because of their poor general medical condition . five and 10-year recurrence - free survival ( rfs ) rate was 62% and 20.5% , respectively . mean time for local recurrence was 24.6 months in patients who treated with pr or biopsy against 105.7 months in patients who treated with gtr ( p=0.012 by log - rank test ) ( fig . 7 ) . regarding adjuvant rt , mean time for local recurrence was 10.7 months in patients who did not received rt against 85.4 months in patients who received rt ( p=0.052 by log - rank test ) ( fig . 8) . there were no distant metastases , based on clinical follow - up and whole body pet - ct scan . the outcomes of treatment were estimated according to the karnofsky performance scale ( kps ) . the mean kps at the final outcome was 64.6 ( initial mean kps was 70.8 ) . the mean follow - up period was 54.3 months ( range : 13 - 185 months ) . five year overall survival rate was 84% and mean overall survival was 158 months ( fig . the patient group consisted of 12 ( 92.3% ) males and 1 ( 7.7% ) female . patient ages at the time of initial diagnosis ranged from 26 to 73 years , with a mean of 48 years . the most common presenting symptom was headache , and four patients had visual field defects due to a tumor in the occipital lobe . the ratio of intracranial hpcs to meningiomas was 1 : 46 , and the proportion of hpcs was only 2.2% . the preferential areas of tumor origin were parasagittal ( 5 patients , 38% ) and falx ( 3 patients , 23% ) . one patient had a homogeneous enhancing pineal gland tumor which turned out to be hpc . distributions of the remaining tumor locations were as follows : two convexity , one sphenoid wing and one cerebellopontine angle . the preoperative contrast - enhanced ct scans of 12 patients ( ct was not available for one patient ) revealed hyperdense and homogeneous or heterogeneous enhancing lesions with no evidence of calcification . radiologic findings of osteolysis of the skull were noted in 2 patients ( 15.4% ) ( fig . enhanced mr images , all patients had well demarcated tumor margins and detailed features of mr images are described in table 1 . preoperative dsas were performed on 4 patients ( 30.8% ) because they had many fluid void signals on the t2-weighted images . each dsa image showed a hypervascular contrast staining mass with an arterial feeder from the middle meningeal artery ( case no . 8) , distal branches of the pericallosal artery and some posterior choroidal arteries ( case no . two patients ( 15.4% ) showed hemorrhage from the hpc on the initial ct scans ( case nos . 1 , 2 ) one day preceding the operation for tumor resection , embolization was performed by dmso and onyx via the left distal mca . post - embolic angiography showed a decrease in the size of the hypervascular contrast staining mass ( fig . 3 ) . an mr image obtained immediately after embolization of the tumor showed no acute infarction or venous congestion . additionally , the patient had no neurological deterioration after embolization . all patients were initially treated with surgical resections and there were no perioperative mortalities . at the initial operation , a grossly total resection ( gtr ) was accomplished in 9 patients ( 69.2% ) , a partial resection ( pr ) in 2 patients ( 15.4% ) and an endoscopic biopsy in 1 patient , while results were not available for one patient . two patients had aborted operations because of intraoperative bleeding that resulted in partial resection . one patient underwent only an endoscopic biopsy because of a deep seated tumor ( pineal lesion , case no . another patient underwent the initial operation at an outside institution , so we could not estimate the extent of the resection . according to the original operation records , ebl ranged from 350 ml to 1200 ml and the mean ebl was 850 ml , for all patients . postoperative external beam radiotherapy ( ebrt ) was delivered in 11 patients ( 84.6% ) regardless of the extent of resection . 9 ) rejected an active treatment after the initial surgery and another patient ( case no . 4 ) was not irradiated postoperatively due to misdiagnosis . however , he received irradiation ( 1500 cgy ) by cyberknife 3 months later . the mean total ebrt dose was 5770 cgy ( range : 5400 - 6000 cgy ) . two patients ( 15.4% ) underwent stereotactic radiosurgery using a cyberknife for treatment of recurrent tumors . his initial pathologic diagnosis was pineal parenchymal neoplasm with intermediate undifferentiation , and he received a total of 2 cycles of ice chemotherapy ( a regimen employing ifosfamide , carboplatin and etoposide ) . but , the tumor progressed during chemotherapy and mental confusion developed due to an obstructed hydrocephalus . one day preceding the operation for tumor resection , embolization was performed by dmso and onyx via the left distal mca . post - embolic angiography showed a decrease in the size of the hypervascular contrast staining mass ( fig . 3 ) . an mr image obtained immediately after embolization of the tumor showed no acute infarction or venous congestion . all patients were initially treated with surgical resections and there were no perioperative mortalities . at the initial operation , a grossly total resection ( gtr ) was accomplished in 9 patients ( 69.2% ) , a partial resection ( pr ) in 2 patients ( 15.4% ) and an endoscopic biopsy in 1 patient , while results were not available for one patient . one patient underwent only an endoscopic biopsy because of a deep seated tumor ( pineal lesion , case no . another patient underwent the initial operation at an outside institution , so we could not estimate the extent of the resection . according to the original operation records , ebl ranged from 350 ml to 1200 ml and the mean ebl was 850 ml , for all patients . postoperative external beam radiotherapy ( ebrt ) was delivered in 11 patients ( 84.6% ) regardless of the extent of resection . 9 ) rejected an active treatment after the initial surgery and another patient ( case no . 4 ) was not irradiated postoperatively due to misdiagnosis . however , he received irradiation ( 1500 cgy ) by cyberknife 3 months later . the mean total ebrt dose was 5770 cgy ( range : 5400 - 6000 cgy ) . two patients ( 15.4% ) underwent stereotactic radiosurgery using a cyberknife for treatment of recurrent tumors . his initial pathologic diagnosis was pineal parenchymal neoplasm with intermediate undifferentiation , and he received a total of 2 cycles of ice chemotherapy ( a regimen employing ifosfamide , carboplatin and etoposide ) . but , the tumor progressed during chemotherapy and mental confusion developed due to an obstructed hydrocephalus . six patients ( 46.1% ) had low grade hpcs ( who grade ii ) and 7 patients ( 53.9% ) had anaplastic hpcs ( who grade iii ) , a percentage that is relatively higher than in other studies . interestingly , the pathologic diagnosis of the resected recurrent tumors changed from low grade to anaplastic hpcs in 2 patients ( 15.4% , case nos . 2 , 4 ) . low grade hpcs ( who grade ii ) showed dense cellularity with branched thin walled vessels ( fig . tumor cells had diffuse cd34 positivity ( fig . 4b ) and the ki-67 proliferative index was 10% ( fig . anaplastic hpcs ( who grade iii ) showed cellular pleomorphism with frequent mitosis and necrosis ( fig . all resected tumors had well developed reticulin fibers , and cd34 was positive in 10 patients ( 83.3% ) and negative in 2 patients ( 16.7% ) . the ki-67 proliferative index ranged from 1% to 30% , wi th a mean of 11.3% . the mean ki-67 proliferative index was 2.2% in the low grade hpc group , and 18.3% in the anaplastic hpc groups . six patients ( 46.2% ) developed local recurrences at 104 , 73 , 19 , 4 and 3 ( two patients ) months after initial surgery . of the 6 patients , one underwent five operations , another underwent four operations and the other two underwent two operations . the two remaining patients underwent only the initial operation because of their poor general medical condition . five and 10-year recurrence - free survival ( rfs ) rate was 62% and 20.5% , respectively . mean time for local recurrence was 24.6 months in patients who treated with pr or biopsy against 105.7 months in patients who treated with gtr ( p=0.012 by log - rank test ) ( fig . regarding adjuvant rt , mean time for local recurrence was 10.7 months in patients who did not received rt against 85.4 months in patients who received rt ( p=0.052 by log - rank test ) ( fig . 8) . there were no distant metastases , based on clinical follow - up and whole body pet - ct scan . the outcomes of treatment were estimated according to the karnofsky performance scale ( kps ) . the mean kps at the final outcome was 64.6 ( initial mean kps was 70.8 ) . the mean follow - up period was 54.3 months ( range : 13 - 185 months ) . five year overall survival rate was 84% and mean overall survival was 158 months ( fig . intracranial hpcs are very rare tumors with an incidence of less than 1% , and they represented 2.2% of all meningiomas diagnosed over the last 18 years in our institute . it is widely known that intracranial hpcs , unlike meningiomas , are more common in males than females , which was also true in our data , where 92% of the patients were males5,18,25 ) . in previous studies on intracranial hpcs , the mean age at the time of diagnosis ranged from 38 to 44.9 years , which was lower than that of meningiomas1,5,22,23,25,48 ) . in our series , our study included two patients ( 15.4% ) with intracerebral hemorrhage on an initial ct scan . the majority of hpcs arise in the supratentorial area with dural attachment23,41 ) . however , non - dural attached hpcs , such as in the pineal region ( case no.4 ) , and purely intraparenchymal hpcs have also been reported28,39,41 ) . distinguishing hpcs from benign meningiomas before surgery can be difficult , but is very important because of the aggressiveness of hpcs and their high rates of local recurrence and distant metastasis . chiechi et al.9 ) reported that intracranial hpcs are more multilobulated than benign meningiomas , and have narrow based dural attachment on ct and conventional mr images . additionally , hyperostosis and intralesional calcification are not found in intracranial hpcs , which is distinct from meningiomas . many other papers have reported that intracranial hpcs may show osteolytic features on plain films , ct and mr images6,11,33 ) . in the current study , osteolysis of the skull on ct scan was noted in 2 patients ( case nos . 5 , 11 ) who were diagnosed with anaplastic hpc . it has been found that osteolysis is more prevalent in anaplastic hpcs than in low grade hpcs9 ) . it has also been found that ct perfusion is valuable in preoperative grading of intracranial gliomas13 ) , in distinguishing between glioblastomas , metastasis and abscesses8,12 ) and in evaluating the prognosis of recurrent brain tumors after stereotactic radiosurgery26 ) . similarly , ren et al.34 ) reported that ct perfusion can provide crucial information on hpcs and benign meningiomas . as mri technology has advanced , liu et al.30 ) demonstrated that minimum apparent diffusion coefficient ( adc ) values may be helpful for distinguishing hpcs from meningiomas . they found that the mean minimum adc values of hpcs were significantly higher than that of meningiomas . the frailty of the conventional surgical approach was intractable intraoperative bleeding from the meningeal artery during the initial stage of the operation . thus , several authors have proposed multi - stage operations , included feeding artery embolization27 ) . matsushige et al.31 ) reported a giant hpc treated with preoperative tumor embolization , where embolization was used to overcome intraoperative bleeding . the use of preoperative tumor embolization has also decreased operation - related mortality and morbidity1 ) . after devascularization of the tumor from the main feeding artery using embolization , we were also able to markedly reduce intraoperative bleeding in case no . 10 . the blood loss during operation was much lower compared to non - embolized patients ( 350 ml vs. 990 ml ) , but we could not draw statistically significant conclusions due to the limited sample size of our study . surgical resection of the tumors offers the advantage of pathologic confirmation and reduction of mass effects . a simpson grade i resection has been found to be associated with a high long - term control rate2,18 ) . similarly , rutkowski et al.36 ) reported that , regardless of adjuvant therapy , gtr was associated with significantly improved survival rates compared with pr . in our series , a total of 9 patients ( 69% ) achieved a gtr at the initial surgery , which was a much higher rate compared to other studies . a literature review on intracranial hpcs showed an average gtr rate of 56.7% ( table 2 ) . we conducted a statistical analysis about correlation between extent of resection and recurrence , and we found statistical significance like other several series ( mean rfs 105.7 months vs. 24.6 months , p=0.012 ) ( fig . interestingly , the average mortality rate in the recent literature was 31.5%1,2,15,16,18,21,28,35,37,42,44 ) , but was only 15.4% in our study . though adjuvant therapy and follow - up period also affect the mortality rate , we cautiously suggest that gtr also contributed to the decreased mortality rate . staples et al.43 ) reported that the 5-year rfs improved from 28% with operation alone to 57% with adjuvant rt after gtr . similarly , we could find that the patients who had been recieved adjuvant rt had tendency of longer rfs ( mean rfs 85.4 months vs. 10.7 months , p=0.052 ) ( fig . a review of the recent literature shows that adjuvant rt is not applied consistently ( range : 17 - 91% , mean : 47.4% ) . on the other hand , we performed adjuvant rt regardless of the pathologic grade and extent of resection in most of the patients , 11 out of 13 patients received adjuvant rt to a mean dose of 5770 cgy . serious side effects from irradiation , such as cerebral edema , leucencephalopathy , radiation - induced necrosis and death have been reported42 ) . in our study , there were no significant radiation - related complications . chemotherapy provided only insignificant benefits3,20 ) , but chamberlain and glantz7 ) reported that chemotherapy ( cyclophosphamide+adriamycin+vincristine , followed by -ifn , followed by ice ) may be helpful for recurrent intractable intracranial hpcs . in our study , one patient was treated with chemotherapy ( ice regimen ) due to an initial misdiagnosis of pineal sarcoma . the histological criteria for anaplastic hpcs ( who grade iii ) include more than five mitoses in 10 high power fields and/or necrosis and moderate to high nuclear atypia and moderate to high cellularity after hemorrhage . moreover , the pathologic features of low grade hpcs ( who grade ii ) show monomorphous round to oval tumor cells having staghorn - like vessels . the tumors contain well developed reticulin fibers and diffuse cd34 immunopositivity . in our data , the mean ki-67 proliferative indexes in low grade and anaplastic hpc were 2.2% and 18.3% , respectively . in recurrent hpc , the mean ki-67 was 14.6% , while in non - recurrent hpc , it was 7.8% . reported that the patients with a ki-67 index less than 5% tended to survive longer , but this finding could not correlate the clinical outcome46,47 ) . also we conducted statistical analysis of rfs depending on the who grade , but we could not find statistical significance ( p=0.644 ) . galanis et al.20 ) found that , out of 32 recurrences , 19 ( 59% ) recurred at the primary site , eleven ( 34% ) recurred at both the primary and other cns sites and the remaining 2 ( 7% ) had diffuse leptomeningeal spread without local recurrence at the primary site . in our series , we would define ' recurrence ' as increased tumor sized by follow - up radiologic study and any apparent deterioration in clinical status to need reoperation . in the strict sense , initially partial resection or biopsy cases should be described as ' progression ' when reoperation were needed , but number of our cases were too small to analyze about progression cases , separately . unfortunately , several authors reported that the extent of tumor resection has been less definitely correlated with the local recurrence of hpcs compared to meningiomas29 ) . this feature of intracranial hpcs might be explained by the strategy to aggressively irradiate partially resected tumors to reduce local recurrence . recently , du et al.14 ) reported only a 3.8% recurrence rate , but their follow - up period was very limited ( mean : 22 months ) . in our series , the recurrence rate was 46.2% ( 6/13 ) , with the mean follow - up period of 54.3 months ( range : 13 - 185 months ) . it is widely known that after the first recurrence , hpc tends to recur at brief intervals . guthrie et al.23 ) reported that the average periods to the second , third and fourth surgeries for recurrence were 38 , 35 and 17 months , respectively . schrder et al.38 ) found 5- , 10- and 15-year survival rates of 65% , 45% and 15% , respectively . in their broad literature review , rutkowski et al.36 ) found 1- , 5- , 10- and 20-year survival rates of 95% , 82% , 60% and 23% , respectively . mena et al.32 ) reported a recurrence rate of 70% ( 57/94 ) , while kochanek et al.29 ) reported 41% ( 7/17 ) . it seems that rarity of intracranial hpcs along with the absence of a well formulated treatment strategy might lead to highly fluctuating survival and local recurrence rates . we believe that adjuvant radiation therapy is helpful , even if maximal tumor resection was performed in low grade hpcs . molecular biologic analysis and studies of the efficacy of chemotherapy are required for better outcomes in relentlessly recurrent hpcs .

objectiveintracranial hemangiopericytomas ( hpcs ) are rare tumors with aggressive behavior , including local recurrence and distant metastasis . we conducted this retrospective study to evaluate the efficacy of grossly total resection and adjuvant radiotherapy ( rt ) for these tumors.methodsa total of 13 patients treated for intracranial hpc from january 1995 through may 2013 were included in this retrospective study . we analyzed the clinical presentations , radiologic appearances , treatment results , and follow - up outcomes , as well as reviewed other studies.resultsthe ages of the patients at the time of diagnosis ranged from 26 to 73 years ( mean : 48 years ) . the majority of the patients were male ( 92.3% ) , and the majority of the tumors were located in the parasagittal and falx . the ratio of intracranial hpcs to meningiomas was 13 : 598 in same period , or 2.2% . seven patients ( 53.8% ) had anaplastic hpcs . nine patients ( 69.2% ) underwent gross total tumor resection in the first operation without mortality . eleven patients ( 84.6% ) underwent postoperative adjuvant rt . follow - up period ranged from 13 to 185 months ( mean : 54.3 months ) . the local recurrence rate was 46.2% ( 6/13 ) , and there were no distant metastases . the 10-year survival rate after initial surgery was 83.9% . the initial mean karnofsky performance scale ( kps ) was 70.8 and the final mean kps was 64.6.conclusiongross total tumor resection upon initial surgery is very important . we believe that adjuvant rt is helpful even with maximal tumor resection . molecular biologic analyses and chemotherapy studies are required to achieve better outcomes in recurrent intracranial hpcs .

INTRODUCTION MATERIALS AND METHODS RESULTS Patient population Incidence of intracranial hemangiopericytoma Tumor characteristics Treatment modalities Preoperative embolization Surgery Radiotherapy Chemotherapy Pathologic results Local recurrences and distant metastases Overall outcome and survival DISCUSSION CONCLUSION

intracranial hemangiopericytoma ( hpc ) was first reported by begg and garret4 ) , who noted its microscopic characteristics including meningiomas . hpc arises from zimmerman pericytes around capillaries and postcapillary venules ; therefore , these tumors can occur anywhere capillaries are found , especially in the lower extremities , retroperitoneum and pelvis24,45 ) . intracranial hpcs rarely present in the central nervous system , and they account for 2.5% of all meningeal tumors and less than 1% of all cns tumors22,23 ) . despite the high rate of local recurrence and distant metastasis , we reviewed our experiences with 13 patients with intracranial hpcs to analyze the disease features , treatment strategies and clinical courses . in particular , we focused on the efficacy of resection and adjuvant radiotherapy ( rt ) . a total of 13 patients underwent microsurgical resection for intracranial hpc at least once at our institute from january 1995 to may 2013 . a total of 20 operations were performed for the resection of either primary or recurrent intracranial hpcs . we reviewed the original operation records to determine the estimated blood loss ( ebl ) , macroscopic features and extent of tumor resection . immunohistochemistry was examined in all cases , including ema , s-100 , cd34 and the ki-67 proliferative index . the mean follow - up period was 54.3 months , with a range of 13 to 185 months . statistical analysis was carried out using kaplan - meier method , and the comparison of recurrence free survival rate between different two groups was conducted using log - rank test . the patient group consisted of 12 ( 92.3% ) males and 1 ( 7.7% ) female . patient ages at the time of initial diagnosis ranged from 26 to 73 years , with a mean of 48 years . the most common presenting symptom was headache , and four patients had visual field defects due to a tumor in the occipital lobe . during the study period , we diagnosed 13 intracranial hpcs and 598 meningiomas . the ratio of intracranial hpcs to meningiomas was 1 : 46 , and the proportion of hpcs was only 2.2% . the preferential areas of tumor origin were parasagittal ( 5 patients , 38% ) and falx ( 3 patients , 23% ) . distributions of the remaining tumor locations were as follows : two convexity , one sphenoid wing and one cerebellopontine angle . the preoperative contrast - enhanced ct scans of 12 patients ( ct was not available for one patient ) revealed hyperdense and homogeneous or heterogeneous enhancing lesions with no evidence of calcification . radiologic findings of osteolysis of the skull were noted in 2 patients ( 15.4% ) ( fig . preoperative dsas were performed on 4 patients ( 30.8% ) because they had many fluid void signals on the t2-weighted images . two patients ( 15.4% ) showed hemorrhage from the hpc on the initial ct scans ( case nos . post - embolic angiography showed a decrease in the size of the hypervascular contrast staining mass ( fig . an mr image obtained immediately after embolization of the tumor showed no acute infarction or venous congestion . all patients were initially treated with surgical resections and there were no perioperative mortalities . at the initial operation , a grossly total resection ( gtr ) was accomplished in 9 patients ( 69.2% ) , a partial resection ( pr ) in 2 patients ( 15.4% ) and an endoscopic biopsy in 1 patient , while results were not available for one patient . another patient underwent the initial operation at an outside institution , so we could not estimate the extent of the resection . according to the original operation records , ebl ranged from 350 ml to 1200 ml and the mean ebl was 850 ml , for all patients . postoperative external beam radiotherapy ( ebrt ) was delivered in 11 patients ( 84.6% ) regardless of the extent of resection . 9 ) rejected an active treatment after the initial surgery and another patient ( case no . two patients ( 15.4% ) underwent stereotactic radiosurgery using a cyberknife for treatment of recurrent tumors . his initial pathologic diagnosis was pineal parenchymal neoplasm with intermediate undifferentiation , and he received a total of 2 cycles of ice chemotherapy ( a regimen employing ifosfamide , carboplatin and etoposide ) . six patients ( 46.1% ) had low grade hpcs ( who grade ii ) and 7 patients ( 53.9% ) had anaplastic hpcs ( who grade iii ) , a percentage that is relatively higher than in other studies . interestingly , the pathologic diagnosis of the resected recurrent tumors changed from low grade to anaplastic hpcs in 2 patients ( 15.4% , case nos . anaplastic hpcs ( who grade iii ) showed cellular pleomorphism with frequent mitosis and necrosis ( fig . all resected tumors had well developed reticulin fibers , and cd34 was positive in 10 patients ( 83.3% ) and negative in 2 patients ( 16.7% ) . the ki-67 proliferative index ranged from 1% to 30% , wi th a mean of 11.3% . the mean ki-67 proliferative index was 2.2% in the low grade hpc group , and 18.3% in the anaplastic hpc groups . six patients ( 46.2% ) developed local recurrences at 104 , 73 , 19 , 4 and 3 ( two patients ) months after initial surgery . of the 6 patients , one underwent five operations , another underwent four operations and the other two underwent two operations . mean time for local recurrence was 24.6 months in patients who treated with pr or biopsy against 105.7 months in patients who treated with gtr ( p=0.012 by log - rank test ) ( fig . regarding adjuvant rt , mean time for local recurrence was 10.7 months in patients who did not received rt against 85.4 months in patients who received rt ( p=0.052 by log - rank test ) ( fig . there were no distant metastases , based on clinical follow - up and whole body pet - ct scan . the outcomes of treatment were estimated according to the karnofsky performance scale ( kps ) . the mean kps at the final outcome was 64.6 ( initial mean kps was 70.8 ) . the mean follow - up period was 54.3 months ( range : 13 - 185 months ) . five year overall survival rate was 84% and mean overall survival was 158 months ( fig . the patient group consisted of 12 ( 92.3% ) males and 1 ( 7.7% ) female . patient ages at the time of initial diagnosis ranged from 26 to 73 years , with a mean of 48 years . the most common presenting symptom was headache , and four patients had visual field defects due to a tumor in the occipital lobe . the ratio of intracranial hpcs to meningiomas was 1 : 46 , and the proportion of hpcs was only 2.2% . radiologic findings of osteolysis of the skull were noted in 2 patients ( 15.4% ) ( fig . preoperative dsas were performed on 4 patients ( 30.8% ) because they had many fluid void signals on the t2-weighted images . two patients ( 15.4% ) showed hemorrhage from the hpc on the initial ct scans ( case nos . post - embolic angiography showed a decrease in the size of the hypervascular contrast staining mass ( fig . an mr image obtained immediately after embolization of the tumor showed no acute infarction or venous congestion . all patients were initially treated with surgical resections and there were no perioperative mortalities . at the initial operation , a grossly total resection ( gtr ) was accomplished in 9 patients ( 69.2% ) , a partial resection ( pr ) in 2 patients ( 15.4% ) and an endoscopic biopsy in 1 patient , while results were not available for one patient . another patient underwent the initial operation at an outside institution , so we could not estimate the extent of the resection . according to the original operation records , ebl ranged from 350 ml to 1200 ml and the mean ebl was 850 ml , for all patients . postoperative external beam radiotherapy ( ebrt ) was delivered in 11 patients ( 84.6% ) regardless of the extent of resection . 9 ) rejected an active treatment after the initial surgery and another patient ( case no . however , he received irradiation ( 1500 cgy ) by cyberknife 3 months later . two patients ( 15.4% ) underwent stereotactic radiosurgery using a cyberknife for treatment of recurrent tumors . his initial pathologic diagnosis was pineal parenchymal neoplasm with intermediate undifferentiation , and he received a total of 2 cycles of ice chemotherapy ( a regimen employing ifosfamide , carboplatin and etoposide ) . post - embolic angiography showed a decrease in the size of the hypervascular contrast staining mass ( fig . all patients were initially treated with surgical resections and there were no perioperative mortalities . at the initial operation , a grossly total resection ( gtr ) was accomplished in 9 patients ( 69.2% ) , a partial resection ( pr ) in 2 patients ( 15.4% ) and an endoscopic biopsy in 1 patient , while results were not available for one patient . another patient underwent the initial operation at an outside institution , so we could not estimate the extent of the resection . according to the original operation records , ebl ranged from 350 ml to 1200 ml and the mean ebl was 850 ml , for all patients . postoperative external beam radiotherapy ( ebrt ) was delivered in 11 patients ( 84.6% ) regardless of the extent of resection . 9 ) rejected an active treatment after the initial surgery and another patient ( case no . 4 ) was not irradiated postoperatively due to misdiagnosis . however , he received irradiation ( 1500 cgy ) by cyberknife 3 months later . two patients ( 15.4% ) underwent stereotactic radiosurgery using a cyberknife for treatment of recurrent tumors . his initial pathologic diagnosis was pineal parenchymal neoplasm with intermediate undifferentiation , and he received a total of 2 cycles of ice chemotherapy ( a regimen employing ifosfamide , carboplatin and etoposide ) . six patients ( 46.1% ) had low grade hpcs ( who grade ii ) and 7 patients ( 53.9% ) had anaplastic hpcs ( who grade iii ) , a percentage that is relatively higher than in other studies . interestingly , the pathologic diagnosis of the resected recurrent tumors changed from low grade to anaplastic hpcs in 2 patients ( 15.4% , case nos . all resected tumors had well developed reticulin fibers , and cd34 was positive in 10 patients ( 83.3% ) and negative in 2 patients ( 16.7% ) . the ki-67 proliferative index ranged from 1% to 30% , wi th a mean of 11.3% . the mean ki-67 proliferative index was 2.2% in the low grade hpc group , and 18.3% in the anaplastic hpc groups . six patients ( 46.2% ) developed local recurrences at 104 , 73 , 19 , 4 and 3 ( two patients ) months after initial surgery . of the 6 patients , one underwent five operations , another underwent four operations and the other two underwent two operations . mean time for local recurrence was 24.6 months in patients who treated with pr or biopsy against 105.7 months in patients who treated with gtr ( p=0.012 by log - rank test ) ( fig . regarding adjuvant rt , mean time for local recurrence was 10.7 months in patients who did not received rt against 85.4 months in patients who received rt ( p=0.052 by log - rank test ) ( fig . there were no distant metastases , based on clinical follow - up and whole body pet - ct scan . the outcomes of treatment were estimated according to the karnofsky performance scale ( kps ) . the mean kps at the final outcome was 64.6 ( initial mean kps was 70.8 ) . the mean follow - up period was 54.3 months ( range : 13 - 185 months ) . five year overall survival rate was 84% and mean overall survival was 158 months ( fig . intracranial hpcs are very rare tumors with an incidence of less than 1% , and they represented 2.2% of all meningiomas diagnosed over the last 18 years in our institute . it is widely known that intracranial hpcs , unlike meningiomas , are more common in males than females , which was also true in our data , where 92% of the patients were males5,18,25 ) . in previous studies on intracranial hpcs , the mean age at the time of diagnosis ranged from 38 to 44.9 years , which was lower than that of meningiomas1,5,22,23,25,48 ) . in our series , our study included two patients ( 15.4% ) with intracerebral hemorrhage on an initial ct scan . the majority of hpcs arise in the supratentorial area with dural attachment23,41 ) . however , non - dural attached hpcs , such as in the pineal region ( case no.4 ) , and purely intraparenchymal hpcs have also been reported28,39,41 ) . distinguishing hpcs from benign meningiomas before surgery can be difficult , but is very important because of the aggressiveness of hpcs and their high rates of local recurrence and distant metastasis . chiechi et al.9 ) reported that intracranial hpcs are more multilobulated than benign meningiomas , and have narrow based dural attachment on ct and conventional mr images . many other papers have reported that intracranial hpcs may show osteolytic features on plain films , ct and mr images6,11,33 ) . in the current study , osteolysis of the skull on ct scan was noted in 2 patients ( case nos . it has been found that osteolysis is more prevalent in anaplastic hpcs than in low grade hpcs9 ) . it has also been found that ct perfusion is valuable in preoperative grading of intracranial gliomas13 ) , in distinguishing between glioblastomas , metastasis and abscesses8,12 ) and in evaluating the prognosis of recurrent brain tumors after stereotactic radiosurgery26 ) . the frailty of the conventional surgical approach was intractable intraoperative bleeding from the meningeal artery during the initial stage of the operation . the blood loss during operation was much lower compared to non - embolized patients ( 350 ml vs. 990 ml ) , but we could not draw statistically significant conclusions due to the limited sample size of our study . surgical resection of the tumors offers the advantage of pathologic confirmation and reduction of mass effects . in our series , a total of 9 patients ( 69% ) achieved a gtr at the initial surgery , which was a much higher rate compared to other studies . we conducted a statistical analysis about correlation between extent of resection and recurrence , and we found statistical significance like other several series ( mean rfs 105.7 months vs. 24.6 months , p=0.012 ) ( fig . interestingly , the average mortality rate in the recent literature was 31.5%1,2,15,16,18,21,28,35,37,42,44 ) , but was only 15.4% in our study . though adjuvant therapy and follow - up period also affect the mortality rate , we cautiously suggest that gtr also contributed to the decreased mortality rate . similarly , we could find that the patients who had been recieved adjuvant rt had tendency of longer rfs ( mean rfs 85.4 months vs. 10.7 months , p=0.052 ) ( fig . a review of the recent literature shows that adjuvant rt is not applied consistently ( range : 17 - 91% , mean : 47.4% ) . on the other hand , we performed adjuvant rt regardless of the pathologic grade and extent of resection in most of the patients , 11 out of 13 patients received adjuvant rt to a mean dose of 5770 cgy . in our study , there were no significant radiation - related complications . chemotherapy provided only insignificant benefits3,20 ) , but chamberlain and glantz7 ) reported that chemotherapy ( cyclophosphamide+adriamycin+vincristine , followed by -ifn , followed by ice ) may be helpful for recurrent intractable intracranial hpcs . reported that the patients with a ki-67 index less than 5% tended to survive longer , but this finding could not correlate the clinical outcome46,47 ) . galanis et al.20 ) found that , out of 32 recurrences , 19 ( 59% ) recurred at the primary site , eleven ( 34% ) recurred at both the primary and other cns sites and the remaining 2 ( 7% ) had diffuse leptomeningeal spread without local recurrence at the primary site . in our series , we would define ' recurrence ' as increased tumor sized by follow - up radiologic study and any apparent deterioration in clinical status to need reoperation . in the strict sense , initially partial resection or biopsy cases should be described as ' progression ' when reoperation were needed , but number of our cases were too small to analyze about progression cases , separately . unfortunately , several authors reported that the extent of tumor resection has been less definitely correlated with the local recurrence of hpcs compared to meningiomas29 ) . this feature of intracranial hpcs might be explained by the strategy to aggressively irradiate partially resected tumors to reduce local recurrence . recently , du et al.14 ) reported only a 3.8% recurrence rate , but their follow - up period was very limited ( mean : 22 months ) . in our series , the recurrence rate was 46.2% ( 6/13 ) , with the mean follow - up period of 54.3 months ( range : 13 - 185 months ) . it is widely known that after the first recurrence , hpc tends to recur at brief intervals . mena et al.32 ) reported a recurrence rate of 70% ( 57/94 ) , while kochanek et al.29 ) reported 41% ( 7/17 ) . it seems that rarity of intracranial hpcs along with the absence of a well formulated treatment strategy might lead to highly fluctuating survival and local recurrence rates . we believe that adjuvant radiation therapy is helpful , even if maximal tumor resection was performed in low grade hpcs . molecular biologic analysis and studies of the efficacy of chemotherapy are required for better outcomes in relentlessly recurrent hpcs .

today , assisted reproductive technology ( art ) has become a well - established and highly efficient therapy for infertility . in art , it is well understood that the most important factors for maximizing the success rate of in vitro fertilization ( ivf ) are retrieving greater numbers of high - quality oocytes using controlled ovarian hyperstimulation ( coh ) and establishing a receptive endometrium . nowadays , the use of long protocols using the gonadotropin - releasing hormone ( gnrh ) analog plus gonadotropins for coh has gained widespread popularity . various gonadotropin preparations are commercially available and used for coh such as human menopausal gonadotropin ( hmg ) , human - derived follicle - stimulating hormone ( hfsh ) , and recombinant fsh ( rfsh ) . hmg contains fsh and luteinizing hormone activity , while rfsh comprises only fsh , and in comparison to hfsh , rfsh includes a high proportion of fewer acidic isoforms with high purity and high in vitro bioactivity . there are many controversies surrounding which kind of exogenous gonadotropin is more suitable and leads to greater ivf success . some studies have demonstrated that a better outcome in terms of oocyte and embryo quality , subsequent pregnancy rates , and live birth rate is obtained when hmg is used for ovarian stimulation , as compared with rfsh . however , other studies have shown that rfsh is as effective as urinary fsh or hmg in terms of the number of oocytes and embryos obtained and the total gonadotropin dose needed . studies that compared hfsh with rfsh noted increased ovarian recruitment of follicles in the rfsh group . daya showed that rfsh was better than hfsh in terms of the pregnancy rate , while van wely et al . illustrated a borderline significant difference of 5% higher clinical pregnancy rate in women stimulated with hfsh compared with rfsh . selman et al . demonstrated that the combination of hfsh / rfsh for ovarian stimulation had a positive effect on follicular development , oocyte quality , embryo development , and clinical outcome in patients with repeated ivf failures . therefore , despite the large number of papers published on coh protocols comparing the efficiency of different exogenous gonadotropins , no confirmed protocol exists , and it is not quite clear which is superior to the others . thus , the objective of the current study was to compare the efficacy of 4 different ovarian stimulation protocols , comprising hfsh , rfsh , hmg , and sequential use of hfsh and rfsh , on oocyte and embryo quality and ivf treatment outcome in patients undergoing ivf or intracytoplasmic sperm injection ( icsi ) . this double - blinded , randomized , clinical trial study was registered in the iranian registry of clinical trials ( code : irct201408116541n7 ) and approved by the institutional review board and the ethics committee of shiraz university of medical sciences , shiraz , iran ( code : ct - p-92 - 7249 ) . the study was performed from january 2014 to may 2014 . written informed consent was obtained from each participant . the consort flow diagram is depicted in figure 2 . the sample size used in this study was determined based on the criteria established by kutner et al . using the following formula : type i error ( ) = 0.05 , power of analysis ( 1-=0.95 ) , effect size and number of groups=4 . study flow chart of the evaluation of the efficacy of different ovarian stimulation protocols , consisting of hfsh , rfsh , hmg , and sequential use of hfsh and rfsh , on oocyte and embryo quality and ivf treatment outcome in patients undergoing ivf or icsi . the study group consisted of 160 women referred to 2 hospital - affiliated ivf centers for infertility treatment in shiraz , iran ( ghadir mother and child hospital and dena hospital ) . patients with unexplained or male factor infertility were included in the study if they met the following criteria : 1 ) age between 20 and 38 years ; 2 ) body mass index ( body weight divided by the square of body height ) between 19 and 29 kg / m ; 3 ) history of regular menstrual cycles , ranging from 2535 days ; 4 ) no relevant systemic disease , severe endometriosis , or uterine or ovarian abnormalities ; 5 ) no more than 3 previous ivf cycles ; and 6 ) no previous ivf cycle with a poor response or the ovarian hyperstimulation syndrome . additionally , patients with fsh > 10 iu / ml , with < 5 follicles in antral follicle count , and anti - mllerian hormone < 1 ng / ml were excluded from the study . after the patients were assessed for eligibility according to the mentioned criteria , a standard downregulation protocol was performed for all of them via a subcutaneous injection of gnrh agonists ( 0.5 mg of buserelin , suprefact , serono ) , on day 21 of their menstrual cycle ( 1 wk before the expected menses ) . subsequently , on day 2 of the next menstrual cycle , after confirming desensitization ( estradiol serum concentration < 50 pg / ml , the absence of follicles 10 mm in diameter , and endometrial thickness < 5 mm ) , the patients were randomized by a person independent of the research team using a computer - generated random - number list . thereafter , ovarian stimulation was commenced for the study population as follows : group a : 40 patients who received hmg ( menogon , ferring pharmaceuticals a / s , copenhagen , denmark ) ; group b : 40 patients who received hfsh ( fostimon , ibsa institut biochimique sa , geneva , switzerland ) ; group c : 40 patients who received rfsh ( gonal - f , merck , serono , rome , italy ) ; and group d : 40 patients who received hfsh ( fostimonx , ibsa institut biochimique sa , geneva , switzerland ) for the first 6 days , followed by rfsh ( gonal - f , merck , serono , rome , italy ) . in all the 4 groups , the gonadotropin administration was continued up to the day of human chorionic gonadotropin injection ( hcg ) ( gonasi hp , ibsa italia , rome , italy ) . it should be mentioned that both the subjects of the study and the investigators performing the study were blind to the type of the gonadotropin each patient received for ovarian stimulation . the monitoring of ovarian responses to gonadotropin stimulation during the treatment cycle began from day 6 , using transvaginal sonography and the measurement of the plasma e2 level every 3 days . each change in the gonadotropin dose was performed according to the follicle size and the plasma e2 level . the treatment was continued until the observation of at least 2 follicles having reached 1718 mm in diameter ( leading follicles ) and some other follicles 1416 mm in diameter . when the leading follicle was 1820 mm , and there were at least 3 follicles of 1617 mm , gonadotropin administration was stopped , and an intramuscular injection of 10,000 iu of hcg was administered for final oocyte maturation . finally , 3436 hours after hcg injection oocyte maturity was tested according to the presence or absence of a germinal vesicle and first polar body and was graded as gv , mi , or mii according to the criteria established by veeck et al . after fertilization , embryo scoring was carried out on the day of embryo transfer ( 3 d after oocyte retrieval ) . the embryos were graded as i , ii , or iii , where i indicates the best - quality embryo and iii indicates the lowest - quality embryo . the luteal phase was supported by an intramuscular injection of 2 vials of progesterone ( 50 mg , iran hormone , tehran , iran ) daily , from the day of oocyte retrieval for 3 days and continued with intravaginal progesterone ( 400 mg , cyclogest , actavis uk ltd . , two weeks after embryo transfer , the chemical pregnancy test was carried out by evaluation of serum -hcg . in addition , clinical pregnancy was evaluated by observing the pregnancy sac 6 weeks after embryo transfer , and the implantation rate was determined by the number of gestational sacs divided by the number of embryos transferred . the secondary endpoints were the duration of stimulation , plasma e2 level on the day of hcg administration , number of used ampoules or vials of gonadotropin , number of large - sized follicles , total number of collected oocytes and transferred embryos , and implantation and miscarriage rates . statistical analysis was performed using spss , version 16 ( ibm , armonk , usa ) . for the analysis of the data , the one - w ay anova test was used followed by the tukey test to compare the means . this double - blinded , randomized , clinical trial study was registered in the iranian registry of clinical trials ( code : irct201408116541n7 ) and approved by the institutional review board and the ethics committee of shiraz university of medical sciences , shiraz , iran ( code : ct - p-92 - 7249 ) . the study was performed from january 2014 to may 2014 . written informed consent was obtained from each participant . the consort flow diagram is depicted in figure 2 . the sample size used in this study was determined based on the criteria established by kutner et al . using the following formula : type i error ( ) = 0.05 , power of analysis ( 1-=0.95 ) , effect size and number of groups=4 . study flow chart of the evaluation of the efficacy of different ovarian stimulation protocols , consisting of hfsh , rfsh , hmg , and sequential use of hfsh and rfsh , on oocyte and embryo quality and ivf treatment outcome in patients undergoing ivf or icsi . the study group consisted of 160 women referred to 2 hospital - affiliated ivf centers for infertility treatment in shiraz , iran ( ghadir mother and child hospital and dena hospital ) . patients with unexplained or male factor infertility were included in the study if they met the following criteria : 1 ) age between 20 and 38 years ; 2 ) body mass index ( body weight divided by the square of body height ) between 19 and 29 kg / m ; 3 ) history of regular menstrual cycles , ranging from 2535 days ; 4 ) no relevant systemic disease , severe endometriosis , or uterine or ovarian abnormalities ; 5 ) no more than 3 previous ivf cycles ; and 6 ) no previous ivf cycle with a poor response or the ovarian hyperstimulation syndrome . additionally , patients with fsh > 10 iu / ml , with < 5 follicles in antral follicle count , and anti - mllerian hormone < 1 ng / ml were excluded from the study . after the patients were assessed for eligibility according to the mentioned criteria , a standard downregulation protocol was performed for all of them via a subcutaneous injection of gnrh agonists ( 0.5 mg of buserelin , suprefact , serono ) , on day 21 of their menstrual cycle ( 1 wk before the expected menses ) . subsequently , on day 2 of the next menstrual cycle , after confirming desensitization ( estradiol serum concentration < 50 pg / ml , the absence of follicles 10 mm in diameter , and endometrial thickness < 5 mm ) , the patients were randomized by a person independent of the research team using a computer - generated random - number list . thereafter , ovarian stimulation was commenced for the study population as follows : group a : 40 patients who received hmg ( menogon , ferring pharmaceuticals a / s , copenhagen , denmark ) ; group b : 40 patients who received hfsh ( fostimon , ibsa institut biochimique sa , geneva , switzerland ) ; group c : 40 patients who received rfsh ( gonal - f , merck , serono , rome , italy ) ; and group d : 40 patients who received hfsh ( fostimonx , ibsa institut biochimique sa , geneva , switzerland ) for the first 6 days , followed by rfsh ( gonal - f , merck , serono , rome , italy ) . in all the 4 groups , the gonadotropin administration was continued up to the day of human chorionic gonadotropin injection ( hcg ) ( gonasi hp , ibsa italia , rome , italy ) . it should be mentioned that both the subjects of the study and the investigators performing the study were blind to the type of the gonadotropin each patient received for ovarian stimulation . the monitoring of ovarian responses to gonadotropin stimulation during the treatment cycle began from day 6 , using transvaginal sonography and the measurement of the plasma e2 level every 3 days . each change in the gonadotropin dose was performed according to the follicle size and the plasma e2 level . the treatment was continued until the observation of at least 2 follicles having reached 1718 mm in diameter ( leading follicles ) and some other follicles 1416 mm in diameter . when the leading follicle was 1820 mm , and there were at least 3 follicles of 1617 mm , gonadotropin administration was stopped , and an intramuscular injection of 10,000 iu of hcg was administered for final oocyte maturation . finally , 3436 hours after hcg injection , transvaginal ultrasound - guided oocyte retrieval was performed . oocyte maturity was tested according to the presence or absence of a germinal vesicle and first polar body and was graded as gv , mi , or mii according to the criteria established by veeck et al . after fertilization , embryo scoring was carried out on the day of embryo transfer ( 3 d after oocyte retrieval ) . the embryos were graded as i , ii , or iii , where i indicates the best - quality embryo and iii indicates the lowest - quality embryo . the luteal phase was supported by an intramuscular injection of 2 vials of progesterone ( 50 mg , iran hormone , tehran , iran ) daily , from the day of oocyte retrieval for 3 days and continued with intravaginal progesterone ( 400 mg , cyclogest , actavis uk ltd . , two weeks after embryo transfer , the chemical pregnancy test was carried out by evaluation of serum -hcg . in addition , clinical pregnancy was evaluated by observing the pregnancy sac 6 weeks after embryo transfer , and the implantation rate was determined by the number of gestational sacs divided by the number of embryos transferred . the secondary endpoints were the duration of stimulation , plasma e2 level on the day of hcg administration , number of used ampoules or vials of gonadotropin , number of large - sized follicles , total number of collected oocytes and transferred embryos , and implantation and miscarriage rates . statistical analysis was performed using spss , version 16 ( ibm , armonk , usa ) . for the analysis of the data , the one - w ay anova test was used followed by the tukey test to compare the means . according to table 1 , age , body mass index , duration of infertility , and endometrial thickness at baseline were similar in all the groups . demographic characteristics of the women receiving hmg , hfsh , rfsh , and sequential hfsh / rfsh the significance was considered at p0.05 . : statistically significant differences between groups a and d ; : statistically significant differences between groups b and d ; : statistically significant differences between groups c and d ; : statistically significant differences between groups a and c the number of ampoules or vials of gonadotropin administered was lower in group d than in the other groups ; this difference was statistically significant compared to groups a , b , and c. the duration of stimulation was longer in group c than in the other 3 groups , and the difference in group c in comparison to groups a and d was statistically significant . endometrial thickness and the estradiol level on the day of hcg administration were higher in group d than in the other groups . apropos the estradiol level , this difference was significant in group d in comparison to groups a and b. as is shown in table 2 , the number of large - sized follicles was high in group d and then in group c , compared to groups a and b. this difference between groups b and d was statistically significant . ovarian response and oocyte maturity in the patients receiving hmg , hfsh , rfsh , or sequential hfsh / rfsh significance was considered at p0.05 . : statistically significant differences between groups b and d ; ns : nonsignificant the number of retrieved oocytes was higher in groups c and d than in groups a and b , but the difference did not constitute statistical significance . the number of degenerated oocytes was higher in group d than in groups a , b , and c ; the difference , however , was not statistically significant . no statistically significant differences were observed in the number of gv and mi oocytes between the studied groups , but the number of mature oocytes ( mii ) was higher in group c and then in group d than in groups a and b ; nevertheless , the difference was not statistically significant . the lowest number of mii oocytes was observed in group b. according to table 3 , the number of transferred embryos was not different between the groups . the highest proportion of grade - i embryos and the lowest proportion of grade - ii and grade - iii embryos were in group d , followed by groups c , b , and a. embryo score of the patients after treatment with hmg , hfsh , rfsh , or sequential hfsh / rfsh significance was considered at p0.05 as is shown in table 4 , the chemical and clinical pregnancy rate , implantation rate , and live birth rate were high in group d , followed by group c , in comparison to the other groups ; nonetheless , the difference was not statistically significant . in addition , the abortion rate was highest in group d. clinical outcome of the patients after treatment with hmg , hfsh , rfsh , or sequential hfsh / rfsh significance was considered at p0.05 among the different protocols for coh , the use of the gnrh analog plus gonadotropins ( long protocol or standard protocol ) is popular , owing to its more favorable results . the literature abounds with studies comparing exogenous gonadotropins for coh , but the issue still remains controversial . exogenous ovarian stimulation increases oocyte yield but may compromise the developmental competence of the oocytes in stimulated cycles . in this study , we evaluated the efficacy of 4 different ovarian stimulation protocols using different gonadotropins in women undergoing ivf or icsi programs . according to our results , the number of ampoules used was significantly lower in the sequential protocol than that in the other 3 protocols and the duration of stimulation in the rfsh - alone protocol was significantly longer than that in the hfsh and hmg protocols . demonstrated that stimulation with the sequential protocol , compared with the rfsh protocol , necessitated a low gonadotropin dose and short duration of stimulation for the stimulation of ovaries . other studies have shown no significant differences between the use of rfsh and hfsh or the sequential protocol in the duration of stimulation and the dose of gonadotropin used . these contradictory results may have originated from diversity not only among the products of pharmaceutical companies but also among patients race and physiological status . the effect of serum estradiol level on the day of hcg on art outcome is controversial . it is said that although the estradiol level increases endometrial proliferation , uterine perfusion , oocyte development and maturation , number of embryos transferred , implantation , delivery , and pregnancy rate , the supraphysiological level of estradiol may not only cause endometrial damage and disrupt the implantation but also exert negative effects on ivf - icsi outcome . gonadotropin stimulation changes in the steroid profile result in modifying the microenvironment of the developing follicle and its oocyte . precise evaluation of follicle size is highly important , and it has been shown that larger follicles at the time of retrieval have consistently mature oocytes with a higher rate of fertilization . we observed that the level of estradiol was significantly higher in the sequential protocol than in the hfsh and hmg protocols , resulting in more large - sized follicles , retrieved oocytes , and mii oocytes in this protocol . nonetheless , it did not lead to a clear increase in the endometrial thickness of these groups compared to the other 2 groups . the sequential use of hfsh / rfsh is the same as the natural physiologic cycle , where more acidic isoforms of fsh are produced in the follicular phase , when the estradiol level is low , and fewer acidic isoforms are produced in the late follicular and periovulatory phase , when estradiol is high . the significantly high number of large - sized follicles in the sequential protocol in comparison to the rfsh protocol may be related to the combined used of acidic ( hfsh ) and less acidic isoforms ( rfsh ) of fsh , which mimics the physiology of the normal menstrual cycle and is an important mechanism for the regulation of the final stages of follicle and oocyte maturation . furthermore , in the rfsh protocol , the level of estradiol was nonsignificantly higher than that with the hfsh and hmg protocols . . showed a significantly high level of estradiol in the rfsh protocol compared with the hfsh protocol . other studies have shown no differences in the estradiol level and endometrial thickness between the sequential , rfsh , hfsh , and hmg protocols . although not significant , the number of retrieved oocytes and mii oocytes was high in the sequential and rfsh protocols compared with the hfsh and hmg protocols . however , the number of degenerated oocytes was nonsignificantly high in the sequential protocol compared to the other 3 groups . in other studies , no significant differences in the number of retrieved oocytes have been seen between the different protocols . we observed no significant differences in the number of retrieved oocytes and mature oocytes between the hfsh and rfsh patients , chiming in with other studies . gerli et al . observed that the number of mii oocytes was significantly higher in the patients who received the sequential protocol than in the patients who received rfsh alone . therefore , using a sequential protocol , our patients reached higher estradiol levels and sufficient numbers of suitable follicles with fewer ampoules and lower durations of stimulation . furthermore , in the rfsh protocol , despite the need for more ampoules and a longer duration of stimulation than in the other groups , more retrieved oocytes and higher numbers of mi and mii oocytes were produced than with the hmg and hfsh protocols , although these differences were not significant . we observed that the use of the sequential and rfsh protocols , by comparison with the hmg or hfsh protocol , nonsignificantly led to more good - quality ( grade i ) embryos . in addition , the number of low - quality embryos was lowest in the sequential protocol , followed by the rfsh , hfsh , and hmg protocols . selman et al . and gerli et al . showed that the number of good - quality embryos was significantly high in the sequential protocol in comparison to the hfsh and rfsh protocols . in other studies , the number of good - quality embryos is similar in the rfsh , hmg , and hfsh protocols . although the total number of transferred embryos was not different between the groups , the implantation and pregnancy and live birth rates were higher in the sequential protocol . this may be related to the higher number of good - quality embryos produced in the patients who received the sequential protocol . selman et al . and gerli et al . showed that the implantation rate and pregnancy and delivery rates were significantly high using the sequential protocol in comparison to the hfsh and rfsh protocols . in the rfsh protocol , these parameters were slightly higher than in the hfsh and hmg protocols . reported that the implantation rate and pregnancy rate were similar between the rfsh and hfsh protocols . in contrast , daya demonstrated that rfsh was better than hfsh in terms of the pregnancy rate , while van wely et al . showed a significantly high clinical pregnancy rate with the hfsh protocol compared with rfsh . ludwig et al . and turhan et al . showed that the pregnancy and live birth rates were similar between the rfsh and hmg protocols . these differences may be due to the heterogeneity of patients in the analysis , their age , type of gnrh analog suppressions , gonadotropin doses , etc . nonetheless , the results of the study by selman et al . and our results showed that the sequential protocol was better than the other protocols in terms of clinical pregnancy and the live birth rate . our results regarding the superiority of rfsh over hfsh differed from their results . in our study , the pregnancy rate was higher in the sequential and rfsh protocols , although the abortion rate was higher in these protocols as well ; however , the overall outcome ( the live birth rate ) stood higher in these 2 groups ( not significantly ) . accordingly , we concluded that the rfsh and sequential hfsh / rfsh protocols yielded more mature oocytes , but the sequential protocol was more valuable in terms of embryo quality , as was seen in implantation , pregnancies , and live birth rate . the sequential use of hfsh / rfsh is the same as the natural physiologic cycle , where more acidic isoforms of fsh are produced in the follicular phase , when the estradiol level is low , and fewer acidic isoforms are produced in the late follicular and periovulatory phase , when estradiol is high . this may be an important mechanism for the regulation of the final stages of follicle and oocyte maturation . therefore , the difference and distribution of exogenously applied gonadotropins should be determined and used for ovarian stimulation . obviously , these differences in the effect of fsh isoforms on follicular development patterns strongly suggest that oocyte development is also likely to be influenced , that normal follicle development and ultimately normal oocyte function depend on an appropriate balance of sequential differentiation , and that this balance is strongly influenced by fsh isoform distribution . in conclusion , the sequential protocol was able to improve the success rate of art and could , as such , be deemed a valuable protocol in ivf programs . further large randomized trials are needed to yield a precise estimation of any difference between the above - mentioned protocols .

background : despite the large number of papers published on the efficiency of different exogenous gonadotropins , no confirmed protocol exists . therefore , the aim of the present study was to compare the efficacy of 4 exogenous gonadotropins in ivf / icsi cycles.methods:this study , performed from january 2014 to may 2014 , recruited 160 women referred to ghadir mother and child hospital and dena hospital , shiraz , iran . the patients underwent standard downregulation and were randomly divided into 4 groups of a , b , c , and d and were administered hmg , hfsh , rfsh , and combined sequential hfsh / rfsh , respectively . then , the duration of stimulation , number of oocytes and embryos as well as their quality , implantation rate , biochemical and clinical pregnancy rate , and live birth rate in each group were evaluated.results:group d patients required significantly fewer ampoules of fsh than did the women in groups a , b , and c ( p=0.004 ) . the duration of stimulation was significantly longer in group c than in groups a and d ( p=0.030 ) . the serum estradiol level was significantly higher in group d than in groups b and c ( p=0.005 ) . a significantly higher number of large - sized follicles was observed in group d than in group b ( p=0.036).conclusion : our data revealed no statistically significant differences in the mean oocyte number , embryo quality , clinical pregnancy rate , or live birth rate between the hmg , hfsh , rfsh , and sequential hfsh / rfsh protocols . however , several differences in the duration of stimulation , serum estradiol levels , and number of large - sized follicles were detected between the groups . trial registration number : irct201408116541n7

Introduction Patients and Methods None None None None None None Results Discussion Conclusion

hmg contains fsh and luteinizing hormone activity , while rfsh comprises only fsh , and in comparison to hfsh , rfsh includes a high proportion of fewer acidic isoforms with high purity and high in vitro bioactivity . some studies have demonstrated that a better outcome in terms of oocyte and embryo quality , subsequent pregnancy rates , and live birth rate is obtained when hmg is used for ovarian stimulation , as compared with rfsh . however , other studies have shown that rfsh is as effective as urinary fsh or hmg in terms of the number of oocytes and embryos obtained and the total gonadotropin dose needed . daya showed that rfsh was better than hfsh in terms of the pregnancy rate , while van wely et al . illustrated a borderline significant difference of 5% higher clinical pregnancy rate in women stimulated with hfsh compared with rfsh . demonstrated that the combination of hfsh / rfsh for ovarian stimulation had a positive effect on follicular development , oocyte quality , embryo development , and clinical outcome in patients with repeated ivf failures . therefore , despite the large number of papers published on coh protocols comparing the efficiency of different exogenous gonadotropins , no confirmed protocol exists , and it is not quite clear which is superior to the others . thus , the objective of the current study was to compare the efficacy of 4 different ovarian stimulation protocols , comprising hfsh , rfsh , hmg , and sequential use of hfsh and rfsh , on oocyte and embryo quality and ivf treatment outcome in patients undergoing ivf or intracytoplasmic sperm injection ( icsi ) . this double - blinded , randomized , clinical trial study was registered in the iranian registry of clinical trials ( code : irct201408116541n7 ) and approved by the institutional review board and the ethics committee of shiraz university of medical sciences , shiraz , iran ( code : ct - p-92 - 7249 ) . the study was performed from january 2014 to may 2014 . using the following formula : type i error ( ) = 0.05 , power of analysis ( 1-=0.95 ) , effect size and number of groups=4 . study flow chart of the evaluation of the efficacy of different ovarian stimulation protocols , consisting of hfsh , rfsh , hmg , and sequential use of hfsh and rfsh , on oocyte and embryo quality and ivf treatment outcome in patients undergoing ivf or icsi . the study group consisted of 160 women referred to 2 hospital - affiliated ivf centers for infertility treatment in shiraz , iran ( ghadir mother and child hospital and dena hospital ) . patients with unexplained or male factor infertility were included in the study if they met the following criteria : 1 ) age between 20 and 38 years ; 2 ) body mass index ( body weight divided by the square of body height ) between 19 and 29 kg / m ; 3 ) history of regular menstrual cycles , ranging from 2535 days ; 4 ) no relevant systemic disease , severe endometriosis , or uterine or ovarian abnormalities ; 5 ) no more than 3 previous ivf cycles ; and 6 ) no previous ivf cycle with a poor response or the ovarian hyperstimulation syndrome . subsequently , on day 2 of the next menstrual cycle , after confirming desensitization ( estradiol serum concentration < 50 pg / ml , the absence of follicles 10 mm in diameter , and endometrial thickness < 5 mm ) , the patients were randomized by a person independent of the research team using a computer - generated random - number list . thereafter , ovarian stimulation was commenced for the study population as follows : group a : 40 patients who received hmg ( menogon , ferring pharmaceuticals a / s , copenhagen , denmark ) ; group b : 40 patients who received hfsh ( fostimon , ibsa institut biochimique sa , geneva , switzerland ) ; group c : 40 patients who received rfsh ( gonal - f , merck , serono , rome , italy ) ; and group d : 40 patients who received hfsh ( fostimonx , ibsa institut biochimique sa , geneva , switzerland ) for the first 6 days , followed by rfsh ( gonal - f , merck , serono , rome , italy ) . in all the 4 groups , the gonadotropin administration was continued up to the day of human chorionic gonadotropin injection ( hcg ) ( gonasi hp , ibsa italia , rome , italy ) . after fertilization , embryo scoring was carried out on the day of embryo transfer ( 3 d after oocyte retrieval ) . in addition , clinical pregnancy was evaluated by observing the pregnancy sac 6 weeks after embryo transfer , and the implantation rate was determined by the number of gestational sacs divided by the number of embryos transferred . the secondary endpoints were the duration of stimulation , plasma e2 level on the day of hcg administration , number of used ampoules or vials of gonadotropin , number of large - sized follicles , total number of collected oocytes and transferred embryos , and implantation and miscarriage rates . for the analysis of the data , the one - w ay anova test was used followed by the tukey test to compare the means . this double - blinded , randomized , clinical trial study was registered in the iranian registry of clinical trials ( code : irct201408116541n7 ) and approved by the institutional review board and the ethics committee of shiraz university of medical sciences , shiraz , iran ( code : ct - p-92 - 7249 ) . the study was performed from january 2014 to may 2014 . the sample size used in this study was determined based on the criteria established by kutner et al . using the following formula : type i error ( ) = 0.05 , power of analysis ( 1-=0.95 ) , effect size and number of groups=4 . study flow chart of the evaluation of the efficacy of different ovarian stimulation protocols , consisting of hfsh , rfsh , hmg , and sequential use of hfsh and rfsh , on oocyte and embryo quality and ivf treatment outcome in patients undergoing ivf or icsi . the study group consisted of 160 women referred to 2 hospital - affiliated ivf centers for infertility treatment in shiraz , iran ( ghadir mother and child hospital and dena hospital ) . patients with unexplained or male factor infertility were included in the study if they met the following criteria : 1 ) age between 20 and 38 years ; 2 ) body mass index ( body weight divided by the square of body height ) between 19 and 29 kg / m ; 3 ) history of regular menstrual cycles , ranging from 2535 days ; 4 ) no relevant systemic disease , severe endometriosis , or uterine or ovarian abnormalities ; 5 ) no more than 3 previous ivf cycles ; and 6 ) no previous ivf cycle with a poor response or the ovarian hyperstimulation syndrome . after the patients were assessed for eligibility according to the mentioned criteria , a standard downregulation protocol was performed for all of them via a subcutaneous injection of gnrh agonists ( 0.5 mg of buserelin , suprefact , serono ) , on day 21 of their menstrual cycle ( 1 wk before the expected menses ) . subsequently , on day 2 of the next menstrual cycle , after confirming desensitization ( estradiol serum concentration < 50 pg / ml , the absence of follicles 10 mm in diameter , and endometrial thickness < 5 mm ) , the patients were randomized by a person independent of the research team using a computer - generated random - number list . thereafter , ovarian stimulation was commenced for the study population as follows : group a : 40 patients who received hmg ( menogon , ferring pharmaceuticals a / s , copenhagen , denmark ) ; group b : 40 patients who received hfsh ( fostimon , ibsa institut biochimique sa , geneva , switzerland ) ; group c : 40 patients who received rfsh ( gonal - f , merck , serono , rome , italy ) ; and group d : 40 patients who received hfsh ( fostimonx , ibsa institut biochimique sa , geneva , switzerland ) for the first 6 days , followed by rfsh ( gonal - f , merck , serono , rome , italy ) . in all the 4 groups , the gonadotropin administration was continued up to the day of human chorionic gonadotropin injection ( hcg ) ( gonasi hp , ibsa italia , rome , italy ) . oocyte maturity was tested according to the presence or absence of a germinal vesicle and first polar body and was graded as gv , mi , or mii according to the criteria established by veeck et al . after fertilization , embryo scoring was carried out on the day of embryo transfer ( 3 d after oocyte retrieval ) . in addition , clinical pregnancy was evaluated by observing the pregnancy sac 6 weeks after embryo transfer , and the implantation rate was determined by the number of gestational sacs divided by the number of embryos transferred . the secondary endpoints were the duration of stimulation , plasma e2 level on the day of hcg administration , number of used ampoules or vials of gonadotropin , number of large - sized follicles , total number of collected oocytes and transferred embryos , and implantation and miscarriage rates . for the analysis of the data , the one - w ay anova test was used followed by the tukey test to compare the means . according to table 1 , age , body mass index , duration of infertility , and endometrial thickness at baseline were similar in all the groups . demographic characteristics of the women receiving hmg , hfsh , rfsh , and sequential hfsh / rfsh the significance was considered at p0.05 . : statistically significant differences between groups a and d ; : statistically significant differences between groups b and d ; : statistically significant differences between groups c and d ; : statistically significant differences between groups a and c the number of ampoules or vials of gonadotropin administered was lower in group d than in the other groups ; this difference was statistically significant compared to groups a , b , and c. the duration of stimulation was longer in group c than in the other 3 groups , and the difference in group c in comparison to groups a and d was statistically significant . endometrial thickness and the estradiol level on the day of hcg administration were higher in group d than in the other groups . apropos the estradiol level , this difference was significant in group d in comparison to groups a and b. as is shown in table 2 , the number of large - sized follicles was high in group d and then in group c , compared to groups a and b. this difference between groups b and d was statistically significant . ovarian response and oocyte maturity in the patients receiving hmg , hfsh , rfsh , or sequential hfsh / rfsh significance was considered at p0.05 . : statistically significant differences between groups b and d ; ns : nonsignificant the number of retrieved oocytes was higher in groups c and d than in groups a and b , but the difference did not constitute statistical significance . the number of degenerated oocytes was higher in group d than in groups a , b , and c ; the difference , however , was not statistically significant . no statistically significant differences were observed in the number of gv and mi oocytes between the studied groups , but the number of mature oocytes ( mii ) was higher in group c and then in group d than in groups a and b ; nevertheless , the difference was not statistically significant . the lowest number of mii oocytes was observed in group b. according to table 3 , the number of transferred embryos was not different between the groups . the highest proportion of grade - i embryos and the lowest proportion of grade - ii and grade - iii embryos were in group d , followed by groups c , b , and a. embryo score of the patients after treatment with hmg , hfsh , rfsh , or sequential hfsh / rfsh significance was considered at p0.05 as is shown in table 4 , the chemical and clinical pregnancy rate , implantation rate , and live birth rate were high in group d , followed by group c , in comparison to the other groups ; nonetheless , the difference was not statistically significant . in addition , the abortion rate was highest in group d. clinical outcome of the patients after treatment with hmg , hfsh , rfsh , or sequential hfsh / rfsh significance was considered at p0.05 among the different protocols for coh , the use of the gnrh analog plus gonadotropins ( long protocol or standard protocol ) is popular , owing to its more favorable results . in this study , we evaluated the efficacy of 4 different ovarian stimulation protocols using different gonadotropins in women undergoing ivf or icsi programs . according to our results , the number of ampoules used was significantly lower in the sequential protocol than that in the other 3 protocols and the duration of stimulation in the rfsh - alone protocol was significantly longer than that in the hfsh and hmg protocols . other studies have shown no significant differences between the use of rfsh and hfsh or the sequential protocol in the duration of stimulation and the dose of gonadotropin used . the effect of serum estradiol level on the day of hcg on art outcome is controversial . it is said that although the estradiol level increases endometrial proliferation , uterine perfusion , oocyte development and maturation , number of embryos transferred , implantation , delivery , and pregnancy rate , the supraphysiological level of estradiol may not only cause endometrial damage and disrupt the implantation but also exert negative effects on ivf - icsi outcome . we observed that the level of estradiol was significantly higher in the sequential protocol than in the hfsh and hmg protocols , resulting in more large - sized follicles , retrieved oocytes , and mii oocytes in this protocol . the sequential use of hfsh / rfsh is the same as the natural physiologic cycle , where more acidic isoforms of fsh are produced in the follicular phase , when the estradiol level is low , and fewer acidic isoforms are produced in the late follicular and periovulatory phase , when estradiol is high . the significantly high number of large - sized follicles in the sequential protocol in comparison to the rfsh protocol may be related to the combined used of acidic ( hfsh ) and less acidic isoforms ( rfsh ) of fsh , which mimics the physiology of the normal menstrual cycle and is an important mechanism for the regulation of the final stages of follicle and oocyte maturation . furthermore , in the rfsh protocol , the level of estradiol was nonsignificantly higher than that with the hfsh and hmg protocols . showed a significantly high level of estradiol in the rfsh protocol compared with the hfsh protocol . other studies have shown no differences in the estradiol level and endometrial thickness between the sequential , rfsh , hfsh , and hmg protocols . although not significant , the number of retrieved oocytes and mii oocytes was high in the sequential and rfsh protocols compared with the hfsh and hmg protocols . however , the number of degenerated oocytes was nonsignificantly high in the sequential protocol compared to the other 3 groups . in other studies , no significant differences in the number of retrieved oocytes have been seen between the different protocols . we observed no significant differences in the number of retrieved oocytes and mature oocytes between the hfsh and rfsh patients , chiming in with other studies . observed that the number of mii oocytes was significantly higher in the patients who received the sequential protocol than in the patients who received rfsh alone . therefore , using a sequential protocol , our patients reached higher estradiol levels and sufficient numbers of suitable follicles with fewer ampoules and lower durations of stimulation . furthermore , in the rfsh protocol , despite the need for more ampoules and a longer duration of stimulation than in the other groups , more retrieved oocytes and higher numbers of mi and mii oocytes were produced than with the hmg and hfsh protocols , although these differences were not significant . we observed that the use of the sequential and rfsh protocols , by comparison with the hmg or hfsh protocol , nonsignificantly led to more good - quality ( grade i ) embryos . in addition , the number of low - quality embryos was lowest in the sequential protocol , followed by the rfsh , hfsh , and hmg protocols . showed that the number of good - quality embryos was significantly high in the sequential protocol in comparison to the hfsh and rfsh protocols . in other studies , the number of good - quality embryos is similar in the rfsh , hmg , and hfsh protocols . although the total number of transferred embryos was not different between the groups , the implantation and pregnancy and live birth rates were higher in the sequential protocol . this may be related to the higher number of good - quality embryos produced in the patients who received the sequential protocol . showed that the implantation rate and pregnancy and delivery rates were significantly high using the sequential protocol in comparison to the hfsh and rfsh protocols . reported that the implantation rate and pregnancy rate were similar between the rfsh and hfsh protocols . in contrast , daya demonstrated that rfsh was better than hfsh in terms of the pregnancy rate , while van wely et al . showed a significantly high clinical pregnancy rate with the hfsh protocol compared with rfsh . showed that the pregnancy and live birth rates were similar between the rfsh and hmg protocols . nonetheless , the results of the study by selman et al . and our results showed that the sequential protocol was better than the other protocols in terms of clinical pregnancy and the live birth rate . in our study , the pregnancy rate was higher in the sequential and rfsh protocols , although the abortion rate was higher in these protocols as well ; however , the overall outcome ( the live birth rate ) stood higher in these 2 groups ( not significantly ) . accordingly , we concluded that the rfsh and sequential hfsh / rfsh protocols yielded more mature oocytes , but the sequential protocol was more valuable in terms of embryo quality , as was seen in implantation , pregnancies , and live birth rate . the sequential use of hfsh / rfsh is the same as the natural physiologic cycle , where more acidic isoforms of fsh are produced in the follicular phase , when the estradiol level is low , and fewer acidic isoforms are produced in the late follicular and periovulatory phase , when estradiol is high . obviously , these differences in the effect of fsh isoforms on follicular development patterns strongly suggest that oocyte development is also likely to be influenced , that normal follicle development and ultimately normal oocyte function depend on an appropriate balance of sequential differentiation , and that this balance is strongly influenced by fsh isoform distribution .

primary intraventricular hemorrhage ( pivh ) is a rare condition defined as bleeding within the ventricular system without a definite parenchymal component , and accounts for 0.31% of all stroke cases and 3.1% of intracerebral hemorrhages ( ichs ) [ 16 ] . intraventricular hemorrhage ( ivh ) occurring with ich , subarachnoid hemorrhage ( sah ) , or other parenchymal hemorrhage , is referred to as secondary ivh . pivh commonly occurs in preterm newborns as a complication of birth , but is relatively rare in adults [ 911 ] . pivh was considered fatal before the modern era of brain imaging , as it could only be diagnosed during postmortem examination . the introduction of brain imaging techniques such as computed tomography ( ct ) and magnetic resonance ( mr ) imaging has allowed for a more rapid and easier diagnosis of pivh . however , due to its rarity , there is relatively little information regarding the clinical manifestations of pivh , its etiology , and its prognosis . the purpose of this study was to review data from 112 patients with pivh admitted to a single tertiary referral hospital over the last 11 years . here , we describe their clinical characteristics , outcomes following treatment , and prognostic factors . we reviewed all patients with ich who were admitted at our institution between january 2004 and december 2014 . exclusion criteria were : neonates ( less than 1 month after birth ) , death within 24 hours of presentation , and life expectancy less than 6 months owing to another medical condition . once pivh was diagnosed , all patients were admitted to a neurosurgical intensive care unit ( icu ) . additional radiological examinations , including ct or mr angiography , and/or transfemoral cerebral angiography ( tfca ) , were considered for all subjects to identify causal vascular pathologies , according to the guidelines provided by the american heart association and the american stroke association . however , these studies were not performed in 13 patients ( 12% ) due to reasons such as poor clinical status or refusal by the patient s family . during the acute phase , systolic blood pressure was maintained below 140 mmhg , and osmotic diuretics were administered to control increased intracranial pressure ( icp ) . patients with a decreased level of consciousness ( glasgow coma scale [ gcs ] < 8) were intubated to maintain the airway and to reduce the gag reflex or the chances of imminent aspiration . mechanical ventilation was used in patients with respiratory insufficiency , as indicated by pco2 > 55 mmhg or po2 < 60 mmhg . in patients with coagulopathy , anticoagulation reversal was performed using fresh - frozen plasma or prothrombin - complex concentrate , and vitamin k. anticoagulant or antiplatelet treatments were discontinued during the acute phase in patients who regularly took the medications . when necessary , informed consent was obtained to perform external ventricular drainage ( evd ) . the laterality of the evd was chosen based on the patient s conditions and the hemorrhagic location , which was observed during the initial ct . indications for evd at our institution were : ( 1 ) initial gcs score of < 12 , ( 2 ) evidence of hydrocephalus defined as an evans ratio of > 0.30 , and ( 3 ) evidence of third or fourth ventricle obstruction with an accompanying prediction that the patient would develop hydrocephalus . neurological outcome at 3 months was assessed using the glasgow outcome scale ( gos ) . patients were divided into 2 groups according to their gos scores : a favorable outcome group ( gos scores of 45 ) and an unfavorable outcome group ( gos scores of 13 ) . additional outcomes measures included mortality 3 months after presentation . the potential prognostic factors discussed in previous reports of intracerebral and/or intraventricular hemorrhage were analyzed to examine their relationships with clinical outcomes [ 5,1317 ] . the presumptive factors were either patient factors or radiologic findings , and were collected from medical records and imaging studies . potential patient factors included : age , sex , initial gcs score , low platelet count ( < 50 000 mm ) , prolonged prothrombin time ( pt ; international normalized ratio [ inr ] > 1.4 ) , use of anticoagulants or antiplatelet agents , the presence of underlying diseases such as hypertension ( determined by previous diagnosis , treatment with antihypertensive medications , or a blood pressure reading of > 140/90 mmhg ) , and diabetes mellitus ( dm ; determined by a fasting glucose reading of > 7 mmol / l or treatment with anti - diabetic medication ) . the severity of illness for the enrolled patients was measured using the new simplified acute physiology score ( saps ii ) , which is calculated based on the worst values taken during the first 24 hours after admission to the icu . the score encompasses 15 parameters ( heart rate ; body temperature ; white blood cell count ; the ratio of partial pressure of oxygen in arterial blood to fractional inspired oxygen ; bicarbonate level ; systolic blood pressure ; urinary output ; serum urea level ; serum potassium , sodium , and bilirubin levels ; gcs score ; age ; type of admission ; and chronic underlying disease ) , and has a range of 0 to 163 . additionally , parameters of the ventricular system were measured , including the transverse diameter of the third ventricle , both anterior - posterior ( ap ) and transverse diameters of the fourth ventricle , and temporal horn size on the dominant side . a dilated fourth ventricle was defined as one with a transverse diameter of > 20 mm or an ap diameter of > 12.5 mm , while third ventricle dilatation was defined as a transverse diameter of > 10 mm . finally , dilatation of the temporal horn was determined as a size of > 6 mm , and hydrocephalus was defined as an evans ratio of > 0.30 . presumptive prognostic factors were compared between the favorable ( gos scores of 45 ) and unfavorable outcome ( gos scores of 13 ) groups using t tests for continuous variables ( age , initial gcs , saps ii score , mgs , diameters of 3 and 4 ventricle , size of dominant side temporal horn ) , and either the chi - square or fisher s exact test for categorical variables ( sex , application of mechanical ventilation within 24 hours of the initial attack , prior medical comorbidities , thrombocytopenia , abnormal coagulation parameters , presence of causative vascular abnormalities , additional treatment for vascular abnormalities , and hydrocephalus ) . the association between presumptive factors and clinical outcomes was analyzed using a logistic regression model , and the odds ratio ( or ) and 95% confidence interval ( ci ) were calculated . all of the statistical calculations were performed using commercially available software ( spss 11.5 , ibm corporation , armonk , ny ) . we reviewed all patients with ich who were admitted at our institution between january 2004 and december 2014 . exclusion criteria were : neonates ( less than 1 month after birth ) , death within 24 hours of presentation , and life expectancy less than 6 months owing to another medical condition . once pivh was diagnosed , all patients were admitted to a neurosurgical intensive care unit ( icu ) . additional radiological examinations , including ct or mr angiography , and/or transfemoral cerebral angiography ( tfca ) , were considered for all subjects to identify causal vascular pathologies , according to the guidelines provided by the american heart association and the american stroke association . however , these studies were not performed in 13 patients ( 12% ) due to reasons such as poor clinical status or refusal by the patient s family . during the acute phase , systolic blood pressure was maintained below 140 mmhg , and osmotic diuretics were administered to control increased intracranial pressure ( icp ) . patients with a decreased level of consciousness ( glasgow coma scale [ gcs ] < 8) were intubated to maintain the airway and to reduce the gag reflex or the chances of imminent aspiration . mechanical ventilation was used in patients with respiratory insufficiency , as indicated by pco2 > 55 mmhg or po2 < 60 mmhg . in patients with coagulopathy , anticoagulation reversal was performed using fresh - frozen plasma or prothrombin - complex concentrate , and vitamin k. anticoagulant or antiplatelet treatments were discontinued during the acute phase in patients who regularly took the medications . when necessary , informed consent was obtained to perform external ventricular drainage ( evd ) . the laterality of the evd was chosen based on the patient s conditions and the hemorrhagic location , which was observed during the initial ct . indications for evd at our institution were : ( 1 ) initial gcs score of < 12 , ( 2 ) evidence of hydrocephalus defined as an evans ratio of > 0.30 , and ( 3 ) evidence of third or fourth ventricle obstruction with an accompanying prediction that the patient would develop hydrocephalus . neurological outcome at 3 months was assessed using the glasgow outcome scale ( gos ) . patients were divided into 2 groups according to their gos scores : a favorable outcome group ( gos scores of 45 ) and an unfavorable outcome group ( gos scores of 13 ) . additional outcomes measures included mortality 3 months after presentation . the potential prognostic factors discussed in previous reports of intracerebral and/or intraventricular hemorrhage were analyzed to examine their relationships with clinical outcomes [ 5,1317 ] . the presumptive factors were either patient factors or radiologic findings , and were collected from medical records and imaging studies . potential patient factors included : age , sex , initial gcs score , low platelet count ( < 50 000 mm ) , prolonged prothrombin time ( pt ; international normalized ratio [ inr ] > 1.4 ) , use of anticoagulants or antiplatelet agents , the presence of underlying diseases such as hypertension ( determined by previous diagnosis , treatment with antihypertensive medications , or a blood pressure reading of > 140/90 mmhg ) , and diabetes mellitus ( dm ; determined by a fasting glucose reading of > 7 mmol / l or treatment with anti - diabetic medication ) . the severity of illness for the enrolled patients was measured using the new simplified acute physiology score ( saps ii ) , which is calculated based on the worst values taken during the first 24 hours after admission to the icu . the score encompasses 15 parameters ( heart rate ; body temperature ; white blood cell count ; the ratio of partial pressure of oxygen in arterial blood to fractional inspired oxygen ; bicarbonate level ; systolic blood pressure ; urinary output ; serum urea level ; serum potassium , sodium , and bilirubin levels ; gcs score ; age ; type of admission ; and chronic underlying disease ) , and has a range of 0 to 163 . additionally , parameters of the ventricular system were measured , including the transverse diameter of the third ventricle , both anterior - posterior ( ap ) and transverse diameters of the fourth ventricle , and temporal horn size on the dominant side . a dilated fourth ventricle was defined as one with a transverse diameter of > 20 mm or an ap diameter of > 12.5 mm , while third ventricle dilatation was defined as a transverse diameter of > 10 mm . finally , dilatation of the temporal horn was determined as a size of > 6 mm , and hydrocephalus was defined as an evans ratio of > 0.30 . presumptive prognostic factors were compared between the favorable ( gos scores of 45 ) and unfavorable outcome ( gos scores of 13 ) groups using t tests for continuous variables ( age , initial gcs , saps ii score , mgs , diameters of 3 and 4 ventricle , size of dominant side temporal horn ) , and either the chi - square or fisher s exact test for categorical variables ( sex , application of mechanical ventilation within 24 hours of the initial attack , prior medical comorbidities , thrombocytopenia , abnormal coagulation parameters , presence of causative vascular abnormalities , additional treatment for vascular abnormalities , and hydrocephalus ) . the association between presumptive factors and clinical outcomes was analyzed using a logistic regression model , and the odds ratio ( or ) and 95% confidence interval ( ci ) were calculated . all of the statistical calculations were performed using commercially available software ( spss 11.5 , ibm corporation , armonk , ny ) . table 1 summarizes the demographic and radiological data of the 112 patients included in the study . angiography ( ct or mr angiography , and/or tfca ) was performed in 99 patients ( 88% ) and presumptive vascular pathologies for pivh were detected in 46 patients ( 46% , 46/99 ) . among them , 28 patients ( 28% ) were diagnosed with moyamoya disease and 16 patients ( 16% ) were found to have arteriovenous malformation ( avm ) . two patients were revealed to have an aneurysm rupture ( 2% , anterior communicating artery aneurysm in one and posterior inferior cerebellar artery aneurysm in the other ) . angiograms for 53 of the patients did not show any significant abnormalities that could be associated with their ventricular hemorrhage . the mean age of those patients who were identified as having underlying vascular pathologies was 43.312.4 years , which was lower than that of patients who were not diagnosed with causal vascular lesions ( mean : 60.820.2 years , p=0.012 ) . based on the angiographic results , previous medical history , and laboratory findings , we identified several possible causes of pivh , which included vascular abnormalities identified in 46 patients ( 41% ) , pre - existing hypertension reported in 31 patients ( 28% ) , and clotting disorders present in 22 patients ( 20% ) . the antecedent vascular abnormalities were managed with medical or surgical interventions , including urgent aneurysm clipping ( 1 patient ) or coiling ( 1 patient ) in the case of a ruptured aneurysm , and radiosurgery ( 11 patients ) , neuro - interventional procedure ( 1 patient ) , or a combination of the 2 ( 2 patients ) to treat avm , for which it had been a mean of 6 weeks ( range : 59 weeks ) since initial presentation . either direct ( 8 patients ) or indirect ( 5 patients ) bypass surgery was utilized to treat patients diagnosed with moyamoya disease , for which it had been an average of 9 weeks since the initial ivh attack ( range : 512 weeks ) . two patients with avm did not undergo treatment for their avm because of refusal by their family members . evd was performed in 46 patients within 3 weeks of the initial attack ( range : 019 days ) . sixteen patients underwent a permanent cerebral spinal fluid diversion procedure within 3 months ( range : 3588 days ) of their initial presentation . all 112 patients were evaluated 3 months after initial presentation , at which point 72 ( 64% ) presented with favorable outcomes as determined by gos scores of 45 , while the other 40 patients ( 36% ) presented with poor outcomes as indicated by gos scores of 13 . eighteen patients ( 16% ) died of intractable intracranial pressure , thought to be a direct consequence of pivh , within a week after ictus . four patients died due to other causes , including a sudden onset of ventricular tachycardia and respiratory arrest , pneumonia , and sepsis of unknown origin . the majority of the survivors ( 72/90 , 80% ) presented with no deficits or mild deficits ( gos 4 ) 3 months after initial presentation . the patient and hemorrhage factors were compared between the favorable and unfavorable groups ( table 2 ) . the mean age of the patients in the unfavorable outcome group was significantly higher than that of the favorable group ( p=0.001 ) . the initial gcs score was 13.32.7 in the favorable outcome group , which was significantly higher than the score of 7.24.1 measured in the unfavorable group ( p<0.001 ) . the mean saps ii score was significantly lower in the favorable group ( 27.816.8 vs. 43.522.2 , p<0.001 ) . the prevalence of pre - existing hypertension was higher in the unfavorable outcome group ( 57% vs. 43% ) . however , the difference between the 2 outcome groups did not reach statistical significance ( p=0.143 ) . a total of 27 patients were taking anticoagulants ( 8 patients ) or antiplatelet agents ( 19 patients ) . however , there was no significant difference between the 2 groups ( p=0.382 for anticoagulant , p=0.245 for antiplatelet ) . the outcomes of patients who presented with abnormal bleeding profiles , including low platelet counts ( < 50 000 mm ) and prolonged pts ( inr > 1.4 ) , were also not significantly different from those of patients with normal bleeding profiles ( p=0.155 and 0.551 , respectively ) . the presence of causative vascular lesions was found not to be likely to affect neurological outcome ( p=0.330 ) . additionally , treatment for underlying vascular pathologies , such as neck clipping or coiling for aneurysms , radiosurgery or neuro - intervention for avm , or direct or indirect bypass surgery for moyamoya disease , did not influence short - term clinical outcomes ( p=0.583 ) . radiologic factors , determined from the initial ct scan , included mgs , the transverse diameter of the third ventricle , the ap and transverse diameters of the fourth ventricle , the size of the temporal horn on the dominant side , the presence of hydrocephalus , and whether there was a need to perform evd in the patient . ventricular hematoma volume ( as assessed by mgs ) , the size of the third and fourth ventricles , and the size of the temporal horn of the dominant lateral ventricle were significantly larger in the unfavorable outcome group than in the favorable group ( p<0.05 for all factors ) . table 3 summarizes the results of the logistic regression analysis performed on the clinical / radiological characteristics and clinical outcomes . the results of the univariate analysis indicate that older age ( 55 years ) , low initial gcs score ( gcs < 13 ) , high saps ii ( saps ii 33 ) , high mgs score , increased transverse diameter ( > 10 mm ) of the third ventricle , increased ap ( > 12.5 mm ) or transverse ( > 20 mm ) diameter of the fourth ventricle , an increase in both the ap ( > 12.5 mm ) and transverse ( > 20 mm ) diameters of the fourth ventricle , a dominant - side temporal horn size of over 6 mm , the presence of hydrocephalus , and performance of evd are all significantly associated with poor outcomes in patients with pivh . results of the multivariate analysis indicate that low gcs scores during initial presentation ( gcs < 13 ) , severe illness within 24 hours of admission ( saps ii 33 ) , and dilatation of the fourth ventricle in both the ap and transverse directions are independent factors associated with clinical outcomes . a 50-year - old man with hypertension presented with sudden onset of headache , followed by although hydrocephalus was not observed ( evans ratio=0.27 ) , the patient presented with fourth ventricle obstruction . evd was performed through the left kocher s point and his gcs score recovered to 15 . tfca did not identify the predisposing vascular pathology . with conservative care and evd management , the patient was discharged 3 weeks later without any neurologic deficits . a 78-year - old woman with a history of hypertension presented with sudden onset of altered mental status ( gcs 3 ) . evd was performed through the right kocher s point . despite administration of osmotic diuretics and evd management , the patient died on the sixth in - patient day due to intractable increased icp . table 1 summarizes the demographic and radiological data of the 112 patients included in the study . angiography ( ct or mr angiography , and/or tfca ) was performed in 99 patients ( 88% ) and presumptive vascular pathologies for pivh were detected in 46 patients ( 46% , 46/99 ) . among them , 28 patients ( 28% ) were diagnosed with moyamoya disease and 16 patients ( 16% ) were found to have arteriovenous malformation ( avm ) . two patients were revealed to have an aneurysm rupture ( 2% , anterior communicating artery aneurysm in one and posterior inferior cerebellar artery aneurysm in the other ) . angiograms for 53 of the patients did not show any significant abnormalities that could be associated with their ventricular hemorrhage . the mean age of those patients who were identified as having underlying vascular pathologies was 43.312.4 years , which was lower than that of patients who were not diagnosed with causal vascular lesions ( mean : 60.820.2 years , p=0.012 ) . based on the angiographic results , previous medical history , and laboratory findings , we identified several possible causes of pivh , which included vascular abnormalities identified in 46 patients ( 41% ) , pre - existing hypertension reported in 31 patients ( 28% ) , and clotting disorders present in 22 patients ( 20% ) . the antecedent vascular abnormalities were managed with medical or surgical interventions , including urgent aneurysm clipping ( 1 patient ) or coiling ( 1 patient ) in the case of a ruptured aneurysm , and radiosurgery ( 11 patients ) , neuro - interventional procedure ( 1 patient ) , or a combination of the 2 ( 2 patients ) to treat avm , for which it had been a mean of 6 weeks ( range : 59 weeks ) since initial presentation . either direct ( 8 patients ) or indirect ( 5 patients ) bypass surgery was utilized to treat patients diagnosed with moyamoya disease , for which it had been an average of 9 weeks since the initial ivh attack ( range : 512 weeks ) . two patients with avm did not undergo treatment for their avm because of refusal by their family members . evd was performed in 46 patients within 3 weeks of the initial attack ( range : 019 days ) . sixteen patients underwent a permanent cerebral spinal fluid diversion procedure within 3 months ( range : 3588 days ) of their initial presentation . all 112 patients were evaluated 3 months after initial presentation , at which point 72 ( 64% ) presented with favorable outcomes as determined by gos scores of 45 , while the other 40 patients ( 36% ) presented with poor outcomes as indicated by gos scores of 13 . eighteen patients ( 16% ) died of intractable intracranial pressure , thought to be a direct consequence of pivh , within a week after ictus . four patients died due to other causes , including a sudden onset of ventricular tachycardia and respiratory arrest , pneumonia , and sepsis of unknown origin . the majority of the survivors ( 72/90 , 80% ) presented with no deficits or mild deficits ( gos 4 ) 3 months after initial presentation . the patient and hemorrhage factors were compared between the favorable and unfavorable groups ( table 2 ) . the mean age of the patients in the unfavorable outcome group was significantly higher than that of the favorable group ( p=0.001 ) . the initial gcs score was 13.32.7 in the favorable outcome group , which was significantly higher than the score of 7.24.1 measured in the unfavorable group ( p<0.001 ) . the mean saps ii score was significantly lower in the favorable group ( 27.816.8 vs. 43.522.2 , p<0.001 ) . the prevalence of pre - existing hypertension was higher in the unfavorable outcome group ( 57% vs. 43% ) . however , the difference between the 2 outcome groups did not reach statistical significance ( p=0.143 ) . a total of 27 patients were taking anticoagulants ( 8 patients ) or antiplatelet agents ( 19 patients ) . however , there was no significant difference between the 2 groups ( p=0.382 for anticoagulant , p=0.245 for antiplatelet ) . the outcomes of patients who presented with abnormal bleeding profiles , including low platelet counts ( < 50 000 mm ) and prolonged pts ( inr > 1.4 ) , were also not significantly different from those of patients with normal bleeding profiles ( p=0.155 and 0.551 , respectively ) . the presence of causative vascular lesions was found not to be likely to affect neurological outcome ( p=0.330 ) . additionally , treatment for underlying vascular pathologies , such as neck clipping or coiling for aneurysms , radiosurgery or neuro - intervention for avm , or direct or indirect bypass surgery for moyamoya disease , did not influence short - term clinical outcomes ( p=0.583 ) . radiologic factors , determined from the initial ct scan , included mgs , the transverse diameter of the third ventricle , the ap and transverse diameters of the fourth ventricle , the size of the temporal horn on the dominant side , the presence of hydrocephalus , and whether there was a need to perform evd in the patient . ventricular hematoma volume ( as assessed by mgs ) , the size of the third and fourth ventricles , and the size of the temporal horn of the dominant lateral ventricle were significantly larger in the unfavorable outcome group than in the favorable group ( p<0.05 for all factors ) . table 3 summarizes the results of the logistic regression analysis performed on the clinical / radiological characteristics and clinical outcomes . the results of the univariate analysis indicate that older age ( 55 years ) , low initial gcs score ( gcs < 13 ) , high saps ii ( saps ii 33 ) , high mgs score , increased transverse diameter ( > 10 mm ) of the third ventricle , increased ap ( > 12.5 mm ) or transverse ( > 20 mm ) diameter of the fourth ventricle , an increase in both the ap ( > 12.5 mm ) and transverse ( > 20 mm ) diameters of the fourth ventricle , a dominant - side temporal horn size of over 6 mm , the presence of hydrocephalus , and performance of evd are all significantly associated with poor outcomes in patients with pivh . results of the multivariate analysis indicate that low gcs scores during initial presentation ( gcs < 13 ) , severe illness within 24 hours of admission ( saps ii 33 ) , and dilatation of the fourth ventricle in both the ap and transverse directions are independent factors associated with clinical outcomes . a 50-year - old man with hypertension presented with sudden onset of headache , followed by altered mental status ( gcs 9 ) . the patient s mgs was 22 . although hydrocephalus was not observed ( evans ratio=0.27 ) , the patient presented with fourth ventricle obstruction . evd was performed through the left kocher s point and his gcs score recovered to 15 . tfca did not identify the predisposing vascular pathology . with conservative care and evd management , the patient was discharged 3 weeks later without any neurologic deficits . a 78-year - old woman with a history of hypertension presented with sudden onset of altered mental status ( gcs 3 ) . evd was performed through the right kocher s point . despite administration of osmotic diuretics and evd management , the patient died on the sixth in - patient day due to intractable increased icp . a 50-year - old man with hypertension presented with sudden onset of headache , followed by altered mental status ( gcs 9 ) . the patient s mgs was 22 . although hydrocephalus was not observed ( evans ratio=0.27 ) , the patient presented with fourth ventricle obstruction . evd was performed through the left kocher s point and his gcs score recovered to 15 . tfca did not identify the predisposing vascular pathology . with conservative care and evd management , a 78-year - old woman with a history of hypertension presented with sudden onset of altered mental status ( gcs 3 ) . evd was performed through the right kocher s point . despite administration of osmotic diuretics and evd management , the patient died on the sixth in - patient day due to intractable increased icp . in the present study , we investigated the prognostic factors that contribute to clinical outcomes in patients with pivh . importantly , we found that low gcs at ictus and an increase in the diameter of the fourth ventricle in both the ap and transverse directions were independent factors that predicted poor outcome . pivh is associated with several possible causative factors , including hypertension , arteriovenous malformation , cerebral aneurysm , tumor , coagulopathy , vasculitis , and choroid plexus cysts [ 1,4,2128 ] . a history of hypertension has been consistently reported as a major risk factor for spontaneous intracranial hemorrhage , and is responsible for 3880% of pivh cases . in the current study , while many patients had a history of hypertension , pre - existing hypertension itself was not found to affect patient outcome . although the prognostic value of premorbid hypertension for outcome determination has not yet been fully investigated , intensive antihypertensive treatment during the acute phase has been noted to improve clinical outcomes . a recent randomized controlled trial in 2839 patients with ich demonstrated that early intensive interventions to lower blood pressure toward a target systolic level of < 140 mmhg improved functional outcomes , although the mortality rate was not affected . therefore , strict blood pressure control during the acute phase of pivh may be critical to improving clinical outcome . in this study , we evaluated the presence of predisposing vascular abnormalities using conventional cerebral angiography and/or ct / mr angiography , and found that nearly half of the angiographically examined patients had presumptive pathologies for their ivh , which included moyamoya disease , avm , and a ruptured cerebral aneurysm . a previous review study including 339 pivh patients in 14 case series reported the rates of avm , aneurysms , and moyamoya disease as 31% , 22% , and 3% , respectively . we found a relatively higher incidence of moyamoya disease than was found in previous series . this might be explained by the fact that this disease is more prevalent among east asian populations . the present study also indicates that patients with underlying vascular pathologies leading to pivh are younger than those with normal angiographic results . this indicates that additional diagnostic imaging should be performed in cases of pivh to detect underlying pathologies , especially in young patients . neither the presence of causative vascular abnormalities nor the treatment of the underlying lesions affected the short - term clinical outcome of pivh in our study . the comparable outcomes observed between patients with causative predisposing vascular lesions and those without obvious vascular pathologies may be attributed to the absence of re - hemorrhage during early period regardless of treatment for underlying vascular etiologies . although the efficacies of the treatments for aneurysms , avm , and moyamoya disease were not defined by measurements of short - term outcomes in the present study , we believe that treatment of the vascular causal factors may enhance long - term outcomes by reducing the risk of re - bleeding in the group of pivh patients who had vascular predisposing factors . how many of the 13 patients who did not have angiographic studies had possible vascular causes for pivh is not known , and this might have affected the outcome results . thus , the lack of difference in neurological outcomes between patients with and without possible vascular etiology of pivh must be interpreted with caution . further studies and additional follow - up are warranted to document the influence of vascular etiologies on the prognosis of pivh . there is considerable variability in the results of previous studies that have attempted to determine factors associated with outcome in cases of pivh . reported the clinical outcomes of 23 patients with intraventricular hemorrhage , showing that 13 patients were dead within a mean of 2.3 days , and the consciousness level during the initial attack was considered to be a major factor affecting neurological outcome . similarly , in a study of 15 patients with pure intraventricular hemorrhage , jayakumar et al . reported 0% mortality in patients with normal or mildly disturbed mental status at presentation , whereas 100% of initially decerebrating patients died . interestingly , the outcomes for the patients were either good or death , indicating that none of the surviving patients had any significant neurological deficits . the lack of parenchymal damage in pivh was considered to be the cause of this extremely different outcome . in addition to initial consciousness level , the authors noted that the amount of hematoma in the ventricle , the occurrence of hematoma in the cistern , hydrocephalus , and brain atrophy are all prognostic factors . , the majority ( 80% ) of patients presented with altered mental status at ictus , and evd was performed in 11 patients ( 73% ) . one patient ( 7% ) died while hospitalized and 8 patients ( 53% ) recovered to an independent neurological status . based on systematic review , the authors noted that patient age and the amount of ivh independently predicted in - hospital mortality . a recent study of 12 patients with pivh indicated a 41.7% mortality rate during a median of 18.5 days of hospital stay . in this study , very old age ( 85 years ) was associated with patient death . in the series , 1 patient ( 8.3% ) underwent surgical intervention ( evd ) , and 6 of the 7 survivors had moderate or severe disability at discharge ( modified rankin scale grade 3 or more . by comparing 133 subcortical hemorrhages , the authors noted that very old age ( 85 years ) , atrial fibrillation , headache , and altered consciousness at onset are independent predictors of pivh . the relatively high mortality and poor neurological outcomes in this series are likely to be associated with the old age of the study population ( mean age : 79 years ) . in another recent analysis of 24 patients with pivh , a low initial gcs score ( 8 ) , low full outline of unresponsiveness score ( 10 ) , and development of early hydrocephalus . found that ivh itself is not a direct cause of death , but rather acts in conjunction with altered cerebrospinal fluid circulation following ventricular hemorrhage , cerebral edema , or increased intracranial pressure . furthermore , the authors state that the level of consciousness during the initial presentation , the cause of ventricular hemorrhage , amount of ventricular dilation , shifting of the midline structure , and hemorrhagic patterns are all key aspects influencing prognosis in patients with pivh . in an analysis of 50 patients with ct - documented fourth ivh , shapiro et al . suggested that the hemorrhagic dilation of the fourth ventricle is an ominous finding , as all patients with this symptom died despite aggressive therapy . autopsies of these patients revealed medullopontine softening on gross examination , and multiple pontine microinfarcts upon histological assessment . consistent with these results , our data indicate that dilation of the fourth ventricle to diameters of over 20 and 12.5 mm in the transverse and ap directions , respectively , is an independent unfavorable prognostic factor , as well as a lower gcs score at ictus . taken together , it seems that hemorrhagic dilation of the fourth ventricle , followed by a sudden increase in intraventricular pressure , places pressure on the brain stem and impairs perforator perfusion . this may promote irreversible ischemic injury to the brain stem , which is the major control center for consciousness and respiration . saps ii is widely used in the general icu for grading derangements in the physiologic homeostasis of individual patients , as well as for prognostic calculations . in the present study , saps ii score , obtained within 24 hours of admission , was significantly associated with neurological outcomes in patients with pivh , which is consistent with the results of other types of stroke [ 3840 ] . this is partly explained by the fact that both gcs and age , which are well known potential prognostic factors of stroke , are major components of the scoring system . to verify how the severity of systemic illness and extracerebral organ dysfunction play important roles in the prognoses of patients with pivh , further studies analyzing the relationships between individual variables that contribute to the overall saps ii and patients outcomes will be necessary . sixteen percent of the patients likely died as a direct consequence of pivh within a week of their initial presentation . three months later , nearly two - thirds ( 64% ) of all patients presented with favorable outcomes . the majority of the survivors ( 80% ) had no deficits or mild deficits ( gos 4 ) . the reported rate of poor outcome following a large series of intracerebral hemorrhages ranges from 49% to 78% . therefore , the neurological prognosis of pivh is likely superior to that of intracerebral hemorrhage . this relatively favorable neurological course for pivh might be associated with comparatively little brain parenchymal damage . coagulopathy , including anticoagulant or antiplatelet agent therapy , is a proven risk factor for hemorrhagic stroke . the number of patients who had coagulopathy or thrombocytopenia in our study was congruent with the results of previous studies . anticoagulant - associated hemorrhagic stroke is associated with an increased risk of hematoma expansion and a higher mortality rate [ 4547 ] . however , we found that a medical history of anticoagulant or antiplatelet agent use , or coagulopathy did not affect clinical outcome . this discrepancy may partly be explained by the aggressive reversal of anticoagulation during the hyper - acute phase in the subjects , which can prevent hematoma expansion or re - bleeding . there are many reports demonstrating that clinical improvement can be achieved through the reversal of thrombocytopenia and coagulopathy [ 30,4850 ] . recently , patients with severe coagulation factor deficiency or severe thrombocytopenia have been recommended to receive appropriate factor replacement therapy or platelets , respectively . therefore , adequate use of hemostatic agents and transfusion of blood should be considered when managing patients with pivh . although the optimal time to resume anticoagulants or antiplatelet agents after ich or ivh is uncertain , current guidelines recommend the avoidance of oral anticoagulation medication for at least 4 weeks to lower the risk of recurrence . a standard treatment plan for pivh although various treatment protocols including supportive medical treatments , steroids , antihypertensive agents , and evd methods are in use , there is still controversy surrounding the effects of these methods . in the present study , many of the patients received evd , of whom the majority displayed unfavorable outcomes ( gos < 4 ) . considering that evd was conducted primarily in those who had poor consciousness levels with significant hydrocephalus , which might be associated with a poor prognosis , it is difficult to judge the effects of the treatment modality by itself . recently , several small studies of intraventricular thrombolysis in combination with evd have shown promising results in patients with ivh [ 5254 ] . these studies suggest that thrombolytic treatment hastens the clearance of ventricular hematomas and normalizes icp . in a review of 7 independent studies using intraventricular thrombolytic agents in patients with ivh however , the procedure was associated with serious complications , including bacterial meningitis ( n=5 ) , increased hematoma volume ( n=1 ) , and extradural hematomas ( n=2 ) . although there is anecdotal evidence for the safety of this treatment and its possible therapeutic value , there is insufficient evidence to support its clinical efficacy therefore , a large , randomized , prospective study of thrombolytic treatment in pivh is needed . second , the number of patients enrolled in this study was relatively small , especially for the group of unfavorable - outcome patients . this limited our ability to subdivide the patient groups into co - affecting area and systemic complication groups . additionally , different surgeons with different treatment preferences undertook the patients care , adding to between - patient variability . other unmeasurable underlying differences between the groups may have confounded our results . a further study in a larger number of patients and a scrupulous prospective set of clinical data is required to gain more accurate knowledge of predictive factors for patients with spontaneous primary intraventricular hemorrhage . most deaths occurred within 1 week of initial presentation and were a direct consequence of pivh . initial gcs score , severity of illness as indicated by saps ii , and dilatation of the fourth ventricle in both the ap and transverse directions were identified as major independent prognostic factors . predisposing vascular abnormalities such as moyamoya disease , avm , or cerebral aneurysm were identified in nearly half of the pivh patients . routine angiography should be included in the examination and diagnosis of patients with pivh to identify potentially treatable causes of the hemorrhage .

backgroundprimary intraventricular hemorrhage ( pivh ) is an uncommon type of intracerebral hemorrhage . owing to its rarity , the clinical and radiological factors affecting outcomes in patients with pivh have not been widely studied.material/methodswe retrospectively reviewed 112 patients ( mean age 53 years ) treated for pivh at our institution from january 2004 to december 2014 . clinical and radiological parameters were analyzed 3 months after initial presentation to identify factors associated with clinical outcomes , as assessed by the glasgow outcome scale ( favorable 4 , unfavorable < 4).resultsof the 99 patients who underwent angiography , causative vascular abnormalities were found in 46% , and included moyamoya disease , arteriovenous malformation , and cerebral aneurysm . at 3 months after initial presentation , 64% and 36% of patients were in the favorable and unfavorable outcome groups , respectively . the mortality rate was 19% . however , most survivors had no or mild deficits . age , initial glasgow coma scale ( gcs ) score , simplified acute physiology score ( saps ii ) , modified graeb score , and various radiological parameters reflecting ventricular dilatation were significantly different between the groups . specifically , a gcs score of less than 13 ( p=0.015 ) , a saps ii score of less than 33 ( p=0.039 ) , and a dilated fourth ventricle ( p=0.043 ) were demonstrated to be independent predictors of an unfavorable clinical outcome.conclusionsin this study we reveal independent predictors of poor outcome in primary intraventricular hemorrhage patients , and show that nearly half of the patients in our study had predisposing vascular abnormalities . routine angiography is recommended in the evaluation of pivh to identify potentially treatable etiologies , which may enhance long - term prognosis .

Background Material and Methods Patient population and inclusion criteria Patient management Outcomes measures Analysis of prognostic factors affecting neurological outcomes Statistical analysis Results Patient characteristics, treatment, and clinical outcomes Patient and hemorrhagic factors Evaluation of prognostic factors Case illustrations Case 1 ( Case 2 ( Discussion Conclusions

primary intraventricular hemorrhage ( pivh ) is a rare condition defined as bleeding within the ventricular system without a definite parenchymal component , and accounts for 0.31% of all stroke cases and 3.1% of intracerebral hemorrhages ( ichs ) [ 16 ] . however , due to its rarity , there is relatively little information regarding the clinical manifestations of pivh , its etiology , and its prognosis . we reviewed all patients with ich who were admitted at our institution between january 2004 and december 2014 . exclusion criteria were : neonates ( less than 1 month after birth ) , death within 24 hours of presentation , and life expectancy less than 6 months owing to another medical condition . indications for evd at our institution were : ( 1 ) initial gcs score of < 12 , ( 2 ) evidence of hydrocephalus defined as an evans ratio of > 0.30 , and ( 3 ) evidence of third or fourth ventricle obstruction with an accompanying prediction that the patient would develop hydrocephalus . neurological outcome at 3 months was assessed using the glasgow outcome scale ( gos ) . the potential prognostic factors discussed in previous reports of intracerebral and/or intraventricular hemorrhage were analyzed to examine their relationships with clinical outcomes [ 5,1317 ] . potential patient factors included : age , sex , initial gcs score , low platelet count ( < 50 000 mm ) , prolonged prothrombin time ( pt ; international normalized ratio [ inr ] > 1.4 ) , use of anticoagulants or antiplatelet agents , the presence of underlying diseases such as hypertension ( determined by previous diagnosis , treatment with antihypertensive medications , or a blood pressure reading of > 140/90 mmhg ) , and diabetes mellitus ( dm ; determined by a fasting glucose reading of > 7 mmol / l or treatment with anti - diabetic medication ) . the severity of illness for the enrolled patients was measured using the new simplified acute physiology score ( saps ii ) , which is calculated based on the worst values taken during the first 24 hours after admission to the icu . presumptive prognostic factors were compared between the favorable ( gos scores of 45 ) and unfavorable outcome ( gos scores of 13 ) groups using t tests for continuous variables ( age , initial gcs , saps ii score , mgs , diameters of 3 and 4 ventricle , size of dominant side temporal horn ) , and either the chi - square or fisher s exact test for categorical variables ( sex , application of mechanical ventilation within 24 hours of the initial attack , prior medical comorbidities , thrombocytopenia , abnormal coagulation parameters , presence of causative vascular abnormalities , additional treatment for vascular abnormalities , and hydrocephalus ) . we reviewed all patients with ich who were admitted at our institution between january 2004 and december 2014 . exclusion criteria were : neonates ( less than 1 month after birth ) , death within 24 hours of presentation , and life expectancy less than 6 months owing to another medical condition . indications for evd at our institution were : ( 1 ) initial gcs score of < 12 , ( 2 ) evidence of hydrocephalus defined as an evans ratio of > 0.30 , and ( 3 ) evidence of third or fourth ventricle obstruction with an accompanying prediction that the patient would develop hydrocephalus . neurological outcome at 3 months was assessed using the glasgow outcome scale ( gos ) . the potential prognostic factors discussed in previous reports of intracerebral and/or intraventricular hemorrhage were analyzed to examine their relationships with clinical outcomes [ 5,1317 ] . potential patient factors included : age , sex , initial gcs score , low platelet count ( < 50 000 mm ) , prolonged prothrombin time ( pt ; international normalized ratio [ inr ] > 1.4 ) , use of anticoagulants or antiplatelet agents , the presence of underlying diseases such as hypertension ( determined by previous diagnosis , treatment with antihypertensive medications , or a blood pressure reading of > 140/90 mmhg ) , and diabetes mellitus ( dm ; determined by a fasting glucose reading of > 7 mmol / l or treatment with anti - diabetic medication ) . the severity of illness for the enrolled patients was measured using the new simplified acute physiology score ( saps ii ) , which is calculated based on the worst values taken during the first 24 hours after admission to the icu . presumptive prognostic factors were compared between the favorable ( gos scores of 45 ) and unfavorable outcome ( gos scores of 13 ) groups using t tests for continuous variables ( age , initial gcs , saps ii score , mgs , diameters of 3 and 4 ventricle , size of dominant side temporal horn ) , and either the chi - square or fisher s exact test for categorical variables ( sex , application of mechanical ventilation within 24 hours of the initial attack , prior medical comorbidities , thrombocytopenia , abnormal coagulation parameters , presence of causative vascular abnormalities , additional treatment for vascular abnormalities , and hydrocephalus ) . angiography ( ct or mr angiography , and/or tfca ) was performed in 99 patients ( 88% ) and presumptive vascular pathologies for pivh were detected in 46 patients ( 46% , 46/99 ) . among them , 28 patients ( 28% ) were diagnosed with moyamoya disease and 16 patients ( 16% ) were found to have arteriovenous malformation ( avm ) . the mean age of those patients who were identified as having underlying vascular pathologies was 43.312.4 years , which was lower than that of patients who were not diagnosed with causal vascular lesions ( mean : 60.820.2 years , p=0.012 ) . based on the angiographic results , previous medical history , and laboratory findings , we identified several possible causes of pivh , which included vascular abnormalities identified in 46 patients ( 41% ) , pre - existing hypertension reported in 31 patients ( 28% ) , and clotting disorders present in 22 patients ( 20% ) . the antecedent vascular abnormalities were managed with medical or surgical interventions , including urgent aneurysm clipping ( 1 patient ) or coiling ( 1 patient ) in the case of a ruptured aneurysm , and radiosurgery ( 11 patients ) , neuro - interventional procedure ( 1 patient ) , or a combination of the 2 ( 2 patients ) to treat avm , for which it had been a mean of 6 weeks ( range : 59 weeks ) since initial presentation . all 112 patients were evaluated 3 months after initial presentation , at which point 72 ( 64% ) presented with favorable outcomes as determined by gos scores of 45 , while the other 40 patients ( 36% ) presented with poor outcomes as indicated by gos scores of 13 . the majority of the survivors ( 72/90 , 80% ) presented with no deficits or mild deficits ( gos 4 ) 3 months after initial presentation . the mean age of the patients in the unfavorable outcome group was significantly higher than that of the favorable group ( p=0.001 ) . the initial gcs score was 13.32.7 in the favorable outcome group , which was significantly higher than the score of 7.24.1 measured in the unfavorable group ( p<0.001 ) . however , the difference between the 2 outcome groups did not reach statistical significance ( p=0.143 ) . the outcomes of patients who presented with abnormal bleeding profiles , including low platelet counts ( < 50 000 mm ) and prolonged pts ( inr > 1.4 ) , were also not significantly different from those of patients with normal bleeding profiles ( p=0.155 and 0.551 , respectively ) . additionally , treatment for underlying vascular pathologies , such as neck clipping or coiling for aneurysms , radiosurgery or neuro - intervention for avm , or direct or indirect bypass surgery for moyamoya disease , did not influence short - term clinical outcomes ( p=0.583 ) . radiologic factors , determined from the initial ct scan , included mgs , the transverse diameter of the third ventricle , the ap and transverse diameters of the fourth ventricle , the size of the temporal horn on the dominant side , the presence of hydrocephalus , and whether there was a need to perform evd in the patient . ventricular hematoma volume ( as assessed by mgs ) , the size of the third and fourth ventricles , and the size of the temporal horn of the dominant lateral ventricle were significantly larger in the unfavorable outcome group than in the favorable group ( p<0.05 for all factors ) . the results of the univariate analysis indicate that older age ( 55 years ) , low initial gcs score ( gcs < 13 ) , high saps ii ( saps ii 33 ) , high mgs score , increased transverse diameter ( > 10 mm ) of the third ventricle , increased ap ( > 12.5 mm ) or transverse ( > 20 mm ) diameter of the fourth ventricle , an increase in both the ap ( > 12.5 mm ) and transverse ( > 20 mm ) diameters of the fourth ventricle , a dominant - side temporal horn size of over 6 mm , the presence of hydrocephalus , and performance of evd are all significantly associated with poor outcomes in patients with pivh . results of the multivariate analysis indicate that low gcs scores during initial presentation ( gcs < 13 ) , severe illness within 24 hours of admission ( saps ii 33 ) , and dilatation of the fourth ventricle in both the ap and transverse directions are independent factors associated with clinical outcomes . angiography ( ct or mr angiography , and/or tfca ) was performed in 99 patients ( 88% ) and presumptive vascular pathologies for pivh were detected in 46 patients ( 46% , 46/99 ) . among them , 28 patients ( 28% ) were diagnosed with moyamoya disease and 16 patients ( 16% ) were found to have arteriovenous malformation ( avm ) . the mean age of those patients who were identified as having underlying vascular pathologies was 43.312.4 years , which was lower than that of patients who were not diagnosed with causal vascular lesions ( mean : 60.820.2 years , p=0.012 ) . based on the angiographic results , previous medical history , and laboratory findings , we identified several possible causes of pivh , which included vascular abnormalities identified in 46 patients ( 41% ) , pre - existing hypertension reported in 31 patients ( 28% ) , and clotting disorders present in 22 patients ( 20% ) . the antecedent vascular abnormalities were managed with medical or surgical interventions , including urgent aneurysm clipping ( 1 patient ) or coiling ( 1 patient ) in the case of a ruptured aneurysm , and radiosurgery ( 11 patients ) , neuro - interventional procedure ( 1 patient ) , or a combination of the 2 ( 2 patients ) to treat avm , for which it had been a mean of 6 weeks ( range : 59 weeks ) since initial presentation . all 112 patients were evaluated 3 months after initial presentation , at which point 72 ( 64% ) presented with favorable outcomes as determined by gos scores of 45 , while the other 40 patients ( 36% ) presented with poor outcomes as indicated by gos scores of 13 . the majority of the survivors ( 72/90 , 80% ) presented with no deficits or mild deficits ( gos 4 ) 3 months after initial presentation . the mean age of the patients in the unfavorable outcome group was significantly higher than that of the favorable group ( p=0.001 ) . the initial gcs score was 13.32.7 in the favorable outcome group , which was significantly higher than the score of 7.24.1 measured in the unfavorable group ( p<0.001 ) . the mean saps ii score was significantly lower in the favorable group ( 27.816.8 vs. 43.522.2 , p<0.001 ) . however , the difference between the 2 outcome groups did not reach statistical significance ( p=0.143 ) . the outcomes of patients who presented with abnormal bleeding profiles , including low platelet counts ( < 50 000 mm ) and prolonged pts ( inr > 1.4 ) , were also not significantly different from those of patients with normal bleeding profiles ( p=0.155 and 0.551 , respectively ) . additionally , treatment for underlying vascular pathologies , such as neck clipping or coiling for aneurysms , radiosurgery or neuro - intervention for avm , or direct or indirect bypass surgery for moyamoya disease , did not influence short - term clinical outcomes ( p=0.583 ) . radiologic factors , determined from the initial ct scan , included mgs , the transverse diameter of the third ventricle , the ap and transverse diameters of the fourth ventricle , the size of the temporal horn on the dominant side , the presence of hydrocephalus , and whether there was a need to perform evd in the patient . ventricular hematoma volume ( as assessed by mgs ) , the size of the third and fourth ventricles , and the size of the temporal horn of the dominant lateral ventricle were significantly larger in the unfavorable outcome group than in the favorable group ( p<0.05 for all factors ) . the results of the univariate analysis indicate that older age ( 55 years ) , low initial gcs score ( gcs < 13 ) , high saps ii ( saps ii 33 ) , high mgs score , increased transverse diameter ( > 10 mm ) of the third ventricle , increased ap ( > 12.5 mm ) or transverse ( > 20 mm ) diameter of the fourth ventricle , an increase in both the ap ( > 12.5 mm ) and transverse ( > 20 mm ) diameters of the fourth ventricle , a dominant - side temporal horn size of over 6 mm , the presence of hydrocephalus , and performance of evd are all significantly associated with poor outcomes in patients with pivh . results of the multivariate analysis indicate that low gcs scores during initial presentation ( gcs < 13 ) , severe illness within 24 hours of admission ( saps ii 33 ) , and dilatation of the fourth ventricle in both the ap and transverse directions are independent factors associated with clinical outcomes . in the present study , we investigated the prognostic factors that contribute to clinical outcomes in patients with pivh . importantly , we found that low gcs at ictus and an increase in the diameter of the fourth ventricle in both the ap and transverse directions were independent factors that predicted poor outcome . pivh is associated with several possible causative factors , including hypertension , arteriovenous malformation , cerebral aneurysm , tumor , coagulopathy , vasculitis , and choroid plexus cysts [ 1,4,2128 ] . a recent randomized controlled trial in 2839 patients with ich demonstrated that early intensive interventions to lower blood pressure toward a target systolic level of < 140 mmhg improved functional outcomes , although the mortality rate was not affected . in this study , we evaluated the presence of predisposing vascular abnormalities using conventional cerebral angiography and/or ct / mr angiography , and found that nearly half of the angiographically examined patients had presumptive pathologies for their ivh , which included moyamoya disease , avm , and a ruptured cerebral aneurysm . neither the presence of causative vascular abnormalities nor the treatment of the underlying lesions affected the short - term clinical outcome of pivh in our study . although the efficacies of the treatments for aneurysms , avm , and moyamoya disease were not defined by measurements of short - term outcomes in the present study , we believe that treatment of the vascular causal factors may enhance long - term outcomes by reducing the risk of re - bleeding in the group of pivh patients who had vascular predisposing factors . there is considerable variability in the results of previous studies that have attempted to determine factors associated with outcome in cases of pivh . reported the clinical outcomes of 23 patients with intraventricular hemorrhage , showing that 13 patients were dead within a mean of 2.3 days , and the consciousness level during the initial attack was considered to be a major factor affecting neurological outcome . in the series , 1 patient ( 8.3% ) underwent surgical intervention ( evd ) , and 6 of the 7 survivors had moderate or severe disability at discharge ( modified rankin scale grade 3 or more . by comparing 133 subcortical hemorrhages , the authors noted that very old age ( 85 years ) , atrial fibrillation , headache , and altered consciousness at onset are independent predictors of pivh . the relatively high mortality and poor neurological outcomes in this series are likely to be associated with the old age of the study population ( mean age : 79 years ) . in another recent analysis of 24 patients with pivh , a low initial gcs score ( 8 ) , low full outline of unresponsiveness score ( 10 ) , and development of early hydrocephalus . furthermore , the authors state that the level of consciousness during the initial presentation , the cause of ventricular hemorrhage , amount of ventricular dilation , shifting of the midline structure , and hemorrhagic patterns are all key aspects influencing prognosis in patients with pivh . suggested that the hemorrhagic dilation of the fourth ventricle is an ominous finding , as all patients with this symptom died despite aggressive therapy . consistent with these results , our data indicate that dilation of the fourth ventricle to diameters of over 20 and 12.5 mm in the transverse and ap directions , respectively , is an independent unfavorable prognostic factor , as well as a lower gcs score at ictus . in the present study , saps ii score , obtained within 24 hours of admission , was significantly associated with neurological outcomes in patients with pivh , which is consistent with the results of other types of stroke [ 3840 ] . to verify how the severity of systemic illness and extracerebral organ dysfunction play important roles in the prognoses of patients with pivh , further studies analyzing the relationships between individual variables that contribute to the overall saps ii and patients outcomes will be necessary . the number of patients who had coagulopathy or thrombocytopenia in our study was congruent with the results of previous studies . in a review of 7 independent studies using intraventricular thrombolytic agents in patients with ivh however , the procedure was associated with serious complications , including bacterial meningitis ( n=5 ) , increased hematoma volume ( n=1 ) , and extradural hematomas ( n=2 ) . a further study in a larger number of patients and a scrupulous prospective set of clinical data is required to gain more accurate knowledge of predictive factors for patients with spontaneous primary intraventricular hemorrhage . initial gcs score , severity of illness as indicated by saps ii , and dilatation of the fourth ventricle in both the ap and transverse directions were identified as major independent prognostic factors . predisposing vascular abnormalities such as moyamoya disease , avm , or cerebral aneurysm were identified in nearly half of the pivh patients . routine angiography should be included in the examination and diagnosis of patients with pivh to identify potentially treatable causes of the hemorrhage .

ninety - percent of head and neck cancers are squamous cell carcinomas ( scchn ) involving the mucosal surfaces of the oral cavity , pharynx , and larynx . the overall relative 5-year survival rates for cancers of the oral cavity / pharynx and larynx are estimated to be 58.3% and 64.5% , respectively . morbidities associated with scchn and its treatments are significant and include eating and swallowing difficulties . targeted therapies for scchn are under active investigation with the goals of reducing scchn morbidity and mortality . targeted therapeutics were conceptualized as a means of exploiting specific molecular alterations associated with cancers in order to selectively kill transformed cells and spare normal , healthy tissues . targeted therapies are anticipated to have fewer associated toxicities than standard chemotherapies , which rely predominately on increased rates of cell division to enhance killing of the tumor cells compared to healthy tissues . for tumors that are treated with radiation and/or surgery , targeted therapies delivered systemically also have the potential to eliminate micrometastases that might not be eliminated with radiation therapy ( rt ) and/or surgery . in addition to reduced toxicity and treatment of undetected disease , it is hypothesized that effective targeted therapy may interfere specifically with processes that the cancer is dependent upon and be more effective than conventional therapies . the epidermal growth factor receptor ( egfr ) was anticipated to be a good drug target for scchn treatment because the majority of scchn overexpress egfr [ 2 , 3 ] , and higher tumor levels of egfr are associated with poorer clinical outcomes [ 4 , 5 ] . egfr participates in scchn autocrine stimulation , and overexpression of egfr and its primary ligand in humans , transforming growth factor alpha ( tgf- ) , have been correlated with poor outcomes for patients receiving therapy . cetuximab ( erbitux ; imclone systems ) , a chimeric monoclonal igg1 antibody directed against egfr , was fda - approved for the treatment of scchn in combination with rt for locally or regionally advanced disease and as a monotherapy for recurrent or metastatic scchn patients who have failed prior platinum - based therapy . in addition to antibodies directed against egfr , small molecule tyrosine kinase inhibitors ( tki ) of egfr and egfr antisense agents are currently under active clinical investigation for scchn treatment . egfr - targeted therapeutics delivered as monotherapies for treatment of scchn have demonstrated fewer toxicities compared to combined modality treatment regimens but only marginal clinical response ( 410% ) [ 7 , 8 ] . in general responses to egfr - targeted therapies in scchn clinical trials improving clinical response rates will involve ( 1 ) identifying scchn patients who are likely to respond to egfr - targeted therapies , ( 2 ) developing effective combinations of targeted therapies , and ( 3 ) correctly identifying patients who will respond to specific targeted agents applied alone or in combination . our understanding of the factors contributing to targeted therapy response now extends beyond the molecular alterations of the tumor to include host genetic variation . in this review , we will summarize molecular data correlated with clinical response to egfr - targeted therapies and discuss factors that may be considered for identifying responsive scchn patients . preclinical evidence suggests that src family kinase - targeted agents administered in combination with egfr - targeted therapies may demonstrate improved clinical response over egfr - targeted agents alone . here we also provide rationale for combining egfr- and src - targeted therapeutics for treatment of scchn , discuss published egfr- and src - combination treatment preclinical data , and summarize completed and ongoing clinical trials in solid tumors evaluating src - targeted therapies in combination with egfr - targeted therapies . there are several egfr - targeted therapies in clinical development for scchn , and these agents are described in table 1 . these inhibitors fall into two primary categories : egfr - directed antibodies and egfr tyrosine kinase inhibitors . egfr - directed antibodies include cetuximab , nimotuzumab ( ym biosciences ) , panitumumab ( amgen ) , and zalutumumab ( genmab ) . egfr - targeted tyrosine kinase inhibitors include erlotinib ( genetech and osi pharmaceuticals ) and gefitinib ( astrazeneca ) . in addition to egfr - targeted kinase inhibitors , inhibitors with broader target specificities are also in phase ii or iii development for scchn including lapatinib ( glaxosmithkline ) , which is a dual egfr / her2 inhibitor , and zactima ( astrazeneca ) , which targets vegfr2 and ret in addition to egfr ( table 1 ) . more recently antisense therapy targeting egfr has been evaluated in a phase i clinical trial by our group . results of clinical trials for egfr - targeted therapies cetuximab , nimotuzumab , gefitinib , and erlotinib used alone or in combination with conventional treatments for scchn have been reviewed by us and others and will not be described in detail here [ 1012 ] . a phase i study of panitumumab in combination with chemoradiotherapy involving 19-treatment - nave patients with stage iii / iv head and neck cancer reported an 87% complete response rate among the 15 evaluable patients and no grade 3 or 4 chronic toxicities . a phase i study of lapatinib in combination with chemoradiation in 31 patients with locally advanced scchn reported an overall response rate of 81% with radiation - associated mucositis , dermatitis , lymphopenia , and neutropenia as the most common grade 3 or 4 adverse events . our phase i study of intratumoral delivery of egfr antisense dna in 17 patients with advanced , refractory scchn was associated with no grade 3 or 4 or dose - limiting toxicities and a clinical response rate ( complete response and partial response by modified recist criteria ) of 29% . the current phase of clinical development for each of these agents is presented in table 1 . to date , no molecular marker has been identified to correlate with scchn response to egfr - targeting in patients . scchn tumor expression of the truncated form of egfr , egfr variant iii ( viii ) , which lacks the ligand binding domain , occurs in up to 40% of scchn tumors and confers resistance to egfr - targeted monoclonal antibodies in scchn preclinical models . however , egfr viii expression and resistance to egfr - targeted therapies in scchn patients has not been described . molecular correlates of clinical response / nonresponse to egfr - targeted therapies have been identified for colon and lung cancers . for example , the treatment of lung cancer with the egfr tyrosine kinase inhibitor gefitinib demonstrated effective responses in a subset of patients whose lung cancers were subsequently found to harbor egfr kinase activating mutations [ 16 , 17 ] . importantly , egfr activating mutations do not appear to be frequent in scchn [ 18 , 19 ] . therefore , some of these molecular correlates , such as the egfr tyrosine kinase activating mutations are not applicable to scchn because the frequency of these mutations is very low and will not be discussed further in this review . however , other molecular correlates of response to egfr - targeting agents have been described for lung cancer and colorectal cancers including egfr gene amplification , other somatic tumor mutations and patient genetic variations . egfr gene amplification occurs in scchn , and the rate of reported egfr gene amplification in scchn varies substantially ( table 2 ) . to date , there are no published reports evaluating egfr gene amplification for association with scchn patient response to egfr - targeted therapies . however , egfr gene amplification has been reported to be positively associated with response to egfr - directed antibody therapies in clinical trials for nonsmall lung cancers ( nsclc ) and colorectal cancers . in a phase ii study of 229 nsclc patients with advanced - stage nsclc treated with cetuximab plus chemotherapy , 76 patient tumors were evaluated for egfr gene amplification by fish and disease controls rate ( complete response / partial response and stable disease ) was found to be significantly higher in patients with fish - positive tumors compared to fish - negative tumors ( 81% versus 55% , p = .02 ) . in this same study , median progression - free survival was also significantly longer for patients with fish - positive tumors compared to fish - negative tumors ( 6 months versus 3 months , p = .0008 ) . several studies have reported positive associations between egfr gene amplification and metastatic colorectal cancer response to egfr - directed antibodies [ 2123 ] . egfr gene amplification in scchn has been reported range between 1058% of scchn ( table 2 ) [ 2432 ] . the range of reported prevalence of egfr gene amplification may be due to differences in expression by tumor anatomical site . however , several methods were used to assess egfr gene amplification , including fluorescence in situ hybridization ( fish ) and quantitative real - time polymerase - chain reaction- ( q - pcr- ) based assays . in addition , different scoring methods were employed in the studies presented in table 2 , some of which included polysomy in the definition for egfr amplification and others did not . these differences in methodologies likely contribute to the variation in reported rates of egfr gene amplification in scchn . the presence of egfr gene amplification in a substantial portion of scchn and the previously reported associations between egfr gene amplification and response to egfr - targeted therapies in other cancers suggest that egfr gene amplification may be a predictive marker for response to egfr - targeted therapies in scchn . when evaluating egfr gene amplification for correlation with response to egfr - targeted agents , it will be important to develop consensus definitions of egfr gene amplification . egfr gene amplification has not consistently been reported to correlate with egfr protein levels although a plausible mechanism for gene amplification without protein overexpression is lacking [ 25 , 2830 ] ( table 2 ) . in nsclc and colon cancers a positive association between egfr gene amplification and protein expression has also not been consistently observed [ 44 , 45 ] . importantly , egfr gene amplification status , but not egfr tumor protein levels , is associated with response to egfr - targeted therapies in nsclc and colorectal cancers . these discrepancies likely reflect the semiquantiatitve and variable methods of assessing gene amplification and protein expression levels in various laboratories . the characterization of egfr gene amplification in scchn patients treated with egfr - directed antibodies and the testing of association with response to therapy will be of interest . ras proteins are small gtpases that regulate signal transduction pathways leading to cell growth , differentiation , and survival . three ras genes produce four ras proteins , kras 4a , kras 4b , h - ras , and n - ras , that are more than 90% homologous but demonstrate a high degree of tumor - type mutation specificity . kras mutations have been reported by several independent groups to be negatively associated with response to egfr tyrosine kinase inhibitors in lung cancer and egfr - directed antibodies in colon cancer . a metaanalysis including 17 nsclc egfr tyrosine kinase inhibitor clinical studies with 1008 patient tumors and 8 metastatic colorectal cancer ( mcrc ) studies of anti - egfr monoclonal antibody - based therapies in 817 mcrc patients found that kras mutations were significantly associated with absence of response to egfr - targeted therapies for these cancers . kras mutations are especially important predictors of unresponsiveness to egfr - directed antibodies in colorectal cancers with egfr gene amplification . reported rates of kras mutations in nsclc range between 820% with higher rates reported for adenocarcinoma compared to squamous cell carcinoma histologies [ 49 , 50 ] . kras mutations are relatively rare in scchn . only one published report has described kras mutations in scchn to date , and in this analysis of 29 oral squamous cell carcinoma tumors , 9 ( 6.9% ) were found to harbor kras mutations . according to the catalogue of somatic mutations in cancer ( cosmic ) database from the sanger institute , kras mutations occur in only approximately 3% of all cancers of the oral cavity , pharynx , or larynx while hras mutations occur in 10% of these cancers ( table 2 ) . these rates are similar to data from the genetic alterations in cancer ( gac ) database presented in table 2 . the recent finding that a mouse knock - in model expressing hras from the kras chromosomal context accumulated hras mutations and resulted in increased lung tumorigenicity suggests that tissue - specific expression of kras and hras likely contributes to tumor - type specificity of mutations in these two genes . to date , only one published study of hras mutations scchn reported a 22% rate of mutations in oral squamous cell carcinoma ( table 2 ) . mutations in hras are likely to be more common in scchn than kras mutations and may be important correlates for lack of response to egfr - targeted therapies in scchn . phosphatidylinositol 3-kinases ( pi3ks ) are heterodimeric kinases composed of regulatory and catalytic subunits that are involved in the control of cell proliferation , survival , and motility . the pi3k catalytic subunit , p110alpha ( pik3ca ) has been reported to be somatically mutated and activated in several cancers including scchn . activation of pik3ca leads to plasma membrane recruitment and activation of akt and downstream survival mechanisms . pik3ca mutations have been reported to be associated with resistance to egfr - targeted monoclonal antibodies in patients with metastatic colorectal cancers ( mcrc ) . in a study involving 110 patients with mcrc , pik3ca mutations were found to be significantly associated with reduced objective response rates following treatment with cetuximab or panitumumab ( p = .038 ) and shorter progression - free survival ( p = .035 ) . pik3ca mutations have been reported to occur in up to 8% of scchn as summarized in table 2 [ 3639 ] . additionally , loss of pten expression occurs by mechanisms including promoter methylation and silencing or loss of heterozygosity . in scchn , pten mutations are not common ( table 2 ) [ 4043 ] , and loss of heterozygocity of pten has been reported to occur in approximately 12% of scchn . though the association with response to egfr - targeted therapy in mcrc and loss of pten expression does not appear to be as strongly correlated as response and pik3ca mutations , the consideration of both tumor pten expression status and pik3ca mutation status may contribute to predicting response to egfr - targeted therapies in scchn . several egfr polymorphisms have been reported to be associated with differential response to egfr - targeted therapies . in lung cancer , shorter egfr intron 1 ca repeat polymorphism has been reported to be associated with improved response to gefitinib in two independent studies [ 54 , 55 ] . in one study involving 70 patients with advanced nsclc , patients with fewer than 17 ca repeats at either allele had significantly longer survival following treatment with gefitinib than patients having both alleles greater than 16 ca repeats ( p = .039 ) . fewer egfr intron 1 ca repeats were also significantly associated with mcrc patient response to cetuximab - based treatment in a study involving 110 mcrc patients receiving combined cetuximab - irinotecan salvage therapy . an independent study of 139 nsclc patients with who performance status of 0 or 1 who received gefitinib reported that patients with the egfr haplotype of 216g/191c had significantly worse survival with a hazard ratio of 1.85 ( 95% ci : 1.09 to 3.12 ) after adjusting for performance status , previous platinum treatment , skin rash , and diarrhea . the egfr intron 1 ca repeat polymorphism has been reported to affect egfr basal transcription with higher transcription rates reported in individuals with fewer ca repeats [ 58 , 59 ] . differential promoter activity has also been reported for the two most common egfr haplotypes at the 216 g > t and 191c > a with the 216g/191c haplotype having lower promoter activity and mrna expression [ 60 , 61 ] . these studies , therefore , indicate that patient egfr polymorphisms associated with higher egfr expression are more likely to respond to egfr - targeted therapies . the presence of the egfr k521r variant has also been found to be associated with significantly improved progression - free survival ( pfs ) and overall survival ( os ) in 32 egfr - positive mcrc patients treated with cetuximab in combination with irinotecan . patients with the k521r variant had significantly longer pfs than patients with wild - type egfr , 5.7 months versus 3.2 months , respectively , ( p = .04 , log rank test ) and os , 20.1 months versus 13.8 months , respectively , ( p = .03 ) . this egfr variant , which resides in the extracellular domain of egfr , has reduced ligand - binding , growth - stimulation , and kinase activity in vitro for the 521k variant . these findings suggest that egfr polymorphisms have the potential to be correlated with response to egfr - targeted therapies in scchn . cetuximab , a chimeric monoclonal igg1 anti - egfr antibody ( table 1 ) , may exert its antitumor effects via several mechanisms including antibody - dependent cell mediated cytotoxicity ( adcc ) . the fragment c ( fc ) portion of igg1 antibodies can be recognized by the fc gamma receptors ( fcr ) on immune effector cells to induce adcc . polymorphisms in fcriiia have been shown to be associated with differential response to cetuximab in mcrc patients in clinical studies and to scchn cell lines in vitro [ 6365 ] . the fcriiia polymorphism - v158f variant 158v was found to have higher cetuximab - mediated adcc in vitro [ 64 , 65 ] . the 158v variant was also associated with longer pfs in a study involving 69 mcrc patients treated with cetuximab plus irinotecan . these findings indicate that fcriii variants may contribute to response to cetuximab in scchn patients . the ability to correctly predict which patients will respond to which egfr - targeted therapy will improve clinical response and reduce treatment - associated toxicities for these patients . however , the minority of scchn patients have responded to egfr - targeted therapies in clinical trials , indicating that even if patients likely to respond to egfr - targeted therapy were identified , they would represent a small portion of scchn patients . even though the majority of scchn cancers overexpress egfr , these tumors are not solely dependent upon egfr activity . this is likely due to the presence of preexisting or treatment - induced compensatory signaling pathways . because egfr is activated in scchn and response to egfr - targeted therapies has been demonstrated in clinical trials , it is reasonable to consider targeted therapies to be used in combination with egfr - targeted therapeutics . molecular signaling pathways in scchn that can be activated independently of egfr include pathways initiated by g - protein - coupled receptors , integrins , and other receptor tyrosine kinases . many of these pathways share src family kinases ( sfk ) as downstream mediators of signaling . for these reasons , the combination of sfk- and egfr - targeted agents for treatment of scchn is anticipated to have improved clinical efficacy compared to egfr - targeting agents alone . eight src nonreceptor protein tyrosine kinase family members are expressed in humans : c - src , blk , fgr , fyn , hck , lck , lyn , and c - yes . c - src , fyn , lyn , and c - yes are broadly expressed , while blk , fgr , hck , and lck expression is primarily restricted to hematopoietic cells . src kinases have been implicated in normal cellular functions including cell adhesion , migration , angiogenesis , survival , proliferation , and differentiation [ 67 , 68 ] . when these processes are inappropriately regulated , they can contribute to tumorigenesis , tumor progression and metastases . in scchn models , src kinases are activated in response to egfr stimulation , associate with egfr , and have reduced activity following egfr inhibition in vitro . src kinases are also upstream activators of egfr and other receptor tyrosine kinases ( rtks ) . following g - protein - coupled receptor ( gpcr ) stimulation , src kinases are activated , resulting in the release of rtk ligands [ 71 , 72 ] . in addition to rtks and gpcrs , src kinases are also activated by integrins in scchn . src kinases , therefore , are involved in the autocrine / paracrine stimulation of scchn and mediate egfr - dependent and egfr - independent signaling events . of the src family kinases ( sfk ) , c - src is the most studied and most often implicated in cancer . elevated c - src protein and/or kinase activity has been reported for cancers of the lung , colon , breast , ovary , and pancreas in addition to head and neck cancers [ 68 , 74 ] . . therefore , increased activity of upstream signaling components and/or decreased activity of c - src negative regulators are likely causes of c - src activation observed in many epithelial cancers . the sfk c - src , fyn , lyn , and c - yes are activated in scchn cell lines in vitro following stimulation with the egfr ligand tgf- , and these sfk likely play roles in scchn . at least one group has reported differential response of sfk to integrin 6 signaling following simulation with fibronectin , the integrin 6 ligand , in oral squamous cell carcinoma cell lines . integrin 6 , which is neoexpressed in scchn , has been found to activate fyn but not c - src or c - yes in scchn upon ligation with fibronectin , leading to fyn - dependent activation of the raf - erk / mapk pathway . the murine knock - out models of specific sfk provide insights into the different roles of the individual sfk . the functions of some of the sfks are redundant , at least regarding mouse development , as evidenced by lack of phenotype for single knock - out models of c - yes , hck , c - fgr , and blk . the single knock - out murine models of lyn and lck had immune impairments , fyn knock - out mice exhibited defective brain development and impaired memory and immune functions , and c - src null mice developed a bone remodeling disease with excess accumulation of bone . therefore , some functions are likely shared between the four sfks with a subset of functions that may be unique to selective sfk . the major site of scchn metastases is locoregional lymph nodes , and presence of neck lymph node metastases is universally accepted as the most important prognostic indicator for scchn . the development of metastases requires that cells move from the primary tumor and invade surrounding tissues . invasion by tumor cells is preceded by the loss of cell adhesion and the gain of mesenchymal features in a process similar to events that occur in development termed epithelial - mesenchymal transition ( emt ) . emt is accompanied by the loss of e - cadherin , which is a principal component of cell adhesion complexes , and the gain of mesenchymal characteristics including expression of vimentin . the activation of src kinases has been shown to be involved in emt in cancer . more recently , a study evaluating 50 primary scchn tumors for activated phospho - src ( p - src ) , e - cadherin , and vimentin expression by immunohistochemical staining found increased p - src , decreased e - cadherin , and presence of vimentin expression in scchn tumors to be significantly associated ( p < .05 ) with morphologies associated with aggressive cancers including penetrating invasive fronts , poor or sarcomatoid differentiation , and lymph node metastases . it is important to note that the p - src antibody used in this study recognizes several activated sfks and is not specific for p - c - src . in addition to studies in scchn tumors , preclinical studies indicate that sfks are involved in scchn migration and invasion . our group found that blockage using an src - specific inhibitor a-419259 resulted in decreased invasion and growth of scchn cell lines in vitro following stimulation with a gpcr ligand , gastrin - releasing peptide . an independent group reported that dasatinib ( sprycel , bms-34825 ; bristol - meyers squibb ) , a dual src / abl kinase inhibitor ( table 3 ) , inhibited migration and invasion in vitro in all 8 scchn cell lines evaluated . together these studies implicate an important role or roles for sfk in tumor migration and invasion , which are associated with increased mortality in scchn . which sfks importantly , epithelial tumor cells including scchn that have undergone emt and acquired mesenchymal characteristics are more resistant to egfr - targeted therapies than tumor cells that have epithelial characteristics . combining src - targeted agents with egfr - targeted therapies may be more effective than egfr - targeted therapies alone for the control of scchn locoregional metastases . several small molecule inhibitors of c - src and sfk are currently in clinical development for solid tumors including dasatinib ( sprycel , bms ) , azd0530 ( astrazeneca ) , bosutinib ( ski-606 ; wyeth ) , xl999 ( exelixix ) , and kx01 ( kinex ) ( table 3 ) . all of these inhibitors are reversible inhibitors , and all except kx01 are atp - competitive inhibitors ( table 3 ) . dasatinib and xl999 target several known kinases in addition to sfk ( table 3 ) , while azd0530 and bosutinib are dual sfk / abl inhibitors . dasatinib , which was fda - approved for treatment of nonsolid tumors in june 2008 , and azd0530 are in phase ii clinical trials for scchn . bosutinib and xl999 are in phase ii clinical trials for other cancers ( table 3 ) . however , xl999 , which inhibits vegfr , pdgfr and fgfr in addition to src kinases , was associated with serious cardiovascular toxicities in phase i and ii clinical trials [ 8284 ] . a new addition to src inhibitors in clinical development includes the c - src substrate competitive inhibitor , kx01 , which is currently being tested in phase i clinical trials . kx01 is exquisitely specific for c - src whereas other src - targeting agents inhibit other sfk in addition to c - src [ 8587 ] . to date there are no reports of src - targeted therapeutics in scchn clinical trials or molecular predictors of response to src - targeted therapies in patients with solid malignancies . combining egfr- and src - targeted therapies for scchn is supported by results from preclinical studies . our group reported that combined azd0530 and gefitinib treatment of scchn cell lines in vitro resulted in significantly reduced cell growth and invasion compared to single agent treatments . de novo and acquired resistance to cetuximab are means by which scchn patients fail therapy . scchn and nsclc preclinical models selected for resistance to cetuximab in vitro have been reported to have high levels of activated sfk and to have decreased pi3k / akt activity following dasatinib treatment . interestingly , these cetuximab resistant cells were found to be resensitized to cetuximab following treatment with dasatinib . these data in addition to our current understanding that many egfr - independent cell signaling pathways , including gpcr- and integrin - initiated pathways , are modulated at least in part by sfk provide the rationale for the combined targeting of egfr and sfk for treatment of scchn . to date , there are no published reports of combined egfr- and src - targeted therapies for treatment of patients with solid tumors . three clinical trials combining egfr- and src - targeted therapies for upper aerodigestive cancers are currently ongoing . a phase i trial combining dasatinib with erlotinib in patients with recurrent nsclc is ongoing at the h. lee moffitt cancer center and research institute ( nct00444015 , clincaltrials.gov ) . a phase i / ii study in nsclc also combining dasatinib with erlotinib is ongoing at m.d . our group will soon open a phase 0 biomarker modulation study combining erlotinib with dasatinib for patients with scchn or nsclc ( nct00779389 , clinicaltrials.gov ) . our group recently completed a phase i trial combining cetuximab with dasatinib for treatment of advanced solid malignancies ( nct00388427 , clinicaltrials.gov ) . seventeen of 25 patients enrolled in our phase i study were evaluable for response , and 9 had stable disease while head ache was a primary toxicity . we have evaluated molecular correlates in these patients and found that p - sfk levels in peripheral blood mononuclear cells were transiently reduced following daily dasatinib dosing ( unpublished data ) . in addition , we found that egfr , tgf- and amphiregulin plasma levels were altered following treatment ( unpublished data ) . a phase ii study combining dasatinib and cetuximab for treating scchn patients is planned at the university of pittsburgh cancer institute . results from molecular correlate studies from this trial and others will be of great importance as the scchn medical and research communities work to identify predictive molecular markers of response to these therapies . despite the ubiquitous expression of egfr in scchn , clinical responses to egfr targeting agents , particularly , when administered as single agents , has been modest . cetuximab was fda - approved in 2006 for the treatment of newly diagnosed scchn in combination with radiation and recently extended to the recurrent / metastatic population in combination with chemoradiotherapy . however , in most of these trials , expression levels of egfr in the tumor have not correlated with response to cetuximab and no single biomarker to date in baseline tissue has been proven to predict response to egfr targeting agents . comprehensive genomic and proteomic studies of baseline tissue are required in the context of clinical trials to begin to identify potential markers of clinical activity . since egfr signaling involves intracellular interactions with other oncogenic pathways in scchn preclinical models , it is plausible that cotargeting of egfr in conjunction with blockade of these pathways may be beneficial . src family kinases represent a potential pathway for targeting , especially given the fda - approval of the src kinase inhibitor dasatinib for hematopoietic malignancies . challenges include : ( 1 ) the difficulty of testing antiinvasive / antimetastatic agents in clinical trial settings , and ( 2 ) the possibility that recist criteria may not reflect decreased tumor proliferation , metabolism , or increased apoptosis as evidence by studies that have incorporated pet tracers . more relevant endpoints in egfr-/src - targeted trials than tumor shrinkage may include time to progression or overall survival

the epidermal growth factor receptor- ( egfr- ) directed antibody , cetuximab , was fda - approved for the treatment of squamous cell carcinoma of the head and neck ( scchn ) in 2006 . additional egfr - targeting agents in clinical development for scchn include other egfr - directed antibodies , tyrosine kinase inhibitors and antisense dna . although the majority of scchn overexpress egfr , scchn clinical responses to egfr - targeting agents have been modest . molecular predictors for scchn response to egfr - targeted therapies have not been identified . however , molecular correlate studies in lung cancer and colon cancer , which have egfr - targeted therapeutics fda - approved for treatment , may provide insights . we describe candidate predictive markers for scchn response to egfr - targeted therapies and their prevalence in scchn . clinical response will likely be improved by targeted therapy combination treatments . src family kinases mediate egfr - dependent and -independent tumor progression pathways in many cancers including scchn . several src - targeting agents are in clinical development for solid malignancies . molecular correlate studies for src - targeting therapies are few and biomarkers correlated with patient response are limited . identifying scchn patients who will respond to combined egfr- and src - targeting will require further characterization of molecular correlates . we discuss rationale for egfr and src co - targeting for scchn treatment and describe recent clinical trials implementing combined src- and egfr - targeted therapeutics .

1. Introduction 2. EGFR-Targeted Therapies for SCCHN 3. Predictors of Response to EGFR-Targeted Therapies 4. Src Family Kinases in SCCHN 5. Src Family Kinases in SCCHN Invasion and Progression 6. Src-Targeting Agents in Clinical Development 7. Cotargeting of EGFR and Src Family Kinases in Patients 8. Summary and Future Directions

ninety - percent of head and neck cancers are squamous cell carcinomas ( scchn ) involving the mucosal surfaces of the oral cavity , pharynx , and larynx . targeted therapies for scchn are under active investigation with the goals of reducing scchn morbidity and mortality . targeted therapies are anticipated to have fewer associated toxicities than standard chemotherapies , which rely predominately on increased rates of cell division to enhance killing of the tumor cells compared to healthy tissues . the epidermal growth factor receptor ( egfr ) was anticipated to be a good drug target for scchn treatment because the majority of scchn overexpress egfr [ 2 , 3 ] , and higher tumor levels of egfr are associated with poorer clinical outcomes [ 4 , 5 ] . egfr participates in scchn autocrine stimulation , and overexpression of egfr and its primary ligand in humans , transforming growth factor alpha ( tgf- ) , have been correlated with poor outcomes for patients receiving therapy . cetuximab ( erbitux ; imclone systems ) , a chimeric monoclonal igg1 antibody directed against egfr , was fda - approved for the treatment of scchn in combination with rt for locally or regionally advanced disease and as a monotherapy for recurrent or metastatic scchn patients who have failed prior platinum - based therapy . in addition to antibodies directed against egfr , small molecule tyrosine kinase inhibitors ( tki ) of egfr and egfr antisense agents are currently under active clinical investigation for scchn treatment . egfr - targeted therapeutics delivered as monotherapies for treatment of scchn have demonstrated fewer toxicities compared to combined modality treatment regimens but only marginal clinical response ( 410% ) [ 7 , 8 ] . in general responses to egfr - targeted therapies in scchn clinical trials improving clinical response rates will involve ( 1 ) identifying scchn patients who are likely to respond to egfr - targeted therapies , ( 2 ) developing effective combinations of targeted therapies , and ( 3 ) correctly identifying patients who will respond to specific targeted agents applied alone or in combination . our understanding of the factors contributing to targeted therapy response now extends beyond the molecular alterations of the tumor to include host genetic variation . in this review , we will summarize molecular data correlated with clinical response to egfr - targeted therapies and discuss factors that may be considered for identifying responsive scchn patients . preclinical evidence suggests that src family kinase - targeted agents administered in combination with egfr - targeted therapies may demonstrate improved clinical response over egfr - targeted agents alone . here we also provide rationale for combining egfr- and src - targeted therapeutics for treatment of scchn , discuss published egfr- and src - combination treatment preclinical data , and summarize completed and ongoing clinical trials in solid tumors evaluating src - targeted therapies in combination with egfr - targeted therapies . there are several egfr - targeted therapies in clinical development for scchn , and these agents are described in table 1 . these inhibitors fall into two primary categories : egfr - directed antibodies and egfr tyrosine kinase inhibitors . egfr - directed antibodies include cetuximab , nimotuzumab ( ym biosciences ) , panitumumab ( amgen ) , and zalutumumab ( genmab ) . egfr - targeted tyrosine kinase inhibitors include erlotinib ( genetech and osi pharmaceuticals ) and gefitinib ( astrazeneca ) . in addition to egfr - targeted kinase inhibitors , inhibitors with broader target specificities are also in phase ii or iii development for scchn including lapatinib ( glaxosmithkline ) , which is a dual egfr / her2 inhibitor , and zactima ( astrazeneca ) , which targets vegfr2 and ret in addition to egfr ( table 1 ) . results of clinical trials for egfr - targeted therapies cetuximab , nimotuzumab , gefitinib , and erlotinib used alone or in combination with conventional treatments for scchn have been reviewed by us and others and will not be described in detail here [ 1012 ] . a phase i study of panitumumab in combination with chemoradiotherapy involving 19-treatment - nave patients with stage iii / iv head and neck cancer reported an 87% complete response rate among the 15 evaluable patients and no grade 3 or 4 chronic toxicities . our phase i study of intratumoral delivery of egfr antisense dna in 17 patients with advanced , refractory scchn was associated with no grade 3 or 4 or dose - limiting toxicities and a clinical response rate ( complete response and partial response by modified recist criteria ) of 29% . the current phase of clinical development for each of these agents is presented in table 1 . to date , no molecular marker has been identified to correlate with scchn response to egfr - targeting in patients . scchn tumor expression of the truncated form of egfr , egfr variant iii ( viii ) , which lacks the ligand binding domain , occurs in up to 40% of scchn tumors and confers resistance to egfr - targeted monoclonal antibodies in scchn preclinical models . however , egfr viii expression and resistance to egfr - targeted therapies in scchn patients has not been described . molecular correlates of clinical response / nonresponse to egfr - targeted therapies have been identified for colon and lung cancers . for example , the treatment of lung cancer with the egfr tyrosine kinase inhibitor gefitinib demonstrated effective responses in a subset of patients whose lung cancers were subsequently found to harbor egfr kinase activating mutations [ 16 , 17 ] . therefore , some of these molecular correlates , such as the egfr tyrosine kinase activating mutations are not applicable to scchn because the frequency of these mutations is very low and will not be discussed further in this review . however , other molecular correlates of response to egfr - targeting agents have been described for lung cancer and colorectal cancers including egfr gene amplification , other somatic tumor mutations and patient genetic variations . to date , there are no published reports evaluating egfr gene amplification for association with scchn patient response to egfr - targeted therapies . however , egfr gene amplification has been reported to be positively associated with response to egfr - directed antibody therapies in clinical trials for nonsmall lung cancers ( nsclc ) and colorectal cancers . several studies have reported positive associations between egfr gene amplification and metastatic colorectal cancer response to egfr - directed antibodies [ 2123 ] . egfr gene amplification in scchn has been reported range between 1058% of scchn ( table 2 ) [ 2432 ] . the presence of egfr gene amplification in a substantial portion of scchn and the previously reported associations between egfr gene amplification and response to egfr - targeted therapies in other cancers suggest that egfr gene amplification may be a predictive marker for response to egfr - targeted therapies in scchn . when evaluating egfr gene amplification for correlation with response to egfr - targeted agents , it will be important to develop consensus definitions of egfr gene amplification . in nsclc and colon cancers a positive association between egfr gene amplification and protein expression has also not been consistently observed [ 44 , 45 ] . importantly , egfr gene amplification status , but not egfr tumor protein levels , is associated with response to egfr - targeted therapies in nsclc and colorectal cancers . the characterization of egfr gene amplification in scchn patients treated with egfr - directed antibodies and the testing of association with response to therapy will be of interest . kras mutations have been reported by several independent groups to be negatively associated with response to egfr tyrosine kinase inhibitors in lung cancer and egfr - directed antibodies in colon cancer . a metaanalysis including 17 nsclc egfr tyrosine kinase inhibitor clinical studies with 1008 patient tumors and 8 metastatic colorectal cancer ( mcrc ) studies of anti - egfr monoclonal antibody - based therapies in 817 mcrc patients found that kras mutations were significantly associated with absence of response to egfr - targeted therapies for these cancers . kras mutations are especially important predictors of unresponsiveness to egfr - directed antibodies in colorectal cancers with egfr gene amplification . reported rates of kras mutations in nsclc range between 820% with higher rates reported for adenocarcinoma compared to squamous cell carcinoma histologies [ 49 , 50 ] . only one published report has described kras mutations in scchn to date , and in this analysis of 29 oral squamous cell carcinoma tumors , 9 ( 6.9% ) were found to harbor kras mutations . to date , only one published study of hras mutations scchn reported a 22% rate of mutations in oral squamous cell carcinoma ( table 2 ) . mutations in hras are likely to be more common in scchn than kras mutations and may be important correlates for lack of response to egfr - targeted therapies in scchn . the pi3k catalytic subunit , p110alpha ( pik3ca ) has been reported to be somatically mutated and activated in several cancers including scchn . pik3ca mutations have been reported to be associated with resistance to egfr - targeted monoclonal antibodies in patients with metastatic colorectal cancers ( mcrc ) . pik3ca mutations have been reported to occur in up to 8% of scchn as summarized in table 2 [ 3639 ] . in scchn , pten mutations are not common ( table 2 ) [ 4043 ] , and loss of heterozygocity of pten has been reported to occur in approximately 12% of scchn . though the association with response to egfr - targeted therapy in mcrc and loss of pten expression does not appear to be as strongly correlated as response and pik3ca mutations , the consideration of both tumor pten expression status and pik3ca mutation status may contribute to predicting response to egfr - targeted therapies in scchn . several egfr polymorphisms have been reported to be associated with differential response to egfr - targeted therapies . in lung cancer , shorter egfr intron 1 ca repeat polymorphism has been reported to be associated with improved response to gefitinib in two independent studies [ 54 , 55 ] . fewer egfr intron 1 ca repeats were also significantly associated with mcrc patient response to cetuximab - based treatment in a study involving 110 mcrc patients receiving combined cetuximab - irinotecan salvage therapy . these studies , therefore , indicate that patient egfr polymorphisms associated with higher egfr expression are more likely to respond to egfr - targeted therapies . the presence of the egfr k521r variant has also been found to be associated with significantly improved progression - free survival ( pfs ) and overall survival ( os ) in 32 egfr - positive mcrc patients treated with cetuximab in combination with irinotecan . this egfr variant , which resides in the extracellular domain of egfr , has reduced ligand - binding , growth - stimulation , and kinase activity in vitro for the 521k variant . these findings suggest that egfr polymorphisms have the potential to be correlated with response to egfr - targeted therapies in scchn . cetuximab , a chimeric monoclonal igg1 anti - egfr antibody ( table 1 ) , may exert its antitumor effects via several mechanisms including antibody - dependent cell mediated cytotoxicity ( adcc ) . polymorphisms in fcriiia have been shown to be associated with differential response to cetuximab in mcrc patients in clinical studies and to scchn cell lines in vitro [ 6365 ] . these findings indicate that fcriii variants may contribute to response to cetuximab in scchn patients . the ability to correctly predict which patients will respond to which egfr - targeted therapy will improve clinical response and reduce treatment - associated toxicities for these patients . however , the minority of scchn patients have responded to egfr - targeted therapies in clinical trials , indicating that even if patients likely to respond to egfr - targeted therapy were identified , they would represent a small portion of scchn patients . even though the majority of scchn cancers overexpress egfr , these tumors are not solely dependent upon egfr activity . because egfr is activated in scchn and response to egfr - targeted therapies has been demonstrated in clinical trials , it is reasonable to consider targeted therapies to be used in combination with egfr - targeted therapeutics . molecular signaling pathways in scchn that can be activated independently of egfr include pathways initiated by g - protein - coupled receptors , integrins , and other receptor tyrosine kinases . many of these pathways share src family kinases ( sfk ) as downstream mediators of signaling . for these reasons , the combination of sfk- and egfr - targeted agents for treatment of scchn is anticipated to have improved clinical efficacy compared to egfr - targeting agents alone . in scchn models , src kinases are activated in response to egfr stimulation , associate with egfr , and have reduced activity following egfr inhibition in vitro . src kinases , therefore , are involved in the autocrine / paracrine stimulation of scchn and mediate egfr - dependent and egfr - independent signaling events . of the src family kinases ( sfk ) , c - src is the most studied and most often implicated in cancer . elevated c - src protein and/or kinase activity has been reported for cancers of the lung , colon , breast , ovary , and pancreas in addition to head and neck cancers [ 68 , 74 ] . at least one group has reported differential response of sfk to integrin 6 signaling following simulation with fibronectin , the integrin 6 ligand , in oral squamous cell carcinoma cell lines . integrin 6 , which is neoexpressed in scchn , has been found to activate fyn but not c - src or c - yes in scchn upon ligation with fibronectin , leading to fyn - dependent activation of the raf - erk / mapk pathway . the murine knock - out models of specific sfk provide insights into the different roles of the individual sfk . the major site of scchn metastases is locoregional lymph nodes , and presence of neck lymph node metastases is universally accepted as the most important prognostic indicator for scchn . more recently , a study evaluating 50 primary scchn tumors for activated phospho - src ( p - src ) , e - cadherin , and vimentin expression by immunohistochemical staining found increased p - src , decreased e - cadherin , and presence of vimentin expression in scchn tumors to be significantly associated ( p < .05 ) with morphologies associated with aggressive cancers including penetrating invasive fronts , poor or sarcomatoid differentiation , and lymph node metastases . in addition to studies in scchn tumors , preclinical studies indicate that sfks are involved in scchn migration and invasion . our group found that blockage using an src - specific inhibitor a-419259 resulted in decreased invasion and growth of scchn cell lines in vitro following stimulation with a gpcr ligand , gastrin - releasing peptide . together these studies implicate an important role or roles for sfk in tumor migration and invasion , which are associated with increased mortality in scchn . which sfks importantly , epithelial tumor cells including scchn that have undergone emt and acquired mesenchymal characteristics are more resistant to egfr - targeted therapies than tumor cells that have epithelial characteristics . combining src - targeted agents with egfr - targeted therapies may be more effective than egfr - targeted therapies alone for the control of scchn locoregional metastases . several small molecule inhibitors of c - src and sfk are currently in clinical development for solid tumors including dasatinib ( sprycel , bms ) , azd0530 ( astrazeneca ) , bosutinib ( ski-606 ; wyeth ) , xl999 ( exelixix ) , and kx01 ( kinex ) ( table 3 ) . dasatinib , which was fda - approved for treatment of nonsolid tumors in june 2008 , and azd0530 are in phase ii clinical trials for scchn . bosutinib and xl999 are in phase ii clinical trials for other cancers ( table 3 ) . however , xl999 , which inhibits vegfr , pdgfr and fgfr in addition to src kinases , was associated with serious cardiovascular toxicities in phase i and ii clinical trials [ 8284 ] . a new addition to src inhibitors in clinical development includes the c - src substrate competitive inhibitor , kx01 , which is currently being tested in phase i clinical trials . kx01 is exquisitely specific for c - src whereas other src - targeting agents inhibit other sfk in addition to c - src [ 8587 ] . to date there are no reports of src - targeted therapeutics in scchn clinical trials or molecular predictors of response to src - targeted therapies in patients with solid malignancies . combining egfr- and src - targeted therapies for scchn is supported by results from preclinical studies . our group reported that combined azd0530 and gefitinib treatment of scchn cell lines in vitro resulted in significantly reduced cell growth and invasion compared to single agent treatments . these data in addition to our current understanding that many egfr - independent cell signaling pathways , including gpcr- and integrin - initiated pathways , are modulated at least in part by sfk provide the rationale for the combined targeting of egfr and sfk for treatment of scchn . to date , there are no published reports of combined egfr- and src - targeted therapies for treatment of patients with solid tumors . three clinical trials combining egfr- and src - targeted therapies for upper aerodigestive cancers are currently ongoing . our group recently completed a phase i trial combining cetuximab with dasatinib for treatment of advanced solid malignancies ( nct00388427 , clinicaltrials.gov ) . results from molecular correlate studies from this trial and others will be of great importance as the scchn medical and research communities work to identify predictive molecular markers of response to these therapies . despite the ubiquitous expression of egfr in scchn , clinical responses to egfr targeting agents , particularly , when administered as single agents , has been modest . cetuximab was fda - approved in 2006 for the treatment of newly diagnosed scchn in combination with radiation and recently extended to the recurrent / metastatic population in combination with chemoradiotherapy . however , in most of these trials , expression levels of egfr in the tumor have not correlated with response to cetuximab and no single biomarker to date in baseline tissue has been proven to predict response to egfr targeting agents . src family kinases represent a potential pathway for targeting , especially given the fda - approval of the src kinase inhibitor dasatinib for hematopoietic malignancies . challenges include : ( 1 ) the difficulty of testing antiinvasive / antimetastatic agents in clinical trial settings , and ( 2 ) the possibility that recist criteria may not reflect decreased tumor proliferation , metabolism , or increased apoptosis as evidence by studies that have incorporated pet tracers .

this study was designed to determine the efficacy and safety of an automated , portable , overnight closed - loop system based on fingerstick meter glucose calibration . the primary end point was the percentage of time spent in euglycemia with overnight closed - loop control ( range 3.98 mmol / l [ 70144 mg / dl ] ) . the secondary end points were 1 ) comparing overnight efficacy of the portable closed - loop system versus open - loop control , as measured by sensor glucose values , and 2 ) evaluating the length of time under closed - loop control without investigator intervention . participants fulfilling the following criteria were recruited : age 1225 years , diagnosis of type 1 diabetes for > 1 year , a1c < 8.5% , and insulin pump therapy use for at least 6 months . written informed consent was obtained from subjects aged 18 years and written parental consent and subject assent for subjects aged < 18 years . the study was approved by the human research ethics committee of princess margaret hospital for children . at the initial outpatient visit , participants underwent insertion of a subcutaneous glucose sensor and ipro2 , professional continuous glucose monitoring device ( medtronic minimed ) to be worn for 6 days to establish baseline overnight glycemic levels . glucose levels from the glucometer were merged with ipro2 sensor data to obtain a retrospective 6-day continuous glucose tracing . participants were asked to proceed with their routine diabetes care . during the week after the baseline open - loop assessment , participants were admitted to the clinical research facility at 1700 h for the overnight closed - loop studies . two subcutaneous glucose sensors were inserted into sites chosen by the participant , and an intravenous catheter was placed in an antecubital vein for blood sampling . the participant s pump was disconnected , and the study pump ( paradigm veo ; medtronic minimed ) containing the subject s usual analog insulin , insulin aspart ( novorapid ; novo nordisk , bagsvaerd , denmark ) , or insulin lispro ( humalog ; eli lilly , indianapolis , in ) , was attached . an evening meal was consumed at 1800 h. an insulin bolus was delivered by the participant , based on his or her usual insulin - to - carbohydrate ratio and insulin sensitivity factor . participants were switched from open - loop to closed - loop control at 2100 h , and this was continued until 0700 h the following morning . participants reverted to open - loop control from 0700 to 2100 h , followed by a second consecutive night of overnight closed - loop control . venous plasma glucose was measured every 30 min on both nights during overnight closed - loop control using the ysi 2300 stat ( yellow springs instruments , yellow springs , oh ) . if the plasma glucose was < 2.8 mmol / l ( 50 mg / dl ) on two occasions 15 min apart or the plasma glucose was > 15 mmol / l ( 270 mg / dl ) for 1 h , closed - loop control was stopped and the system reverted to open - loop control . blood samples for plasma insulin determination were collected hourly during overnight closed - loop control . samples were assayed using a one - step chemiluminescent noncompetitive immunoassay ( architect ; abbott diagnostics , abbott park , il ) with coefficients of variation of < 2% for means of 17 , 46 , and 130 raw sensor data were transmitted to the blackberry by way of the radiofrequency ( rf ) translator module that translates the medtronic proprietary 916-mhz rf protocol to the bluetooth communication protocol , and vice versa . after sensor data are processed , the blackberry calculates pump strokes based on the closed - loop control algorithm and sends pump delivery commands over the bluetooth interface to the translator . insulin is delivered as a series of microboluses in multiples of 0.025 units every min . the deviation of sensor signal is continuously evaluated to ensure that both sensors track within predetermined parameters . if the sensor deviation falls outside these parameters , the fault detection will be triggered to request another blood glucose level to be entered for calibration . remote monitoring of the subject was accomplished by packaging real - time data into compressed files and sending them to a remote monitoring station over the wireless cellular communication network . during this study , experiments were remotely monitored in real - time in perth , western australia , and in northridge , california . historical data were also available daily directly from blackberry downloads and remotely in archived form . insulin delivery was modeled on the multiphasic insulin response of the -cell and consisted of three principal components proportional , integral , and derivative ( pid)as used in other studies ( 11,12 ) , with a modification to include feedback of a model - predicted insulin profile ( 19 ) . insulin feedback ( ifb ) allows for more physiologic replication of a functioning -cell , which is widely believed to reduce insulin secretion as plasma insulin levels increase ( 20 ) . the pgcs used fault detection settings regarding 1 ) correlation of sensor signals , 2 ) deviation of sensor glucose ( sg1 and sg2 ) from the mean ( sg ) , 3 ) mutual difference in rate of glucose change ( direction of trend ) , 4 ) loss of sensor signal , rf , or bluetooth connectivity , and 5 ) insulin delivery limits . a system of triggers was developed to allow appropriate temporal tolerance for the faults to clear . the system required a minimum of one calibration every 12 h and could request a calibration in the event of scenario 1 , 2 , or 3 above . if a calibration is not entered within 15 min of the request , or both sensor signals or the bluetooth connection are lost for 15 min , the system automatically reverts to open - loop mode . as the system reverts to open - loop mode , the pump rate is set to deliver no insulin for 2 h. this is a temporary basal rate setting before it reverts to the preprogrammed basal rate after 2 h. this will ensure that a hypoglycemic patient will not receive further basal insulin for 2 h. depending on distribution , data are expressed as mean sd or as median and range or interquartile range ( 25th75th centile ) , where appropriate . comparisons between overnight closed - loop and open - loop control were made using a mann - whitney u test for nonparametric data and a student t test for data conforming to a normal distribution . at the initial outpatient visit , participants underwent insertion of a subcutaneous glucose sensor and ipro2 , professional continuous glucose monitoring device ( medtronic minimed ) to be worn for 6 days to establish baseline overnight glycemic levels . glucose levels from the glucometer were merged with ipro2 sensor data to obtain a retrospective 6-day continuous glucose tracing . participants were asked to proceed with their routine diabetes care . during the week after the baseline open - loop assessment , participants were admitted to the clinical research facility at 1700 h for the overnight closed - loop studies . two subcutaneous glucose sensors were inserted into sites chosen by the participant , and an intravenous catheter was placed in an antecubital vein for blood sampling . the participant s pump was disconnected , and the study pump ( paradigm veo ; medtronic minimed ) containing the subject s usual analog insulin , insulin aspart ( novorapid ; novo nordisk , bagsvaerd , denmark ) , or insulin lispro ( humalog ; eli lilly , indianapolis , in ) , was attached . an evening meal was consumed at 1800 h. an insulin bolus was delivered by the participant , based on his or her usual insulin - to - carbohydrate ratio and insulin sensitivity factor . participants were switched from open - loop to closed - loop control at 2100 h , and this was continued until 0700 h the following morning . participants reverted to open - loop control from 0700 to 2100 h , followed by a second consecutive night of overnight closed - loop control . venous plasma glucose was measured every 30 min on both nights during overnight closed - loop control using the ysi 2300 stat ( yellow springs instruments , yellow springs , oh ) . if the plasma glucose was < 2.8 mmol / l ( 50 mg / dl ) on two occasions 15 min apart or the plasma glucose was > 15 mmol / l ( 270 mg / dl ) for 1 h , closed - loop control was stopped and the system reverted to open - loop control . blood samples for plasma insulin determination were collected hourly during overnight closed - loop control . samples were assayed using a one - step chemiluminescent noncompetitive immunoassay ( architect ; abbott diagnostics , abbott park , il ) with coefficients of variation of < 2% for means of 17 , 46 , and 130 mu / l . at the initial outpatient visit , participants underwent insertion of a subcutaneous glucose sensor and ipro2 , professional continuous glucose monitoring device ( medtronic minimed ) to be worn for 6 days to establish baseline overnight glycemic levels . glucose levels from the glucometer were merged with ipro2 sensor data to obtain a retrospective 6-day continuous glucose tracing . during the week after the baseline open - loop assessment , participants were admitted to the clinical research facility at 1700 h for the overnight closed - loop studies . two subcutaneous glucose sensors were inserted into sites chosen by the participant , and an intravenous catheter was placed in an antecubital vein for blood sampling . the participant s pump was disconnected , and the study pump ( paradigm veo ; medtronic minimed ) containing the subject s usual analog insulin , insulin aspart ( novorapid ; novo nordisk , bagsvaerd , denmark ) , or insulin lispro ( humalog ; eli lilly , indianapolis , in ) , was attached . an evening meal was consumed at 1800 h. an insulin bolus was delivered by the participant , based on his or her usual insulin - to - carbohydrate ratio and insulin sensitivity factor . participants were switched from open - loop to closed - loop control at 2100 h , and this was continued until 0700 h the following morning . participants reverted to open - loop control from 0700 to 2100 h , followed by a second consecutive night of overnight closed - loop control . venous plasma glucose was measured every 30 min on both nights during overnight closed - loop control using the ysi 2300 stat ( yellow springs instruments , yellow springs , oh ) . if the plasma glucose was < 2.8 mmol / l ( 50 mg / dl ) on two occasions 15 min apart or the plasma glucose was > 15 mmol / l ( 270 mg / dl ) for 1 h , closed - loop control was stopped and the system reverted to open - loop control . blood samples for plasma insulin determination were collected hourly during overnight closed - loop control . samples were assayed using a one - step chemiluminescent noncompetitive immunoassay ( architect ; abbott diagnostics , abbott park , il ) with coefficients of variation of < 2% for means of 17 , 46 , and 130 mu / l . raw sensor data were transmitted to the blackberry by way of the radiofrequency ( rf ) translator module that translates the medtronic proprietary 916-mhz rf protocol to the bluetooth communication protocol , and vice versa . after sensor data are processed , the blackberry calculates pump strokes based on the closed - loop control algorithm and sends pump delivery commands over the bluetooth interface to the translator . insulin is delivered as a series of microboluses in multiples of 0.025 units every min . the deviation of sensor signal is continuously evaluated to ensure that both sensors track within predetermined parameters . if the sensor deviation falls outside these parameters , the fault detection will be triggered to request another blood glucose level to be entered for calibration . remote monitoring of the subject was accomplished by packaging real - time data into compressed files and sending them to a remote monitoring station over the wireless cellular communication network . during this study , experiments were remotely monitored in real - time in perth , western australia , and in northridge , california . historical data were also available daily directly from blackberry downloads and remotely in archived form . insulin delivery was modeled on the multiphasic insulin response of the -cell and consisted of three principal components proportional , integral , and derivative ( pid)as used in other studies ( 11,12 ) , with a modification to include feedback of a model - predicted insulin profile ( 19 ) . insulin feedback ( ifb ) allows for more physiologic replication of a functioning -cell , which is widely believed to reduce insulin secretion as plasma insulin levels increase ( 20 ) . the pgcs used fault detection settings regarding 1 ) correlation of sensor signals , 2 ) deviation of sensor glucose ( sg1 and sg2 ) from the mean ( sg ) , 3 ) mutual difference in rate of glucose change ( direction of trend ) , 4 ) loss of sensor signal , rf , or bluetooth connectivity , and 5 ) insulin delivery limits . a system of triggers was developed to allow appropriate temporal tolerance for the faults to clear . the system required a minimum of one calibration every 12 h and could request a calibration in the event of scenario 1 , 2 , or 3 above . if a calibration is not entered within 15 min of the request , or both sensor signals or the bluetooth connection are lost for 15 min , the system automatically reverts to open - loop mode . as the system reverts to open - loop mode , the pump rate is set to deliver no insulin for 2 h. this is a temporary basal rate setting before it reverts to the preprogrammed basal rate after 2 h. this will ensure that a hypoglycemic patient will not receive further basal insulin for 2 h. depending on distribution , data are expressed as mean sd or as median and range or interquartile range ( 25th75th centile ) , where appropriate . comparisons between overnight closed - loop and open - loop control were made using a mann - whitney u test for nonparametric data and a student t test for data conforming to a normal distribution . eight subjects ( four males ) underwent 145 h of closed - loop control over 16 nights . median age was 14.8 years ( range 12.624 ) , duration of diabetes was 6.9 years ( range 3.821.5 ) , duration of pump therapy was 3.8 years ( range 2.610.8 ) , and bmi was 21 kg / m ( range 1830 ) . mean a1c was 7.3 1.1% , and total daily dose of insulin was 0.9 0.1 units / kg . interstitial glucose values tracked venous plasma glucose levels effectively during overnight closed - loop control . point accuracy of the sensor , expressed as median ( interquartile range ) and mean sd relative absolute difference from the venous plasma glucose were 7.5% ( 3.318.5 ) and 12.3 12.7% , respectively , similar to previously published studies ( 9,10,12,15 ) and in keeping with the published accuracy of this sensor ( 21 ) . mean plasma glucose during overnight closed - loop control was 6.4 1.7 mmol / l ( 115 31 mg / dl ) . the proportion of time with venous plasma glucose < 3.9 , between 3.9 and 8 ( 70 and 144 mg / dl ) , and > 8 mmol / l was 7 , 78 , and 15% respectively . mmol / l ) was significantly higher after midnight than before midnight ( 85 vs. 66% ; p = 0.001 ) . plasma glucose < 3.3 mmol / l occurred in 3 of 16 study nights and within the first 3.5 h. plasma glucose readings < 3.9 mmol / l were significantly more common in the first 3 h of closed - loop control than thereafter ( 13.9 vs. 4% ; p = 0.005 ) . sensor and venous plasma glucose levels during closed - loop control are shown in fig . glycemic control during closed - loop insulin delivery was superior to prestudy home overnight open - loop control ( fig . an evening meal and prandial bolus were administered at 1800 , and the closed - loop mode commenced at 2100 . b : pump delivery rates are represented as a black dashed line , and hourly plasma insulin measurements are represented in green . comparison of overnight closed - loop ( red ) with prestudy open - loop ( blue ) control.*p < 0.0001 . sensor fault detection requested fingerstick calibration on both closed - loop nights for two subjects . these subjects had the greatest relative - absolute difference between sensor glucose and plasma glucose , and between them accounted for 46% of plasma glucose levels out of target range for the entire study . the sensors were inserted in the left and right abdomen in subject 5 and in the left and right buttocks in subject 6 . these subjects had similar bmis , 22 and 26 kg / m , respectively ; thus , patient phenotype did not explain the difference between sensor and plasma glucose . investigator intervention during automated , portable , closed - loop control for one further subject ( 2 nights ) , the bluetooth link was lost for > 15 min and needed to be re - established . one patient ( age 24 years , weight 94 kg , total daily dose 105 units ) required rescue carbohydrates for plasma glucose 2.5 mmol / l ( sensor 3.5 mmol / l ) . he commenced closed - loop 3 h after an evening meal where he gave himself a preprandial bolus of 22.7 units but did not finish the meal . his usual basal rate was 2.7 units / h , and during closed - loop , he received only 0.67 units of insulin over 3 h before the study night was terminated , as per protocol . interstitial glucose values tracked venous plasma glucose levels effectively during overnight closed - loop control . point accuracy of the sensor , expressed as median ( interquartile range ) and mean sd relative absolute difference from the venous plasma glucose were 7.5% ( 3.318.5 ) and 12.3 12.7% , respectively , similar to previously published studies ( 9,10,12,15 ) and in keeping with the published accuracy of this sensor ( 21 ) . mean plasma glucose during overnight closed - loop control was 6.4 1.7 mmol / l ( 115 31 mg / dl ) . the proportion of time with venous plasma glucose < 3.9 , between 3.9 and 8 ( 70 and 144 mg / dl ) , and > 8 mmol / l was 7 , 78 , and 15% respectively . mmol / l ) was significantly higher after midnight than before midnight ( 85 vs. 66% ; p = 0.001 ) . plasma glucose < 3.3 mmol / l occurred in 3 of 16 study nights and within the first 3.5 h. plasma glucose readings < 3.9 mmol / l were significantly more common in the first 3 h of closed - loop control than thereafter ( 13.9 vs. 4% ; p = 0.005 ) . sensor and venous plasma glucose levels during closed - loop control are shown in fig . glycemic control during closed - loop insulin delivery was superior to prestudy home overnight open - loop control ( fig . an evening meal and prandial bolus were administered at 1800 , and the closed - loop mode commenced at 2100 . b : pump delivery rates are represented as a black dashed line , and hourly plasma insulin measurements are represented in green . comparison of overnight closed - loop ( red ) with prestudy open - loop ( blue ) control.*p < 0.0001 . sensor fault detection requested fingerstick calibration on both closed - loop nights for two subjects . these subjects had the greatest relative - absolute difference between sensor glucose and plasma glucose , and between them accounted for 46% of plasma glucose levels out of target range for the entire study . the sensors were inserted in the left and right abdomen in subject 5 and in the left and right buttocks in subject 6 . these subjects had similar bmis , 22 and 26 kg / m , respectively ; thus , patient phenotype did not explain the difference between sensor and plasma glucose . investigator intervention during automated , portable , closed - loop control for one further subject ( 2 nights ) , the bluetooth link was lost for > 15 min and needed to be re - established . one patient ( age 24 years , weight 94 kg , total daily dose 105 units ) required rescue carbohydrates for plasma glucose 2.5 mmol / l ( sensor 3.5 mmol / l ) . he commenced closed - loop 3 h after an evening meal where he gave himself a preprandial bolus of 22.7 units but did not finish the meal . his usual basal rate was 2.7 units / h , and during closed - loop , he received only 0.67 units of insulin over 3 h before the study night was terminated , as per protocol . this study demonstrates the feasibility and safety features of a portable , automated , closed - loop system for overnight glucose control in adolescents and young adults with type 1 diabetes . this represents a further step toward making such technology available to patients in the outpatient setting . monitoring of system operation from a remote location using data transmission over a wireless network is an achievable goal and is an additional safety feature , paving the way for physician - supervised home studies . automated fault detection requested fingerstick calibration of the system when sensor correlation was below a specified threshold or sensor glucose values deviated significantly from the mean . further study is necessary to determine the optimum fault detection settings to maximize patient safety yet minimize disruption to the operation of the closed - loop system . initial commercially available fully closed - loop systems will likely focus on overnight glucose control to minimize nocturnal hypoglycemia . the effects of intensive management of type 1 diabetes on the incidence of hypoglycemia are well documented . in the diabetes control and complications trial ( 1 ) , 55% of severe hypoglycemia occurred during sleep , and in the pediatric population , 75% of episodes of severe hypoglycemia occurred overnight ( 22 ) . although arousal thresholds are not impaired by hypoglycemia ( 23 ) , 71% of low - glucose alarms are not responded to during sleep ( 24 ) . an overnight closed - loop system that automatically suspends insulin delivery in response to impending hypoglycemia would minimize exposure to prolonged hypoglycemia and adverse outcomes such as seizures ( 25 ) . in our study , although plasma glucose < 3.3 mmol / l ( 60 mg / dl ) occurred in 3 of 16 study nights , the sensor readings indicated that hypoglycemia was significantly less common during closed - loop compared with open - loop therapy . episodes of hypoglycemia during closed - loop therapy occurred during the first 3.5 h and likely related to start - up algorithm is necessary to smooth the transition between open - loop and closed - loop sessions and account for insulin delivered before initiation of the closed - loop program to further reduce exposure to hypoglycemia . the other main concern regarding nocturnal hypoglycemia pertains to the use of glucose sensors that may overread the true plasma glucose value , leading to an overestimation of insulin requirements . in this series of experiments , as previously ( 12 ) , the overnight glucose target was set at 6.6 mmol / l ( 120 mg / dl ) to provide a margin of error . the median error of currently available real - time continuous glucose monitors is between 10 and 16% when plasma glucose levels are between 70 and 180 mg / dl ( 9,10,26,27 ) . a sensor error overreading the plasma glucose by 33% would drive the true glucose to only 90 mg / dl . furthermore , the veo calibration algorithm was recently compared with that in the paradigm real - time ( prt ) sensor - augmented pump ( 28 ) . sensor error reduced significantly in the 40120 mg / dl range , and hypoglycemia detection was improved from 55% in the prt to 82% in the veo , while retaining accuracy at higher glucose levels . this study represents progress toward the development of an automated , portable , closed - loop system for outpatient use . further research is needed to optimize automated fault detection settings and to ensure current mechanisms are effective . in the current study , fault detection settings successfully identified sensor failure . despite the limitations of current transcutaneous sensors , this portable glucose control system reduced exposure to hypoglycemia and hyperglycemia in adolescents and young adults with type 1 diabetes . . the combination sensor - infusion set requiring only one insertion site is currently undergoing clinical trials and will also be incorporated into this system to minimize insertion sites for the patient .

objectivea key milestone in progress towards providing an efficacious and safe closed - loop artificial pancreas system for outpatient use is the development of fully automated , portable devices with fault detection capabilities to ensure patient safety . the ability to remotely monitor the operation of the closed - loop system would facilitate future physician - supervised home studies.research design and methodsthis study was designed to investigate the efficacy and safety of a fully automated , portable , closed - loop system . the medtronic portable glucose control system ( pgcs ) consists of two subcutaneous glucose sensors , a control algorithm based on proportional - integral - derivative with insulin feedback operating from a blackberry storm smartphone platform , bluetooth radiofrequency translator , and an off - the - shelf medtronic paradigm veo insulin pump . participants with type 1 diabetes using insulin pump therapy underwent two consecutive nights of in - clinic , overnight , closed - loop control after a baseline open - loop assessment.resultseight participants attended for 16 overnight studies . the pgcs maintained mean overnight plasma glucose levels of 6.4 1.7 mmol / l ( 115 31 mg / dl ) . the proportion of time with venous plasma glucose < 3.9 , between 3.9 and 8 ( 70 and 144 mg / dl ) , and > 8 mmol / l was 7 , 78 , and 15% , respectively . the proportion of time the sensor glucose values were maintained between 3.9 and 8 mmol / l was greater for closed - loop than open - loop ( 84.5 vs. 46.7% ; p < 0.0001 ) , and time spent < 3.3 mmol / l was also reduced ( 0.9 vs. 3% ; p < 0.0001).conclusionsthese results suggest that the pgcs , an automated closed - loop device , is safe and effective in achieving overnight glucose control in patients with type 1 diabetes .

RESEARCH DESIGN AND METHODS Study procedures Baseline open-loop assessment. Overnight in-clinic closed-loop studies. System considerations Control algorithm Statistics RESULTS Sensor performance Glycemic control Investigator intervention CONCLUSIONS

this study was designed to determine the efficacy and safety of an automated , portable , overnight closed - loop system based on fingerstick meter glucose calibration . the primary end point was the percentage of time spent in euglycemia with overnight closed - loop control ( range 3.98 mmol / l [ 70144 mg / dl ] ) . the secondary end points were 1 ) comparing overnight efficacy of the portable closed - loop system versus open - loop control , as measured by sensor glucose values , and 2 ) evaluating the length of time under closed - loop control without investigator intervention . participants fulfilling the following criteria were recruited : age 1225 years , diagnosis of type 1 diabetes for > 1 year , a1c < 8.5% , and insulin pump therapy use for at least 6 months . at the initial outpatient visit , participants underwent insertion of a subcutaneous glucose sensor and ipro2 , professional continuous glucose monitoring device ( medtronic minimed ) to be worn for 6 days to establish baseline overnight glycemic levels . during the week after the baseline open - loop assessment , participants were admitted to the clinical research facility at 1700 h for the overnight closed - loop studies . two subcutaneous glucose sensors were inserted into sites chosen by the participant , and an intravenous catheter was placed in an antecubital vein for blood sampling . the participant s pump was disconnected , and the study pump ( paradigm veo ; medtronic minimed ) containing the subject s usual analog insulin , insulin aspart ( novorapid ; novo nordisk , bagsvaerd , denmark ) , or insulin lispro ( humalog ; eli lilly , indianapolis , in ) , was attached . participants were switched from open - loop to closed - loop control at 2100 h , and this was continued until 0700 h the following morning . participants reverted to open - loop control from 0700 to 2100 h , followed by a second consecutive night of overnight closed - loop control . venous plasma glucose was measured every 30 min on both nights during overnight closed - loop control using the ysi 2300 stat ( yellow springs instruments , yellow springs , oh ) . if the plasma glucose was < 2.8 mmol / l ( 50 mg / dl ) on two occasions 15 min apart or the plasma glucose was > 15 mmol / l ( 270 mg / dl ) for 1 h , closed - loop control was stopped and the system reverted to open - loop control . blood samples for plasma insulin determination were collected hourly during overnight closed - loop control . samples were assayed using a one - step chemiluminescent noncompetitive immunoassay ( architect ; abbott diagnostics , abbott park , il ) with coefficients of variation of < 2% for means of 17 , 46 , and 130 raw sensor data were transmitted to the blackberry by way of the radiofrequency ( rf ) translator module that translates the medtronic proprietary 916-mhz rf protocol to the bluetooth communication protocol , and vice versa . after sensor data are processed , the blackberry calculates pump strokes based on the closed - loop control algorithm and sends pump delivery commands over the bluetooth interface to the translator . if the sensor deviation falls outside these parameters , the fault detection will be triggered to request another blood glucose level to be entered for calibration . insulin delivery was modeled on the multiphasic insulin response of the -cell and consisted of three principal components proportional , integral , and derivative ( pid)as used in other studies ( 11,12 ) , with a modification to include feedback of a model - predicted insulin profile ( 19 ) . the pgcs used fault detection settings regarding 1 ) correlation of sensor signals , 2 ) deviation of sensor glucose ( sg1 and sg2 ) from the mean ( sg ) , 3 ) mutual difference in rate of glucose change ( direction of trend ) , 4 ) loss of sensor signal , rf , or bluetooth connectivity , and 5 ) insulin delivery limits . if a calibration is not entered within 15 min of the request , or both sensor signals or the bluetooth connection are lost for 15 min , the system automatically reverts to open - loop mode . as the system reverts to open - loop mode , the pump rate is set to deliver no insulin for 2 h. this is a temporary basal rate setting before it reverts to the preprogrammed basal rate after 2 h. this will ensure that a hypoglycemic patient will not receive further basal insulin for 2 h. depending on distribution , data are expressed as mean sd or as median and range or interquartile range ( 25th75th centile ) , where appropriate . comparisons between overnight closed - loop and open - loop control were made using a mann - whitney u test for nonparametric data and a student t test for data conforming to a normal distribution . at the initial outpatient visit , participants underwent insertion of a subcutaneous glucose sensor and ipro2 , professional continuous glucose monitoring device ( medtronic minimed ) to be worn for 6 days to establish baseline overnight glycemic levels . during the week after the baseline open - loop assessment , participants were admitted to the clinical research facility at 1700 h for the overnight closed - loop studies . two subcutaneous glucose sensors were inserted into sites chosen by the participant , and an intravenous catheter was placed in an antecubital vein for blood sampling . the participant s pump was disconnected , and the study pump ( paradigm veo ; medtronic minimed ) containing the subject s usual analog insulin , insulin aspart ( novorapid ; novo nordisk , bagsvaerd , denmark ) , or insulin lispro ( humalog ; eli lilly , indianapolis , in ) , was attached . participants were switched from open - loop to closed - loop control at 2100 h , and this was continued until 0700 h the following morning . participants reverted to open - loop control from 0700 to 2100 h , followed by a second consecutive night of overnight closed - loop control . venous plasma glucose was measured every 30 min on both nights during overnight closed - loop control using the ysi 2300 stat ( yellow springs instruments , yellow springs , oh ) . if the plasma glucose was < 2.8 mmol / l ( 50 mg / dl ) on two occasions 15 min apart or the plasma glucose was > 15 mmol / l ( 270 mg / dl ) for 1 h , closed - loop control was stopped and the system reverted to open - loop control . blood samples for plasma insulin determination were collected hourly during overnight closed - loop control . samples were assayed using a one - step chemiluminescent noncompetitive immunoassay ( architect ; abbott diagnostics , abbott park , il ) with coefficients of variation of < 2% for means of 17 , 46 , and 130 mu / l . at the initial outpatient visit , participants underwent insertion of a subcutaneous glucose sensor and ipro2 , professional continuous glucose monitoring device ( medtronic minimed ) to be worn for 6 days to establish baseline overnight glycemic levels . during the week after the baseline open - loop assessment , participants were admitted to the clinical research facility at 1700 h for the overnight closed - loop studies . two subcutaneous glucose sensors were inserted into sites chosen by the participant , and an intravenous catheter was placed in an antecubital vein for blood sampling . the participant s pump was disconnected , and the study pump ( paradigm veo ; medtronic minimed ) containing the subject s usual analog insulin , insulin aspart ( novorapid ; novo nordisk , bagsvaerd , denmark ) , or insulin lispro ( humalog ; eli lilly , indianapolis , in ) , was attached . participants were switched from open - loop to closed - loop control at 2100 h , and this was continued until 0700 h the following morning . participants reverted to open - loop control from 0700 to 2100 h , followed by a second consecutive night of overnight closed - loop control . venous plasma glucose was measured every 30 min on both nights during overnight closed - loop control using the ysi 2300 stat ( yellow springs instruments , yellow springs , oh ) . if the plasma glucose was < 2.8 mmol / l ( 50 mg / dl ) on two occasions 15 min apart or the plasma glucose was > 15 mmol / l ( 270 mg / dl ) for 1 h , closed - loop control was stopped and the system reverted to open - loop control . blood samples for plasma insulin determination were collected hourly during overnight closed - loop control . raw sensor data were transmitted to the blackberry by way of the radiofrequency ( rf ) translator module that translates the medtronic proprietary 916-mhz rf protocol to the bluetooth communication protocol , and vice versa . after sensor data are processed , the blackberry calculates pump strokes based on the closed - loop control algorithm and sends pump delivery commands over the bluetooth interface to the translator . if the sensor deviation falls outside these parameters , the fault detection will be triggered to request another blood glucose level to be entered for calibration . insulin delivery was modeled on the multiphasic insulin response of the -cell and consisted of three principal components proportional , integral , and derivative ( pid)as used in other studies ( 11,12 ) , with a modification to include feedback of a model - predicted insulin profile ( 19 ) . the pgcs used fault detection settings regarding 1 ) correlation of sensor signals , 2 ) deviation of sensor glucose ( sg1 and sg2 ) from the mean ( sg ) , 3 ) mutual difference in rate of glucose change ( direction of trend ) , 4 ) loss of sensor signal , rf , or bluetooth connectivity , and 5 ) insulin delivery limits . if a calibration is not entered within 15 min of the request , or both sensor signals or the bluetooth connection are lost for 15 min , the system automatically reverts to open - loop mode . as the system reverts to open - loop mode , the pump rate is set to deliver no insulin for 2 h. this is a temporary basal rate setting before it reverts to the preprogrammed basal rate after 2 h. this will ensure that a hypoglycemic patient will not receive further basal insulin for 2 h. depending on distribution , data are expressed as mean sd or as median and range or interquartile range ( 25th75th centile ) , where appropriate . comparisons between overnight closed - loop and open - loop control were made using a mann - whitney u test for nonparametric data and a student t test for data conforming to a normal distribution . eight subjects ( four males ) underwent 145 h of closed - loop control over 16 nights . median age was 14.8 years ( range 12.624 ) , duration of diabetes was 6.9 years ( range 3.821.5 ) , duration of pump therapy was 3.8 years ( range 2.610.8 ) , and bmi was 21 kg / m ( range 1830 ) . mean a1c was 7.3 1.1% , and total daily dose of insulin was 0.9 0.1 units / kg . interstitial glucose values tracked venous plasma glucose levels effectively during overnight closed - loop control . point accuracy of the sensor , expressed as median ( interquartile range ) and mean sd relative absolute difference from the venous plasma glucose were 7.5% ( 3.318.5 ) and 12.3 12.7% , respectively , similar to previously published studies ( 9,10,12,15 ) and in keeping with the published accuracy of this sensor ( 21 ) . mean plasma glucose during overnight closed - loop control was 6.4 1.7 mmol / l ( 115 31 mg / dl ) . the proportion of time with venous plasma glucose < 3.9 , between 3.9 and 8 ( 70 and 144 mg / dl ) , and > 8 mmol / l was 7 , 78 , and 15% respectively . mmol / l ) was significantly higher after midnight than before midnight ( 85 vs. 66% ; p = 0.001 ) . plasma glucose < 3.3 mmol / l occurred in 3 of 16 study nights and within the first 3.5 h. plasma glucose readings < 3.9 mmol / l were significantly more common in the first 3 h of closed - loop control than thereafter ( 13.9 vs. 4% ; p = 0.005 ) . sensor and venous plasma glucose levels during closed - loop control are shown in fig . glycemic control during closed - loop insulin delivery was superior to prestudy home overnight open - loop control ( fig . an evening meal and prandial bolus were administered at 1800 , and the closed - loop mode commenced at 2100 . comparison of overnight closed - loop ( red ) with prestudy open - loop ( blue ) control. sensor fault detection requested fingerstick calibration on both closed - loop nights for two subjects . these subjects had the greatest relative - absolute difference between sensor glucose and plasma glucose , and between them accounted for 46% of plasma glucose levels out of target range for the entire study . investigator intervention during automated , portable , closed - loop control for one further subject ( 2 nights ) , the bluetooth link was lost for > 15 min and needed to be re - established . one patient ( age 24 years , weight 94 kg , total daily dose 105 units ) required rescue carbohydrates for plasma glucose 2.5 mmol / l ( sensor 3.5 mmol / l ) . he commenced closed - loop 3 h after an evening meal where he gave himself a preprandial bolus of 22.7 units but did not finish the meal . his usual basal rate was 2.7 units / h , and during closed - loop , he received only 0.67 units of insulin over 3 h before the study night was terminated , as per protocol . interstitial glucose values tracked venous plasma glucose levels effectively during overnight closed - loop control . point accuracy of the sensor , expressed as median ( interquartile range ) and mean sd relative absolute difference from the venous plasma glucose were 7.5% ( 3.318.5 ) and 12.3 12.7% , respectively , similar to previously published studies ( 9,10,12,15 ) and in keeping with the published accuracy of this sensor ( 21 ) . mean plasma glucose during overnight closed - loop control was 6.4 1.7 mmol / l ( 115 31 mg / dl ) . the proportion of time with venous plasma glucose < 3.9 , between 3.9 and 8 ( 70 and 144 mg / dl ) , and > 8 mmol / l was 7 , 78 , and 15% respectively . mmol / l ) was significantly higher after midnight than before midnight ( 85 vs. 66% ; p = 0.001 ) . plasma glucose < 3.3 mmol / l occurred in 3 of 16 study nights and within the first 3.5 h. plasma glucose readings < 3.9 mmol / l were significantly more common in the first 3 h of closed - loop control than thereafter ( 13.9 vs. 4% ; p = 0.005 ) . sensor and venous plasma glucose levels during closed - loop control are shown in fig . glycemic control during closed - loop insulin delivery was superior to prestudy home overnight open - loop control ( fig . an evening meal and prandial bolus were administered at 1800 , and the closed - loop mode commenced at 2100 . comparison of overnight closed - loop ( red ) with prestudy open - loop ( blue ) control. sensor fault detection requested fingerstick calibration on both closed - loop nights for two subjects . these subjects had the greatest relative - absolute difference between sensor glucose and plasma glucose , and between them accounted for 46% of plasma glucose levels out of target range for the entire study . these subjects had similar bmis , 22 and 26 kg / m , respectively ; thus , patient phenotype did not explain the difference between sensor and plasma glucose . investigator intervention during automated , portable , closed - loop control for one further subject ( 2 nights ) , the bluetooth link was lost for > 15 min and needed to be re - established . one patient ( age 24 years , weight 94 kg , total daily dose 105 units ) required rescue carbohydrates for plasma glucose 2.5 mmol / l ( sensor 3.5 mmol / l ) . he commenced closed - loop 3 h after an evening meal where he gave himself a preprandial bolus of 22.7 units but did not finish the meal . his usual basal rate was 2.7 units / h , and during closed - loop , he received only 0.67 units of insulin over 3 h before the study night was terminated , as per protocol . this study demonstrates the feasibility and safety features of a portable , automated , closed - loop system for overnight glucose control in adolescents and young adults with type 1 diabetes . monitoring of system operation from a remote location using data transmission over a wireless network is an achievable goal and is an additional safety feature , paving the way for physician - supervised home studies . automated fault detection requested fingerstick calibration of the system when sensor correlation was below a specified threshold or sensor glucose values deviated significantly from the mean . further study is necessary to determine the optimum fault detection settings to maximize patient safety yet minimize disruption to the operation of the closed - loop system . initial commercially available fully closed - loop systems will likely focus on overnight glucose control to minimize nocturnal hypoglycemia . the effects of intensive management of type 1 diabetes on the incidence of hypoglycemia are well documented . in the diabetes control and complications trial ( 1 ) , 55% of severe hypoglycemia occurred during sleep , and in the pediatric population , 75% of episodes of severe hypoglycemia occurred overnight ( 22 ) . an overnight closed - loop system that automatically suspends insulin delivery in response to impending hypoglycemia would minimize exposure to prolonged hypoglycemia and adverse outcomes such as seizures ( 25 ) . in our study , although plasma glucose < 3.3 mmol / l ( 60 mg / dl ) occurred in 3 of 16 study nights , the sensor readings indicated that hypoglycemia was significantly less common during closed - loop compared with open - loop therapy . episodes of hypoglycemia during closed - loop therapy occurred during the first 3.5 h and likely related to start - up algorithm is necessary to smooth the transition between open - loop and closed - loop sessions and account for insulin delivered before initiation of the closed - loop program to further reduce exposure to hypoglycemia . the other main concern regarding nocturnal hypoglycemia pertains to the use of glucose sensors that may overread the true plasma glucose value , leading to an overestimation of insulin requirements . in this series of experiments , as previously ( 12 ) , the overnight glucose target was set at 6.6 mmol / l ( 120 mg / dl ) to provide a margin of error . the median error of currently available real - time continuous glucose monitors is between 10 and 16% when plasma glucose levels are between 70 and 180 mg / dl ( 9,10,26,27 ) . a sensor error overreading the plasma glucose by 33% would drive the true glucose to only 90 mg / dl . sensor error reduced significantly in the 40120 mg / dl range , and hypoglycemia detection was improved from 55% in the prt to 82% in the veo , while retaining accuracy at higher glucose levels . this study represents progress toward the development of an automated , portable , closed - loop system for outpatient use . further research is needed to optimize automated fault detection settings and to ensure current mechanisms are effective . despite the limitations of current transcutaneous sensors , this portable glucose control system reduced exposure to hypoglycemia and hyperglycemia in adolescents and young adults with type 1 diabetes .

pantoprazole [ 5 - ( difluoromethoxy -2 - [ [ ( 3,4-dimethoxy-2-pyridinyl ) methyl ] sulfinyl]-1h - benzimidazol ] is a weakly basic prodrug which accumulates in highly acidic environments and becomes rapidly activated in cationic sulfonamide ( raffin et al . , 2007 ; vishvakarma and singh , 2011 ) . ( 2004 ) , it has a low potential for metabolic interaction with cytochrome p450 ( cyp450 ) oxidation systems and is , for this reason , especially suitable for patients treated with other medications . ( 2010 ) found that pantoprazole is completely metabolized by the hepatic cytochrome p450 system , and more than 80% of the inactive metabolites are eliminated via renal excretion . 2008 ) tested the effects of several drugs used in the treatment of gastric cell tumors . they demonstrated that the cytotoxic effect of pantoprazole triggers mitochondria - dependent apoptosis in the cells of the tumor . the long - term use of pantoprazole , however , may result in hypergastrinemia , possible hyperplasia of the cells of the enteric nervous system , carcinoid tumors of the stomach , liver cell adenoma and other carcinomas as well as thyroid neoplasms ( pantoloc , 2003 ) . genetic toxicology is an important field that studies the genotoxic / mutagenic properties of agents ( chemical , physical and biological ) to which organisms are exposed , using various assays to assess the damage that these may cause to dna in the presence or absence of mass metabolic systems . these assays include the smart ( somatic mutation and recombination test ) developed by graf et al . the use of smart on drosophila melanogaster wings can detect a wide spectrum of genetic abnormalities , such as mutation , deletion and recombination ( graf et al . , 1984 ) . the test is based on the fact that during early embryonic development of d. melanogaster , groups of cells composing imaginal discs proliferate mitotically until during metamorphosis they become differentiated into body structures of the adult fly . if there is a genetic alteration in an imaginal wing disc , a clone of mutant cells will be formed and detected as a spot on the wings of the mutant adult fly ( guzmn - rincn and graf , 1995 ) . the analysis of these spots determines the phenotypic expression of the marker genes flr or mwh , responsible for changes in the shape of wing hairs or trichomes ( graf et al . the worldwide growing consumption of pantoprazole and easy access to this drug , which no longer requires a medical prescription , have generated increased interest in assessing possible genotoxic effects associated with its use . hence , the objective of the present study was to evaluate genotoxic effects of pantoprazole by applying the drosophila melanogaster wing spot test . differences in the levels of cytochrome p450 on pantoprazole genotoxic activity was evaluated by way of standard ( st ) and high - bioactivation ( hb ) crosses of drosophila . an hb cross is characterized by an increased cytochrome p450-dependent bioactivation capacity for promutagens when compared with an st cross . ( cas 102625 - 70 - 7 ; total impurity : 0.69% ; density : 0.88 g / ml ) , obtained from the university pharmacy of the university center of patos de minas ( unipam ) , patos de minas , brazil , was prepared in three concentrations ( 2.5 , 5.0 and 10.0 m ) , based on research previously published by masubuchi and okazaki ( 1999 ) on primary cultured hepatocytes from female sprague - dawley rats . 83520 ) was produced by eurofarma laboratories so paulo , brazil and distributed by zodiac pharmaceuticals sa , sao paulo , brazil . in the present research , doxolen was used at a concentration of 0.4 mm . for testing with smart ( graf et al . , 1984 ) , mutant strains of d. melanogaster were provided by dr . urich graf of the institute of toxicology , university of zurich , schwerzenbach , switzerland . three mutant strains of drosophila melanogaster with genetic markers were used in the study : multiple wing hairs ( mwh , 30.33 ) , flare-3 ( flr , 338.8 ) and orr ; flare-3 ( orr ; flr ) . stocks of these strains were kept in a bod incubator 411d new ethics ( nova tica indstria comrcio e servios ltda , so paulo , brazil ) at a temperature of about 25 c 2 and 60% humidity in 250 ml flasks containing a medium prepared with 820 ml of water , 11 g agar , 156 g of banana , 1 g of nipagin ( fagron do brasil farmacutica , so paulo , brazil ) and 25 g of biological yeast saccharomyces cerevisiae . two types of crosses were performed : ( 1 ) a standard cross ( st ) , in which virgin females flr/ in(3lr)tm3 , ri p sep l(3)89aa bx bd were crossed with mwh/ mwh males , and ( 2 ) a high bioactivation cross ( hb ) , with virgin females orr ; flr/ in(3lr)tm3 , ri p sep l(3)89aa bx bd crossed with mwh / mwh males . in both crosses , two types of offspring were obtained : trans - marker heterozygous ( mh ) with the ( mwh + /+ flr ) genotype and wings phenotypically of the wild type ; and heterozygous balancer ( bh ) with the ( mwh + /+ the larvae , of both genotypes from these crosses were treated with three concentrations of the chemical agent to be tested . eggs were collected for a period of 8 hin flasks containing solid agar ( 4% agar in water ) and a layer of yeast ( s. cerevisiae ) supplemented with sugar . after 72 4 h , the third instar larvae were washed with reverse osmosis water and collected in a fine mesh steel sieve . groups of approximately 100 larvae were transferred to glass tubes ( 2.5 cm in diameter and 8.0 cm in height ) containing 1.5 g of a culture medium of instant mashed potatoes ( hikari , so paulo brazil ) and 5.0 ml of each of three concentrations of the agent to be tested . the 72-hour old larvae from both crosses ( st and hb ) were transferred to 2.5 cm 8.0 cm high glass tubes containing 1.5 g of instant mashed potatoes with three concentrations of pantoprazole : 2.5 m , 5.0 m or 10.0 m . the larvae subsequently continued on to develop through the pupal stage ( 48 h ) . reverse osmosis water was used as a solvent and negative control and doxorubicin ( dxr , 0.4 mm ) as a positive control . dxr was used as positive control , because in smart assays with d. melanogaster it was classified as a strong mutagen , inducing all types of wing spots ( orsolin et al . , 2012 ) . the wings of adult flies preserved in 70% ethanol were removed with entomological forceps under a stereo - microscope . they were soaked in faure solution ( 30 g of gum arabic , 20 ml of glycerol , 1.5 g chloral hydrate and 50 ml distilled water ) , and stretched on slides . the slides were dried for approximately 2 h on a hot plate ( 40 c ) . finally , a cover slip was applied and the wings were coded . wing spot analysis was performed using a light optical microscope at 400x magnification ( 40 ) . the number , types ( single or twin ) , position and size of the spots were calculated and recorded . statistical analysis of the experiment was performed using a chi square test as described by frei and wrgler ( 1988 ) . the non - parametric u test of mann - whitney and a wilcoxon test were used to exclude false positive results . for the analysis of anti - mutagenicity , the frequencies of each type of spot were compared in pairs , using the u test ( frei and wrgler , 1995 ) at a significance level of = 0.05 . ( cas 102625 - 70 - 7 ; total impurity : 0.69% ; density : 0.88 g / ml ) , obtained from the university pharmacy of the university center of patos de minas ( unipam ) , patos de minas , brazil , was prepared in three concentrations ( 2.5 , 5.0 and 10.0 m ) , based on research previously published by masubuchi and okazaki ( 1999 ) on primary cultured hepatocytes from female sprague - dawley rats . 83520 ) was produced by eurofarma laboratories so paulo , brazil and distributed by zodiac pharmaceuticals sa , sao paulo , brazil . in the present research urich graf of the institute of toxicology , university of zurich , schwerzenbach , switzerland . three mutant strains of drosophila melanogaster with genetic markers were used in the study : multiple wing hairs ( mwh , 30.33 ) , flare-3 ( flr , 338.8 ) and orr ; flare-3 ( orr ; flr ) . stocks of these strains were kept in a bod incubator 411d new ethics ( nova tica indstria comrcio e servios ltda , so paulo , brazil ) at a temperature of about 25 c 2 and 60% humidity in 250 ml flasks containing a medium prepared with 820 ml of water , 11 g agar , 156 g of banana , 1 g of nipagin ( fagron do brasil farmacutica , so paulo , brazil ) and 25 g of biological yeast saccharomyces cerevisiae . two types of crosses were performed : ( 1 ) a standard cross ( st ) , in which virgin females flr/ in(3lr)tm3 , ri p sep l(3)89aa bx bd were crossed with mwh/ mwh males , and ( 2 ) a high bioactivation cross ( hb ) , with virgin females orr ; flr/ in(3lr)tm3 , ri p sep l(3)89aa bx bd crossed with mwh / mwh males . in both crosses , two types of offspring were obtained : trans - marker heterozygous ( mh ) with the ( mwh + /+ flr ) genotype and wings phenotypically of the wild type ; and heterozygous balancer ( bh ) with the ( mwh + /+ the larvae , of both genotypes from these crosses were treated with three concentrations of the chemical agent to be tested . eggs were collected for a period of 8 hin flasks containing solid agar ( 4% agar in water ) and a layer of yeast ( s. cerevisiae ) supplemented with sugar . after 72 4 h , the third instar larvae were washed with reverse osmosis water and collected in a fine mesh steel sieve . groups of approximately 100 larvae were transferred to glass tubes ( 2.5 cm in diameter and 8.0 cm in height ) containing 1.5 g of a culture medium of instant mashed potatoes ( hikari , so paulo brazil ) and 5.0 ml of each of three concentrations of the agent to be tested . the 72-hour old larvae from both crosses ( st and hb ) were transferred to 2.5 cm 8.0 cm high glass tubes containing 1.5 g of instant mashed potatoes with three concentrations of pantoprazole : 2.5 m , 5.0 m or 10.0 m . the larvae subsequently continued on to develop through the pupal stage ( 48 h ) . reverse osmosis water was used as a solvent and negative control and doxorubicin ( dxr , 0.4 mm ) as a positive control . dxr was used as positive control , because in smart assays with d. melanogaster it was classified as a strong mutagen , inducing all types of wing spots ( orsolin et al . , 2012 ) . the wings of adult flies preserved in 70% ethanol were removed with entomological forceps under a stereo - microscope . they were soaked in faure solution ( 30 g of gum arabic , 20 ml of glycerol , 1.5 g chloral hydrate and 50 ml distilled water ) , and stretched on slides . the slides were dried for approximately 2 h on a hot plate ( 40 c ) . finally , a cover slip was applied and the wings were coded . wing spot analysis was performed using a light optical microscope at 400x magnification ( 40 ) . the number , types ( single or twin ) , position and size of the spots were calculated and recorded . urich graf of the institute of toxicology , university of zurich , schwerzenbach , switzerland . three mutant strains of drosophila melanogaster with genetic markers were used in the study : multiple wing hairs ( mwh , 30.33 ) , flare-3 ( flr , 338.8 ) and orr ; flare-3 ( orr ; flr ) . stocks of these strains were kept in a bod incubator 411d new ethics ( nova tica indstria comrcio e servios ltda , so paulo , brazil ) at a temperature of about 25 c 2 and 60% humidity in 250 ml flasks containing a medium prepared with 820 ml of water , 11 g agar , 156 g of banana , 1 g of nipagin ( fagron do brasil farmacutica , so paulo , brazil ) and 25 g of biological yeast saccharomyces cerevisiae . two types of crosses were performed : ( 1 ) a standard cross ( st ) , in which virgin females flr/ in(3lr)tm3 , ri p sep l(3)89aa bx bd were crossed with mwh/ mwh males , and ( 2 ) a high bioactivation cross ( hb ) , with virgin females orr ; flr/ in(3lr)tm3 , ri p sep l(3)89aa bx bd crossed with mwh / mwh males . in both crosses , two types of offspring were obtained : trans - marker heterozygous ( mh ) with the ( mwh + /+ flr ) genotype and wings phenotypically of the wild type ; and heterozygous balancer ( bh ) with the ( mwh + /+ the larvae , of both genotypes from these crosses were treated with three concentrations of the chemical agent to be tested . eggs were collected for a period of 8 hin flasks containing solid agar ( 4% agar in water ) and a layer of yeast ( s. cerevisiae ) supplemented with sugar . after 72 4 h , the third instar larvae were washed with reverse osmosis water and collected in a fine mesh steel sieve . groups of approximately 100 larvae were transferred to glass tubes ( 2.5 cm in diameter and 8.0 cm in height ) containing 1.5 g of a culture medium of instant mashed potatoes ( hikari , so paulo brazil ) and 5.0 ml of each of three concentrations of the agent to be tested . the 72-hour old larvae from both crosses ( st and hb ) were transferred to 2.5 cm 8.0 cm high glass tubes containing 1.5 g of instant mashed potatoes with three concentrations of pantoprazole : 2.5 m , 5.0 m or 10.0 m . the larvae subsequently continued on to develop through the pupal stage ( 48 h ) . reverse osmosis water was used as a solvent and negative control and doxorubicin ( dxr , 0.4 mm ) as a positive control . dxr was used as positive control , because in smart assays with d. melanogaster it was classified as a strong mutagen , inducing all types of wing spots ( orsolin et al . , 2012 ) . the wings of adult flies preserved in 70% ethanol were removed with entomological forceps under a stereo - microscope . they were soaked in faure solution ( 30 g of gum arabic , 20 ml of glycerol , 1.5 g chloral hydrate and 50 ml distilled water ) , and stretched on slides . the slides were dried for approximately 2 h on a hot plate ( 40 c ) . finally , a cover slip was applied and the wings were coded . wing spot analysis was performed using a light optical microscope at 400x magnification ( 40 ) . the number , types ( single or twin ) , position and size of the spots were calculated and recorded . statistical analysis of the experiment was performed using a chi square test as described by frei and wrgler ( 1988 ) . the non - parametric u test of mann - whitney and a wilcoxon test were used to exclude false positive results . for the analysis of anti - mutagenicity , the frequencies of each type of spot were compared in pairs , using the u test ( frei and wrgler , 1995 ) at a significance level of = 0.05 . the data were pooled after verifying that the two independent experiments were in agreement with acceptable reproducibility . no significant decreases in the survival rates of larvae submitted to treatments were observed when compared to the negative control . the maximum concentration used in our study corresponded to plasma levels found in patients treated with single oral dose of 40 mg of pantoprazole ( kamdi and palkar , 2013 ) . the maximum values found by the authors were seen at 2 h 56 min after exposure . table 1 shows the results of mutant spot frequencies observed in the bh and mh descendants of the standard cross ( st ) , treated with pantoprazole in three different concentrations ( 2.5 , 5.0 or 10.0 m ) . the positive control ( dxr 0.4 mm ) and negative control ( reverse osmosis water ) are also presented . table 2 shows the results of mutant spot frequencies observed in the bh and mh descendants of the high bioactivation cross ( hb ) , for the same concentrations of pantoprazole , as well as the positive and negative controls . statistical diagnostic according to frei and wrgler ( 1988 ) : ( + ) positive ( compared to the negative control ) ; ( ) negative ; ( i ) inconclusive . including rare single flr spots . considering the mwh clones for the single spots and mwh for the twin spots . frequency of clone formation : clones / flies/48,800 cells ( without size correction ) . statistical diagnostic according to frei and wrgler ( 1988 ) : ( + ) positive ( compared to the negative control ) ; ( ) negative ; ( i ) inconclusive . including rare single flr spots . considering the mwh clones for the single spots and mwh for the twin spots . frequency of clone formation : clones / flies/48,800 cells ( without size correction ) . when compared to the negative control , pantoprazole caused a significant increase in the frequency of small , simple spots at all concentrations . the total number of spots , however , was only statistically significant at 2.5 and 5.0 m . results for the hb cross in terms of the potential mutagenic properties of pantoprazole are presented in table 2 . the total number of mutant spots among the mh descendants , compared to the negative control , was significantly increased in all concentrations . the analysis of flies with the bh genotype ( mwh / tm3 ) was carried out for the purpose of calculating the portion of recombinogenic and mutagenic events . it is possible to separate mutational events from recombinational events , because recombinational events are eliminated in flies with this genotype . a comparison of clone - induction frequencies obtained for pantoprazole in both genotypes indicated that in st flies , 49.76% of mutant clones produced by pantoprazole were due to mutation and 50.24% due to recombination at the 2.5 m concentration . clone - induction frequencies for pantoprazole ( 5.0 m ) indicated that 52.85% of the mutant clones produced were due to mutation and 47.15% to recombination . however , the very same analysis showed that in hb flies , 31.30% of spots induced by pantoprazole ( 2.5 m ) were due to mutation and 68.70% to recombination ; 37.56% of spots induced by pantoprazole ( 5.0 m ) were due to mutation and 62.44% to recombination ; 35.48% mutant clones produced by pantoprazole ( 10 m ) were due to mutation and 64.52 to recombination . thus , our results indicate that recombinogenicity was the major genotoxic effect of pantoprazole in hb flies . many compounds are converted to highly reactive metabolites by oxidative enzymes , principally cytochrome p450 . thus , by introducing one or more hydroxyl groups on a substrate , a pre - carcinogen can become a carcinogen ( gregory , 1986 ) . the genetic control of xenobiotic metabolism in drosophila is complex , and multiple forms of p450 as well as other enzymes ( e.g. , amine oxidases ) are known to be involved in the activation of certain promutagens ( frlich and wrgler , 1989 ) . according to our findings , pantoprazole has a clear recombinogenic potential in drosophila , and the stock differences demonstrate a strong dependence on levels of metabolic activation ( hb flies ) as the increased cytochrome p450-dependent biocativation capacity present in these hb larvae leads to significantly increased recombinogenicity . therefore , the metabolic pathway in the induction of recombinogenicity most probably involves cytochrome p450-dependent enzyme activity . in accordance with these findings , mathews et al . ( 2010 ) showed that pantoprazole is completely metabolized by the hepatic cytochrome p450 system . although homologous recombination is an important pathway in dna repair , there is growing evidence that deleterious genomic rearrangements may result from homologous recombination , which means that homologous recombination events may play a causative role in carcinogenesis ( arossi et al . , 2009 ) . the transformation of normal cells into cancer cells is a multistep process , and mitotic recombination can be a mechanism involved in such transformation ( nowell , 1976 ; barrett , 1993 ) . in heterozygous cells bearing a mutant and normal alleles for a tumor suppressor gene , somatic recombination may turn out to be a promoter of neoplasms by inducing homozygosis of the mutant tumor suppressor allele ( maher et al . , 1993 ; sengstag , 1994 ) . kuipers ( 2006 ) has stated that prolonged use of proton pump inhibitors ( ppis ) may be related to the development of gastric cancer . it is also known that treatment with ppis does not protect against this type of cancer , but the increased risk of cancer due to prolonged use remains unknown . ( 2010 ) have reported that among patients diagnosed as negative for h. pylori and without pre - existing gastritis , ppis did not cause chronic gastritis . in contrast , people infected by h. pylori were found to have chronic , persistent gastritis , atrophy and metaplasia , which may progress to gastric atrophy , intestinal metaplasia and gastric cancer . ppis used in the treatment of this infection may also cause an acceleration of the progression or development of gastritis ( thomson et al . , 2010 ) . nonetheless , until now there is no evidence that ppis increase the risk of gastric cancer . persistent , predominant gastritis and atrophic gastritis of the gastric body mucosa , however , are considered important risk factors for the development of gastric cancer , and , clearly , more studies are needed to reach a definitive conclusion ( kuipers , 2006 ) . by means of in vivo experiments ( 2012 ) showed that pantoprazole pretreatment could enhance the anti - tumor effects of adriamycin on xenografted tumor in nude mice and also improve the apoptotic index in xenografted tumor tissues . according to the authors , pantoprazole pretreatment enhances the cytotoxic effects of anti - tumor drugs on human gastric adenocarcinoma cells ( sgc7901 ) and reverts multidrug resistance of sgc7901/adr cells by down - regulating the v - atpases / mtor / hif-1/p - gp and mrp1 signaling pathway . ( 2013 ) also showed that pantoprazole inhibits the proliferation and induced apoptosis of sgc7901 human gastric cancer cells . ( 2013 ) , the use of pantoprazole to enhance the distribution and cytotoxicity of anticancer drugs in solid tumors might be a novel treatment strategy to improve their therapeutic indices . it is worthy of note that tests for mutagenic evaluation are generally limited to such specific effects and that not every change in genetic material is a mutation . for this reason , smart is an important tool for mutagenic assessment . it provides an evaluation of mutational events , as well as recombinogenic events , as shown in the present study . it is known that substances that cause dna damage also induce recombination , which generates more dna damage ( hoffman , 1994 ) . recombination can promote loss of heterozygosity in somatic cells and germ cells which , in turn , may influence cancer progression ( happle , 1999 ) . ( 2010 ) reviewed the genotoxic and carcinogenic effects of 71 gastrointestinal drugs , demonstrating that pantoprazole was found to be genotoxic and carcinogenic in several assays , in addition to causing chromosomal damage . these results are consistent with those presented herein , where pantoprazole caused an increase in the frequency of mutant spots for somatic cells , revealing its genotoxic characteristics . the genotoxicity of a particular substance is , thus , not exclusively caused by its association with mutation but also with recombination events , which may cause chromosomal damage . at current , the published studies are conflicting in their results , warranting further examination by means of additional assays and test organisms . in conclusion , the present study indicates that pantoprazole possesses recombinogenic activity in the drosophila wing spot test . nonetheless , although there was an increase in mutant spots in the st descendants , the increase in recombinogenic activity was observed only in the high bioactivation ( hb ) descendants , this suggesting the interaction of their constituents ( pantoprazole ) with cytochrome p450 .

pantoprazole is one of the leading proton pump inhibitors ( ppis ) used in the treatment of a variety of diseases related to the upper gastrointestinal tract . however , studies have shown an increased risk of developing gastric cancer , intestinal metaplasia and hyperplasia of endocrine cells with prolonged use . in the present study , the somatic mutation and recombination test ( smart ) was employed to determine the mutagenic effects of pantoprazole on drosophila melanogaster . repeated treatments with pantoprazole were performed on 72-hour larvae of the standard ( st ) and high bioactivation ( hb ) crosses at concentrations of 2.5 , 5.0 , and 10.0 m . in addition , doxorubicin ( dxr ) was administered at 0.4 mm , as a positive control . when administered to st descendants , total number of spots were statistically significant at 2.5 and 5.0 m concentrations . for hb descendants , a significant increase in the total number of spots was observed among the marked transheterozygous ( mh ) flies . through analysis of balancer heterozygous ( bh ) descendants , recombinogenic effects were observed at all concentrations in descendants of the hb cross . in view of these experimental conditions and results , it was concluded that pantoprazole is associated with recombinogenic effects in drosophila melanogaster .

Introduction Material and Methods Chemical compounds Somatic Mutation And Recombination Test (SMART) in somatic cells of Strain stock crosses and treatment Preparation and microscopic analysis of the wings Statistical analysis Results and Discussion

( 2010 ) found that pantoprazole is completely metabolized by the hepatic cytochrome p450 system , and more than 80% of the inactive metabolites are eliminated via renal excretion . 2008 ) tested the effects of several drugs used in the treatment of gastric cell tumors . they demonstrated that the cytotoxic effect of pantoprazole triggers mitochondria - dependent apoptosis in the cells of the tumor . the long - term use of pantoprazole , however , may result in hypergastrinemia , possible hyperplasia of the cells of the enteric nervous system , carcinoid tumors of the stomach , liver cell adenoma and other carcinomas as well as thyroid neoplasms ( pantoloc , 2003 ) . these assays include the smart ( somatic mutation and recombination test ) developed by graf et al . the use of smart on drosophila melanogaster wings can detect a wide spectrum of genetic abnormalities , such as mutation , deletion and recombination ( graf et al . if there is a genetic alteration in an imaginal wing disc , a clone of mutant cells will be formed and detected as a spot on the wings of the mutant adult fly ( guzmn - rincn and graf , 1995 ) . the analysis of these spots determines the phenotypic expression of the marker genes flr or mwh , responsible for changes in the shape of wing hairs or trichomes ( graf et al . the worldwide growing consumption of pantoprazole and easy access to this drug , which no longer requires a medical prescription , have generated increased interest in assessing possible genotoxic effects associated with its use . hence , the objective of the present study was to evaluate genotoxic effects of pantoprazole by applying the drosophila melanogaster wing spot test . differences in the levels of cytochrome p450 on pantoprazole genotoxic activity was evaluated by way of standard ( st ) and high - bioactivation ( hb ) crosses of drosophila . an hb cross is characterized by an increased cytochrome p450-dependent bioactivation capacity for promutagens when compared with an st cross . ( cas 102625 - 70 - 7 ; total impurity : 0.69% ; density : 0.88 g / ml ) , obtained from the university pharmacy of the university center of patos de minas ( unipam ) , patos de minas , brazil , was prepared in three concentrations ( 2.5 , 5.0 and 10.0 m ) , based on research previously published by masubuchi and okazaki ( 1999 ) on primary cultured hepatocytes from female sprague - dawley rats . in the present research , doxolen was used at a concentration of 0.4 mm . three mutant strains of drosophila melanogaster with genetic markers were used in the study : multiple wing hairs ( mwh , 30.33 ) , flare-3 ( flr , 338.8 ) and orr ; flare-3 ( orr ; flr ) . stocks of these strains were kept in a bod incubator 411d new ethics ( nova tica indstria comrcio e servios ltda , so paulo , brazil ) at a temperature of about 25 c 2 and 60% humidity in 250 ml flasks containing a medium prepared with 820 ml of water , 11 g agar , 156 g of banana , 1 g of nipagin ( fagron do brasil farmacutica , so paulo , brazil ) and 25 g of biological yeast saccharomyces cerevisiae . two types of crosses were performed : ( 1 ) a standard cross ( st ) , in which virgin females flr/ in(3lr)tm3 , ri p sep l(3)89aa bx bd were crossed with mwh/ mwh males , and ( 2 ) a high bioactivation cross ( hb ) , with virgin females orr ; flr/ in(3lr)tm3 , ri p sep l(3)89aa bx bd crossed with mwh / mwh males . in both crosses , two types of offspring were obtained : trans - marker heterozygous ( mh ) with the ( mwh + /+ flr ) genotype and wings phenotypically of the wild type ; and heterozygous balancer ( bh ) with the ( mwh + /+ the larvae , of both genotypes from these crosses were treated with three concentrations of the chemical agent to be tested . groups of approximately 100 larvae were transferred to glass tubes ( 2.5 cm in diameter and 8.0 cm in height ) containing 1.5 g of a culture medium of instant mashed potatoes ( hikari , so paulo brazil ) and 5.0 ml of each of three concentrations of the agent to be tested . the 72-hour old larvae from both crosses ( st and hb ) were transferred to 2.5 cm 8.0 cm high glass tubes containing 1.5 g of instant mashed potatoes with three concentrations of pantoprazole : 2.5 m , 5.0 m or 10.0 m . reverse osmosis water was used as a solvent and negative control and doxorubicin ( dxr , 0.4 mm ) as a positive control . dxr was used as positive control , because in smart assays with d. melanogaster it was classified as a strong mutagen , inducing all types of wing spots ( orsolin et al . the number , types ( single or twin ) , position and size of the spots were calculated and recorded . statistical analysis of the experiment was performed using a chi square test as described by frei and wrgler ( 1988 ) . for the analysis of anti - mutagenicity , the frequencies of each type of spot were compared in pairs , using the u test ( frei and wrgler , 1995 ) at a significance level of = 0.05 . ( cas 102625 - 70 - 7 ; total impurity : 0.69% ; density : 0.88 g / ml ) , obtained from the university pharmacy of the university center of patos de minas ( unipam ) , patos de minas , brazil , was prepared in three concentrations ( 2.5 , 5.0 and 10.0 m ) , based on research previously published by masubuchi and okazaki ( 1999 ) on primary cultured hepatocytes from female sprague - dawley rats . in the present research urich graf of the institute of toxicology , university of zurich , schwerzenbach , switzerland . three mutant strains of drosophila melanogaster with genetic markers were used in the study : multiple wing hairs ( mwh , 30.33 ) , flare-3 ( flr , 338.8 ) and orr ; flare-3 ( orr ; flr ) . two types of crosses were performed : ( 1 ) a standard cross ( st ) , in which virgin females flr/ in(3lr)tm3 , ri p sep l(3)89aa bx bd were crossed with mwh/ mwh males , and ( 2 ) a high bioactivation cross ( hb ) , with virgin females orr ; flr/ in(3lr)tm3 , ri p sep l(3)89aa bx bd crossed with mwh / mwh males . in both crosses , two types of offspring were obtained : trans - marker heterozygous ( mh ) with the ( mwh + /+ flr ) genotype and wings phenotypically of the wild type ; and heterozygous balancer ( bh ) with the ( mwh + /+ the larvae , of both genotypes from these crosses were treated with three concentrations of the chemical agent to be tested . groups of approximately 100 larvae were transferred to glass tubes ( 2.5 cm in diameter and 8.0 cm in height ) containing 1.5 g of a culture medium of instant mashed potatoes ( hikari , so paulo brazil ) and 5.0 ml of each of three concentrations of the agent to be tested . the 72-hour old larvae from both crosses ( st and hb ) were transferred to 2.5 cm 8.0 cm high glass tubes containing 1.5 g of instant mashed potatoes with three concentrations of pantoprazole : 2.5 m , 5.0 m or 10.0 m . reverse osmosis water was used as a solvent and negative control and doxorubicin ( dxr , 0.4 mm ) as a positive control . dxr was used as positive control , because in smart assays with d. melanogaster it was classified as a strong mutagen , inducing all types of wing spots ( orsolin et al . the number , types ( single or twin ) , position and size of the spots were calculated and recorded . three mutant strains of drosophila melanogaster with genetic markers were used in the study : multiple wing hairs ( mwh , 30.33 ) , flare-3 ( flr , 338.8 ) and orr ; flare-3 ( orr ; flr ) . stocks of these strains were kept in a bod incubator 411d new ethics ( nova tica indstria comrcio e servios ltda , so paulo , brazil ) at a temperature of about 25 c 2 and 60% humidity in 250 ml flasks containing a medium prepared with 820 ml of water , 11 g agar , 156 g of banana , 1 g of nipagin ( fagron do brasil farmacutica , so paulo , brazil ) and 25 g of biological yeast saccharomyces cerevisiae . two types of crosses were performed : ( 1 ) a standard cross ( st ) , in which virgin females flr/ in(3lr)tm3 , ri p sep l(3)89aa bx bd were crossed with mwh/ mwh males , and ( 2 ) a high bioactivation cross ( hb ) , with virgin females orr ; flr/ in(3lr)tm3 , ri p sep l(3)89aa bx bd crossed with mwh / mwh males . in both crosses , two types of offspring were obtained : trans - marker heterozygous ( mh ) with the ( mwh + /+ flr ) genotype and wings phenotypically of the wild type ; and heterozygous balancer ( bh ) with the ( mwh + /+ the larvae , of both genotypes from these crosses were treated with three concentrations of the chemical agent to be tested . groups of approximately 100 larvae were transferred to glass tubes ( 2.5 cm in diameter and 8.0 cm in height ) containing 1.5 g of a culture medium of instant mashed potatoes ( hikari , so paulo brazil ) and 5.0 ml of each of three concentrations of the agent to be tested . the 72-hour old larvae from both crosses ( st and hb ) were transferred to 2.5 cm 8.0 cm high glass tubes containing 1.5 g of instant mashed potatoes with three concentrations of pantoprazole : 2.5 m , 5.0 m or 10.0 m . reverse osmosis water was used as a solvent and negative control and doxorubicin ( dxr , 0.4 mm ) as a positive control . dxr was used as positive control , because in smart assays with d. melanogaster it was classified as a strong mutagen , inducing all types of wing spots ( orsolin et al . the number , types ( single or twin ) , position and size of the spots were calculated and recorded . statistical analysis of the experiment was performed using a chi square test as described by frei and wrgler ( 1988 ) . for the analysis of anti - mutagenicity , the frequencies of each type of spot were compared in pairs , using the u test ( frei and wrgler , 1995 ) at a significance level of = 0.05 . no significant decreases in the survival rates of larvae submitted to treatments were observed when compared to the negative control . table 1 shows the results of mutant spot frequencies observed in the bh and mh descendants of the standard cross ( st ) , treated with pantoprazole in three different concentrations ( 2.5 , 5.0 or 10.0 m ) . the positive control ( dxr 0.4 mm ) and negative control ( reverse osmosis water ) are also presented . table 2 shows the results of mutant spot frequencies observed in the bh and mh descendants of the high bioactivation cross ( hb ) , for the same concentrations of pantoprazole , as well as the positive and negative controls . when compared to the negative control , pantoprazole caused a significant increase in the frequency of small , simple spots at all concentrations . the total number of spots , however , was only statistically significant at 2.5 and 5.0 m . results for the hb cross in terms of the potential mutagenic properties of pantoprazole are presented in table 2 . the total number of mutant spots among the mh descendants , compared to the negative control , was significantly increased in all concentrations . the analysis of flies with the bh genotype ( mwh / tm3 ) was carried out for the purpose of calculating the portion of recombinogenic and mutagenic events . a comparison of clone - induction frequencies obtained for pantoprazole in both genotypes indicated that in st flies , 49.76% of mutant clones produced by pantoprazole were due to mutation and 50.24% due to recombination at the 2.5 m concentration . clone - induction frequencies for pantoprazole ( 5.0 m ) indicated that 52.85% of the mutant clones produced were due to mutation and 47.15% to recombination . however , the very same analysis showed that in hb flies , 31.30% of spots induced by pantoprazole ( 2.5 m ) were due to mutation and 68.70% to recombination ; 37.56% of spots induced by pantoprazole ( 5.0 m ) were due to mutation and 62.44% to recombination ; 35.48% mutant clones produced by pantoprazole ( 10 m ) were due to mutation and 64.52 to recombination . the genetic control of xenobiotic metabolism in drosophila is complex , and multiple forms of p450 as well as other enzymes ( e.g. according to our findings , pantoprazole has a clear recombinogenic potential in drosophila , and the stock differences demonstrate a strong dependence on levels of metabolic activation ( hb flies ) as the increased cytochrome p450-dependent biocativation capacity present in these hb larvae leads to significantly increased recombinogenicity . therefore , the metabolic pathway in the induction of recombinogenicity most probably involves cytochrome p450-dependent enzyme activity . ( 2010 ) showed that pantoprazole is completely metabolized by the hepatic cytochrome p450 system . kuipers ( 2006 ) has stated that prolonged use of proton pump inhibitors ( ppis ) may be related to the development of gastric cancer . it is also known that treatment with ppis does not protect against this type of cancer , but the increased risk of cancer due to prolonged use remains unknown . in contrast , people infected by h. pylori were found to have chronic , persistent gastritis , atrophy and metaplasia , which may progress to gastric atrophy , intestinal metaplasia and gastric cancer . ppis used in the treatment of this infection may also cause an acceleration of the progression or development of gastritis ( thomson et al . nonetheless , until now there is no evidence that ppis increase the risk of gastric cancer . persistent , predominant gastritis and atrophic gastritis of the gastric body mucosa , however , are considered important risk factors for the development of gastric cancer , and , clearly , more studies are needed to reach a definitive conclusion ( kuipers , 2006 ) . according to the authors , pantoprazole pretreatment enhances the cytotoxic effects of anti - tumor drugs on human gastric adenocarcinoma cells ( sgc7901 ) and reverts multidrug resistance of sgc7901/adr cells by down - regulating the v - atpases / mtor / hif-1/p - gp and mrp1 signaling pathway . ( 2013 ) , the use of pantoprazole to enhance the distribution and cytotoxicity of anticancer drugs in solid tumors might be a novel treatment strategy to improve their therapeutic indices . it provides an evaluation of mutational events , as well as recombinogenic events , as shown in the present study . ( 2010 ) reviewed the genotoxic and carcinogenic effects of 71 gastrointestinal drugs , demonstrating that pantoprazole was found to be genotoxic and carcinogenic in several assays , in addition to causing chromosomal damage . these results are consistent with those presented herein , where pantoprazole caused an increase in the frequency of mutant spots for somatic cells , revealing its genotoxic characteristics . at current , the published studies are conflicting in their results , warranting further examination by means of additional assays and test organisms . in conclusion , the present study indicates that pantoprazole possesses recombinogenic activity in the drosophila wing spot test . nonetheless , although there was an increase in mutant spots in the st descendants , the increase in recombinogenic activity was observed only in the high bioactivation ( hb ) descendants , this suggesting the interaction of their constituents ( pantoprazole ) with cytochrome p450 .

the stability of the outcome of orthodontic treatment is a major clinical concern , as retraction of periodontal fibers might cause many cases to eventually relapse , particularly after alignment of the anterior teeth of the mandible.12 many clinicians consider permanent or long - term retention to be the only way to maintain a proper post - treatment alignment.23 one method for maintaining long - term retention is to use fixed retainers that remain permanently in the mouth and are invisible , well tolerated , and do not require patient compliance after application by an orthodontist.24 nevertheless , approximately 10% to 47% of fixed retainers fail because of wire fractures or bond failures.2456 previously , fixed retainers were fabricated from thick stainless steel round wires and were later made from thinner coaxial or braided round wires.245 recently , fiber - reinforced composite ( frc ) resins were introduced as an alternative to stainless steel archwires to reduce the bulk of the lingual retainer.2789 frc retainers are flexible and able to easily conform to tooth surfaces . additionally , they are esthetically accepted , nickel - free , biocompatible , and easily repairable.210111213 however , the main disadvantage is that frc retainers produce a rigid splint that limits physiological tooth movement and may contribute to a higher failure rate.26 the development of a method for long - term retention is important for treatment stability and to prevent further problems , such as incisor crowding.21415 the most appropriate method for assessing retainer success is to perform a long - term randomized clinical trial . however , only a limited number of studies have been conducted to assess frc retainers,2131617 and few studies have extended longer than 1 year.18 furthermore , the results of previous studies do not favor any specific method , and there is no consensus on which method is the best.192021 both conventional wire retainers and frc splints have specific disadvantages . conventional retainers are made of active orthodontic wires and are thus rather technique - sensitive and time - consuming to position passively on the lingual surface . additionally , these types of retainers can either debond or exert undesirable orthodontic forces on aligned teeth . they may have higher failure rates , they are more difficult to repair , and they may cause additional periodontal conditions.26172223 therefore , a more flexible yet strong wire design might be advantageous over the abovementioned types in terms of passivity ( i.e. , theability to conform to the patient 's dentition without exerting orthodontic force ) , the likeliness of debonding , cost , and chairside time . this 2-year prospective preliminary randomized clinical trial was conducted to compare the success rate of an experimental dead soft twisted wire ( tw ) retainer , which has not been assessed previously , with frc and flexible spiral wire ( fsw ) retainers . the null hypothesis was that there would be no difference in the success rate between the three retainer types . success was defined as the lack of any failure , ranging from a single - tooth debond to a total retainer breakage . this explorative , prospective preliminary single - blind randomized clinical trial originally enrolled 150 ( 50 3 ) fixed orthodontic patients who were monitored over 2 years . the sample size was predetermined as n = 40 3 based on previous clinical research on retainers . to compensate for potential dropouts during the study period , three groups of 50 patients each the study protocol was approved by the research committee of the research department of the university , in accordance with the helsinki declaration ( ethical approval # 2318 ) . all patients ( or their parents ) were verbally informed of the purpose of the study , and they all signed routine informed consent forms . the subjects could request to change or remove the retainer ( and leave the study ) at any time during the study . the selected patients had been previously treated in a private office with standard mbt 0.022-inch ( in ) slot fixed appliances . the subjects were sequentially included based on the criteria described below and randomly assigned to one of the groups until each group reached 50 patients . we included healthy participants who provided consent and had no history of previous dental extraction or orthognathic surgery , with an original indication for non - extraction treatment ( class i crowding , incisor mandibular plane angle [ impa ] < 92 , vertically normal or horizontal and no pattern of vertical excess , and with a crowding extent and soft tissue characteristics appropriate for non - extraction treatment ) . after the treatment period , patients were included if they had ideally aligned dentition , a class i relationship with an overbite / overjet between 1 and 3 mm , no issues contraindicating or interfering with retention , clinical signs suggesting bruxism or clenching , advanced dental abrasion / attrition / erosion , and a need for mandibular canine - to - canine fixed retention . we included patients who had good oral hygiene , healthy periodontal condition , and no previous history of using bonded retainers . the probing pocket depth was assessed and radiological examinations were performed to detect any periodontal problems . patients with widespread probing depths more than 3 mm and radiographic evidence of periodontal bone loss were excluded . random allocation was accomplished by the same orthodontist who enrolled the participants using a random number table . the study was single - blind : each patient had layperson knowledge of his / her own retainer , but he / she had no knowledge ( either technically or in layperson terms ) of the other designs involved in the study . the experiments were performed using lingual canine - to - canine retainers , all bonded in a similar fashion , as described previously.2224 in the first group , 0.0175-in fsw ( ortosmail ; krugg spa , milan , italy ) was used . in the second group , an frc splint ( everstick ortho ; stick tech , turku , finland ) was used . in the third group , as described here for the first time , the retainers were fabricated by a left - handed orthodontist by carefully twisting two 0.009-in dead soft wires ( ligature wire ; 3 m unitek , monrovia , ca , usa ) using a mathieu plier to form a passive yet sufficiently strong bundle . the extent to which the wires were twisted ( the number of twists per millimeter of wire ) was similar between all specimens . the bundle was formed against the lingual surface of the anterior teeth on a dental plaster . after scaling and polishing ( using fluoride - free pumice ) the lingual surface of the mandibular anterior teeth , full isolation was carried out using cheek retractors and cotton rolls in the labial and lingual areas . the surfaces were etched with 37% phosphoric acid ( 3 m unitek ) for 40 seconds followed by 30 seconds of rinsing and then thorough air - drying with an oil - fee syringe . two layers of light - curable primer ( ormco , orange , ca , usa ) were added to the surface . each layer was light - cured for 20 seconds ( optilux 501 ; kerr corp . , orange , ca , usa ; light intensity , 930 mw / cm ; wavelength range , 400 - 505 nm ) . a layer of resin ( transbond xt ; 3 m unitek ) was applied after placement to stabilize the retainer on the lingual surface , followed by light - curing for 20 seconds . m unitek ) was placed over the retainer and light - cured for 40 seconds . finally , excess resin was removed from the interproximal areas using a scaler , and the restorations were polished.2224 each patient was instructed not to bite on hard or sticky foods with the anterior teeth to avoid disruption of the bonded retainers . each patient was followed up once a month for 2 years or until the failure of the retainer . retainer failure was defined as the first detachment of any composite pad or breakage / distortion of the retainer . debonded retainers were routinely re - bonded or replaced depending on the severity of the case . the failure time was recorded for the survival analysis . if a patient missed a follow - up session and visited the next month with an intact retainer , he / she remained in the study . however , if the retainer had failed during any period of absence lasting more than a month , the patient was excluded . the exclusion of a patient or the failure of a retainer did result in the discontinuation of treatment , as the patients continued to receive routine healthcare . the failure rates of the three groups of retainers were calculated using a kaplan - meier estimate and compared using a chi - squared test and a cox proportional hazard regression using ibm spss statistics ver . the study protocol was approved by the research committee of the research department of the university , in accordance with the helsinki declaration ( ethical approval # 2318 ) . all patients ( or their parents ) were verbally informed of the purpose of the study , and they all signed routine informed consent forms . the subjects could request to change or remove the retainer ( and leave the study ) at any time during the study . the selected patients had been previously treated in a private office with standard mbt 0.022-inch ( in ) slot fixed appliances . the subjects were sequentially included based on the criteria described below and randomly assigned to one of the groups until each group reached 50 patients . we included healthy participants who provided consent and had no history of previous dental extraction or orthognathic surgery , with an original indication for non - extraction treatment ( class i crowding , incisor mandibular plane angle [ impa ] < 92 , vertically normal or horizontal and no pattern of vertical excess , and with a crowding extent and soft tissue characteristics appropriate for non - extraction treatment ) . after the treatment period , patients were included if they had ideally aligned dentition , a class i relationship with an overbite / overjet between 1 and 3 mm , no issues contraindicating or interfering with retention , clinical signs suggesting bruxism or clenching , advanced dental abrasion / attrition / erosion , and a need for mandibular canine - to - canine fixed retention . we included patients who had good oral hygiene , healthy periodontal condition , and no previous history of using bonded retainers . the probing pocket depth was assessed and radiological examinations were performed to detect any periodontal problems . patients with widespread probing depths more than 3 mm and radiographic evidence of periodontal bone loss were excluded . random allocation was accomplished by the same orthodontist who enrolled the participants using a random number table . the study was single - blind : each patient had layperson knowledge of his / her own retainer , but he / she had no knowledge ( either technically or in layperson terms ) of the other designs involved in the study . the experiments were performed using lingual canine - to - canine retainers , all bonded in a similar fashion , as described previously.2224 in the first group , 0.0175-in fsw ( ortosmail ; krugg spa , milan , italy ) was used . in the second group , an frc splint ( everstick ortho ; stick tech , turku , finland ) was used . in the third group , as described here for the first time , the retainers were fabricated by a left - handed orthodontist by carefully twisting two 0.009-in dead soft wires ( ligature wire ; 3 m unitek , monrovia , ca , usa ) using a mathieu plier to form a passive yet sufficiently strong bundle . the extent to which the wires were twisted ( the number of twists per millimeter of wire ) was similar between all specimens . the bundle was formed against the lingual surface of the anterior teeth on a dental plaster . after scaling and polishing ( using fluoride - free pumice ) the lingual surface of the mandibular anterior teeth , full isolation was carried out using cheek retractors and cotton rolls in the labial and lingual areas . the surfaces were etched with 37% phosphoric acid ( 3 m unitek ) for 40 seconds followed by 30 seconds of rinsing and then thorough air - drying with an oil - fee syringe . two layers of light - curable primer ( ormco , orange , ca , usa ) were added to the surface . each layer was light - cured for 20 seconds ( optilux 501 ; kerr corp . , orange , ca , usa ; light intensity , 930 mw / cm ; wavelength range , 400 - 505 nm ) . a layer of resin ( transbond xt ; 3 m unitek ) was applied after placement to stabilize the retainer on the lingual surface , followed by light - curing for 20 seconds . afterwards , a layer of restorative composite ( z100 ; 3 m unitek ) was placed over the retainer and light - cured for 40 seconds . finally , excess resin was removed from the interproximal areas using a scaler , and the restorations were polished.2224 each patient was instructed not to bite on hard or sticky foods with the anterior teeth to avoid disruption of the bonded retainers . in the first group , 0.0175-in fsw ( ortosmail ; krugg spa , milan , italy ) was used . in the second group , an frc splint ( everstick ortho ; stick tech , turku , finland ) was used . in the third group , as described here for the first time , the retainers were fabricated by a left - handed orthodontist by carefully twisting two 0.009-in dead soft wires ( ligature wire ; 3 m unitek , monrovia , ca , usa ) using a mathieu plier to form a passive yet sufficiently strong bundle . the extent to which the wires were twisted ( the number of twists per millimeter of wire ) was similar between all specimens . the bundle was formed against the lingual surface of the anterior teeth on a dental plaster . after scaling and polishing ( using fluoride - free pumice ) the lingual surface of the mandibular anterior teeth , full isolation was carried out using cheek retractors and cotton rolls in the labial and lingual areas . the surfaces were etched with 37% phosphoric acid ( 3 m unitek ) for 40 seconds followed by 30 seconds of rinsing and then thorough air - drying with an oil - fee syringe . two layers of light - curable primer ( ormco , orange , ca , usa ) were added to the surface . each layer was light - cured for 20 seconds ( optilux 501 ; kerr corp . , orange , ca , usa ; light intensity , 930 mw / cm ; wavelength range , 400 - 505 nm ) . the retainers were prepared and contoured on plaster casts . a layer of resin ( transbond xt ; 3 m unitek ) was applied after placement to stabilize the retainer on the lingual surface , followed by light - curing for 20 seconds . afterwards , a layer of restorative composite ( z100 ; 3 m unitek ) was placed over the retainer and light - cured for 40 seconds . finally , excess resin was removed from the interproximal areas using a scaler , and the restorations were polished.2224 each patient was instructed not to bite on hard or sticky foods with the anterior teeth to avoid disruption of the bonded retainers . each patient was followed up once a month for 2 years or until the failure of the retainer . retainer failure was defined as the first detachment of any composite pad or breakage / distortion of the retainer . debonded retainers were routinely re - bonded or replaced depending on the severity of the case . the failure time was recorded for the survival analysis . if a patient missed a follow - up session and visited the next month with an intact retainer , he / she remained in the study . however , if the retainer had failed during any period of absence lasting more than a month , the patient was excluded . the exclusion of a patient or the failure of a retainer did result in the discontinuation of treatment , as the patients continued to receive routine healthcare . descriptive statistics were calculated for each group . the ages were compared using an analysis of variance ( anova ) . the failure rates of the three groups of retainers were calculated using a kaplan - meier estimate and compared using a chi - squared test and a cox proportional hazard regression using ibm spss statistics ver . more than 180 patients were assessed until 50 3 patients were included . of the 150 enrolled patients , 22 were removed from the study because of failure to regularly attend the follow - up sessions . finally , 42 , 41 , and 45 patients who received an frc splint , an fsw retainer , or an experimental tdw retainer , respectively , were included in the analysis . the mean age of the included patients was 18.0 3.6 years ( range , 1325 years ) . the average ages were 18.5 3.6 , 18.4 3.7 , and 17.0 3.4 years , respectively , in the frc , spiral flex , and tdw groups . the average ages were similar between the three groups , according to the anova ( p = 0.102 ) . the frc splint was used for 23 men and 19 women , the fsw retainer was used for 17 men and 24 women , and the tw retainer was used for 20 men and 25 women . the difference between the gender distributions of the groups was not significant ( chi - squared p = 0.441 ) . of the frc , fsw retainer , and tdw retainers , 15 ( 35.7% ) , 11 ( 26.8% ) , and 8 ( 17.8% ) failed , respectively . three frc retainers , one fsw retainer , and one tw retainer broke during the stud period , and the rest of the failures were caused by detachment . the chi - squared test failed to detect a significant difference in the failure rates between the groups ( p = 0.167 , table 1 ) . the average duration of success was approximately 21 months , according to the kaplan - meier estimates ( table 1 ) . the cox regression analysis showed no significant overall difference between the treatments ( p = 0.146 , figure 1 , table 2 ) , although a marginally significant difference was detected in the survival rates between the frc and tdw retainers . a hazard ratio of 2.3 indicated that the risk of failure may be two times higher in the case of frc retainers compared to tdw retainers , with a non - statistical trend ( p = 0.057 , table 2 ) . the risk of failure was approximately 50% less for tdw retainers compared to fsw retainers , though it was not statistically significant ( p = 0.317 ) . fsw retainer , and tdw retainers , 15 ( 35.7% ) , 11 ( 26.8% ) , and 8 ( 17.8% ) failed , respectively . three frc retainers , one fsw retainer , and one tw retainer broke during the stud period , and the rest of the failures were caused by detachment . the chi - squared test failed to detect a significant difference in the failure rates between the groups ( p = 0.167 , table 1 ) . the average duration of success was approximately 21 months , according to the kaplan - meier estimates ( table 1 ) . the cox regression analysis showed no significant overall difference between the treatments ( p = 0.146 , figure 1 , table 2 ) , although a marginally significant difference was detected in the survival rates between the frc and tdw retainers . a hazard ratio of 2.3 indicated that the risk of failure may be two times higher in the case of frc retainers compared to tdw retainers , with a non - statistical trend ( p = 0.057 , table 2 ) . the risk of failure was approximately 50% less for tdw retainers compared to fsw retainers , though it was not statistically significant ( p = 0.317 ) . we did not observe an overall statistically significant difference between the success rates of the three retainer types . nonetheless , the experimental retainers showed better survival results compared to the frc retainers . similar to metallic alloys , fibers might offer superior mechanical properties , while composite resins provide esthetic benefits.6222526 considering the increasing number of adult patients seeking orthodontic treatment , esthetics is now a major factor contributing to patient satisfaction.2227 frc retainers have advantages , including biocom patibility ( no metal content ) , making them safe for patients who are allergic to metals or who are undergoing mri assessments , and frc retainers can be bonded to dental tissues.2228 frcs have been intro duced in dental practice in recent years . the possibility of reinforcing composite resins with fibers such as aramid , polyethylene , carbon , or glass has numerous clinical applications , including the replacement of missing teeth , repair of complete dentures , preparation of overdenture parts , splinting of periodontal teeth , use in maryland bridges , or the direct construction of posts and cores.132229 the flexural strength of frcs might be similar to that of gold and cr - co alloys and greater than that of stainless steel.222530 in orthodontic treatment , frcs have passive and active applications , such as post - orthodontic tooth retention and anchorage unit increases.62226 glass frcs are recommended for post - orthodontic fixed lingual retention in the anterior segment.6222526 the clinical efficacy of frc retainers may be based on the internal structure of the complex . the homogeneous structure of integrated resin matrix , adhesive , and fiber might absorb and dissipate mechanical stresses.622252630 conventional retainers use mechanical retention between non - bondable materials , such as metal and composite adhesives , creating a point of weakness at the junction between different materials.6222526 multistranded fsws are broadly accepted for routine use in modern orthodontics.6172226 both frcs and multi - stranded wires used for postorthodontic retention have shown 2-year success rates of approximately 50% to 90%.17222830 in this regard , our findings are consistent with studies reporting better results with multi - stranded wires compared to frc retainers , such as polyethylene ribbon - reinforced or glass fiber - reinforced ( gfr ) retainers , in vivo1317 or in vitro.16 for instance , the average success durations were approximately 24 and 12 months for multi - stranded and frc retainers , respectively.13 another study showed significantly higher success rates for multi - stranded retainers ( 88% ) compared to gfr retainers ( 49%).17 frc retainers might have a higher failure rate because of their lower flexibility , which results in higher strain in the inter - dental areas under loading.231 this can lead to a higher probability of microfracture or debonding , particularly in the case of teeth that have become more mobile after orthodontic treatment.213173132 other chemomechanical properties , such as water sorption and thermal expansion of polyethylene materials , might also contribute to the higher failure rates of frc retainers.213 capillary forces might cause water to enter non - polymerized voids along the woven fibers and alter the mechanical characteristics.213 moreover , the lingual placement of frc , which is necessary for esthetic reasons , is not the best option to reinforce a composite.17333435 furthermore , frc retainers are technique - sensitive , which may indicate higher rate of human error.17 multi - stranded retainers have suitable efficacy and reliability ; hence , they are considered a standard treatment option in modern orthodontics.245 despite the success of multi - stranded retainers , splinting the teeth with an frc is also a popular choice.212 on the other hand , the results of the present study are in contrast to some previous studies showing insignificant differences between multi - stranded metal wires and frc retainers.22228 the discrepancies might be attributable to the use of different materials and methodologies , including differences in the method used for light curing ( light - emitting diode versus quartz - tungsten halogen ) , which might affect the polymerization , or differences in the interval for follow - up ( 6-month follow - up visits versus monthly follow - up visits).22 this study was limited by several factors . it would be preferable to include more specimens ( based on power calculations ) and to evaluate other frc designs and maxillary retainers for a longer duration . in addition , if the sample size were increased , a p - value less than 0.05 might be obtained in the comparison between frc and tdw , which resulted in a p - value of 0.057 in this study . it should be noted , however , that conducting a 2-year pilot study for a retainer type that has not been previously investigated could postpone such a project for another 2 or 3 years . moreover , although we considered numerous inclusion / exclusion criteria to ensure a uniform sample , the control of patient factors was lacking . it would be preferable to also control for chewing habits , vertical skeletal patterns , and inclination of the lower incisors , which might influence the success rate . this study was advantageous in terms of the use of standardized materials for bonding,22 the well - balanced groups and homogenous randomization of the treatments,17 and the introduction of a successful retainer , which might be more flexible than the traditional spiral wires . nevertheless , the mechanical properties of this new retainer should be assessed and optimized in follow - up in vitro studies . moreover , although the orthodontist made efforts to exert a similar force while twisting the dead wires , it was impossible to standardize this factor without the use of automatic machinery . still , the wires were twisted the same number of times per unit of length . the generalizability of this study is improved due to the recruitment of subjects of both genders and of different ages . however , the findings are only generalizable to the mandible and to the material brands used ( results achieved with a particular brand are less likely to be generalizable to other brands ) . within the limitations of this preliminary clinical trial , the three types of retainers , including the experimental retainer created by twisting two dead soft wires , appear to present similar success rates within a 2-year follow - up period . the survival rate of the experimental tw retainers might be better than that of the frc retainers , although we can not decisively conclude this from a statistical point of view , and future studies with larger samples are necessary to confirm or reject the success of this retainer and to assess the gingival response . although the superiority of the tdw retainer over the frc could not be confirmed , the results show that the tw retainer was at least equivalent to the frc .

objectivetraditional retainers ( both metal and fiber - reinforced composite [ frc ] ) have limitations , and a retainer made from more flexible ligature wires might be advantageous . we aimed to compare an experimental design with two traditional retainers.methodsin this prospective preliminary clinical trial , 150 post - treatment patients were enrolled and randomly divided into three groups of 50 patients each to receive mandibular canine - to - canine retainers made of frc , flexible spiral wire ( fsw ) , and twisted wire ( tw ) . the patients were monitored monthly . the time at which the first signs of breakage / debonding were detected was recorded . the success rates of the retainers were compared using chi - squared , kaplan - meier , and cox proportional - hazard regression analyses ( = 0.05).resultsin total , 42 patients in the frc group , 41 in the fsw group , and 45 in the tw group completed the study . the 2-year failure rates were 35.7% in the frc group , 26.8% in the fsw group , and 17.8% in the tw group . these rates differed insignificantly ( chi - squared p = 0.167 ) . according to the kaplan - meier analysis , failure occurred at 19.95 months in the frc group , 21.37 months in the fsw group , and 22.36 months in the tw group . the differences between the survival rates in the three groups were not significant ( cox regression p = 0.146).conclusionsalthough the failure rate of the experimental retainer was two times lower than that of the frc retainer , the difference was not statistically significant . the experimental tw retainer was successful , and larger studies are warranted to verify these results .

INTRODUCTION MATERIALS AND METHODS Ethical considerations and potential harms Subject screening and eligibility criteria Randomization and blinding Interventions Retainer types Uniform bonding protocols Follow-up and retainer failure Statistical analysis RESULTS Failure rates Duration of successful retainer use DISCUSSION CONCLUSION

the stability of the outcome of orthodontic treatment is a major clinical concern , as retraction of periodontal fibers might cause many cases to eventually relapse , particularly after alignment of the anterior teeth of the mandible.12 many clinicians consider permanent or long - term retention to be the only way to maintain a proper post - treatment alignment.23 one method for maintaining long - term retention is to use fixed retainers that remain permanently in the mouth and are invisible , well tolerated , and do not require patient compliance after application by an orthodontist.24 nevertheless , approximately 10% to 47% of fixed retainers fail because of wire fractures or bond failures.2456 previously , fixed retainers were fabricated from thick stainless steel round wires and were later made from thinner coaxial or braided round wires.245 recently , fiber - reinforced composite ( frc ) resins were introduced as an alternative to stainless steel archwires to reduce the bulk of the lingual retainer.2789 frc retainers are flexible and able to easily conform to tooth surfaces . additionally , they are esthetically accepted , nickel - free , biocompatible , and easily repairable.210111213 however , the main disadvantage is that frc retainers produce a rigid splint that limits physiological tooth movement and may contribute to a higher failure rate.26 the development of a method for long - term retention is important for treatment stability and to prevent further problems , such as incisor crowding.21415 the most appropriate method for assessing retainer success is to perform a long - term randomized clinical trial . however , only a limited number of studies have been conducted to assess frc retainers,2131617 and few studies have extended longer than 1 year.18 furthermore , the results of previous studies do not favor any specific method , and there is no consensus on which method is the best.192021 both conventional wire retainers and frc splints have specific disadvantages . they may have higher failure rates , they are more difficult to repair , and they may cause additional periodontal conditions.26172223 therefore , a more flexible yet strong wire design might be advantageous over the abovementioned types in terms of passivity ( i.e. , theability to conform to the patient 's dentition without exerting orthodontic force ) , the likeliness of debonding , cost , and chairside time . this 2-year prospective preliminary randomized clinical trial was conducted to compare the success rate of an experimental dead soft twisted wire ( tw ) retainer , which has not been assessed previously , with frc and flexible spiral wire ( fsw ) retainers . the null hypothesis was that there would be no difference in the success rate between the three retainer types . this explorative , prospective preliminary single - blind randomized clinical trial originally enrolled 150 ( 50 3 ) fixed orthodontic patients who were monitored over 2 years . to compensate for potential dropouts during the study period , three groups of 50 patients each the study protocol was approved by the research committee of the research department of the university , in accordance with the helsinki declaration ( ethical approval # 2318 ) . all patients ( or their parents ) were verbally informed of the purpose of the study , and they all signed routine informed consent forms . the subjects were sequentially included based on the criteria described below and randomly assigned to one of the groups until each group reached 50 patients . after the treatment period , patients were included if they had ideally aligned dentition , a class i relationship with an overbite / overjet between 1 and 3 mm , no issues contraindicating or interfering with retention , clinical signs suggesting bruxism or clenching , advanced dental abrasion / attrition / erosion , and a need for mandibular canine - to - canine fixed retention . the study was single - blind : each patient had layperson knowledge of his / her own retainer , but he / she had no knowledge ( either technically or in layperson terms ) of the other designs involved in the study . the experiments were performed using lingual canine - to - canine retainers , all bonded in a similar fashion , as described previously.2224 in the first group , 0.0175-in fsw ( ortosmail ; krugg spa , milan , italy ) was used . in the second group , an frc splint ( everstick ortho ; stick tech , turku , finland ) was used . in the third group , as described here for the first time , the retainers were fabricated by a left - handed orthodontist by carefully twisting two 0.009-in dead soft wires ( ligature wire ; 3 m unitek , monrovia , ca , usa ) using a mathieu plier to form a passive yet sufficiently strong bundle . after scaling and polishing ( using fluoride - free pumice ) the lingual surface of the mandibular anterior teeth , full isolation was carried out using cheek retractors and cotton rolls in the labial and lingual areas . finally , excess resin was removed from the interproximal areas using a scaler , and the restorations were polished.2224 each patient was instructed not to bite on hard or sticky foods with the anterior teeth to avoid disruption of the bonded retainers . each patient was followed up once a month for 2 years or until the failure of the retainer . retainer failure was defined as the first detachment of any composite pad or breakage / distortion of the retainer . debonded retainers were routinely re - bonded or replaced depending on the severity of the case . the failure time was recorded for the survival analysis . if a patient missed a follow - up session and visited the next month with an intact retainer , he / she remained in the study . the exclusion of a patient or the failure of a retainer did result in the discontinuation of treatment , as the patients continued to receive routine healthcare . the failure rates of the three groups of retainers were calculated using a kaplan - meier estimate and compared using a chi - squared test and a cox proportional hazard regression using ibm spss statistics ver . the study protocol was approved by the research committee of the research department of the university , in accordance with the helsinki declaration ( ethical approval # 2318 ) . all patients ( or their parents ) were verbally informed of the purpose of the study , and they all signed routine informed consent forms . the subjects were sequentially included based on the criteria described below and randomly assigned to one of the groups until each group reached 50 patients . after the treatment period , patients were included if they had ideally aligned dentition , a class i relationship with an overbite / overjet between 1 and 3 mm , no issues contraindicating or interfering with retention , clinical signs suggesting bruxism or clenching , advanced dental abrasion / attrition / erosion , and a need for mandibular canine - to - canine fixed retention . the study was single - blind : each patient had layperson knowledge of his / her own retainer , but he / she had no knowledge ( either technically or in layperson terms ) of the other designs involved in the study . the experiments were performed using lingual canine - to - canine retainers , all bonded in a similar fashion , as described previously.2224 in the first group , 0.0175-in fsw ( ortosmail ; krugg spa , milan , italy ) was used . in the second group , an frc splint ( everstick ortho ; stick tech , turku , finland ) was used . in the third group , as described here for the first time , the retainers were fabricated by a left - handed orthodontist by carefully twisting two 0.009-in dead soft wires ( ligature wire ; 3 m unitek , monrovia , ca , usa ) using a mathieu plier to form a passive yet sufficiently strong bundle . after scaling and polishing ( using fluoride - free pumice ) the lingual surface of the mandibular anterior teeth , full isolation was carried out using cheek retractors and cotton rolls in the labial and lingual areas . finally , excess resin was removed from the interproximal areas using a scaler , and the restorations were polished.2224 each patient was instructed not to bite on hard or sticky foods with the anterior teeth to avoid disruption of the bonded retainers . in the first group , 0.0175-in fsw ( ortosmail ; krugg spa , milan , italy ) was used . in the second group , an frc splint ( everstick ortho ; stick tech , turku , finland ) was used . in the third group , as described here for the first time , the retainers were fabricated by a left - handed orthodontist by carefully twisting two 0.009-in dead soft wires ( ligature wire ; 3 m unitek , monrovia , ca , usa ) using a mathieu plier to form a passive yet sufficiently strong bundle . after scaling and polishing ( using fluoride - free pumice ) the lingual surface of the mandibular anterior teeth , full isolation was carried out using cheek retractors and cotton rolls in the labial and lingual areas . the retainers were prepared and contoured on plaster casts . finally , excess resin was removed from the interproximal areas using a scaler , and the restorations were polished.2224 each patient was instructed not to bite on hard or sticky foods with the anterior teeth to avoid disruption of the bonded retainers . each patient was followed up once a month for 2 years or until the failure of the retainer . retainer failure was defined as the first detachment of any composite pad or breakage / distortion of the retainer . debonded retainers were routinely re - bonded or replaced depending on the severity of the case . the failure time was recorded for the survival analysis . if a patient missed a follow - up session and visited the next month with an intact retainer , he / she remained in the study . the exclusion of a patient or the failure of a retainer did result in the discontinuation of treatment , as the patients continued to receive routine healthcare . the ages were compared using an analysis of variance ( anova ) . the failure rates of the three groups of retainers were calculated using a kaplan - meier estimate and compared using a chi - squared test and a cox proportional hazard regression using ibm spss statistics ver . of the 150 enrolled patients , 22 were removed from the study because of failure to regularly attend the follow - up sessions . finally , 42 , 41 , and 45 patients who received an frc splint , an fsw retainer , or an experimental tdw retainer , respectively , were included in the analysis . the average ages were 18.5 3.6 , 18.4 3.7 , and 17.0 3.4 years , respectively , in the frc , spiral flex , and tdw groups . the average ages were similar between the three groups , according to the anova ( p = 0.102 ) . the frc splint was used for 23 men and 19 women , the fsw retainer was used for 17 men and 24 women , and the tw retainer was used for 20 men and 25 women . the difference between the gender distributions of the groups was not significant ( chi - squared p = 0.441 ) . of the frc , fsw retainer , and tdw retainers , 15 ( 35.7% ) , 11 ( 26.8% ) , and 8 ( 17.8% ) failed , respectively . three frc retainers , one fsw retainer , and one tw retainer broke during the stud period , and the rest of the failures were caused by detachment . the chi - squared test failed to detect a significant difference in the failure rates between the groups ( p = 0.167 , table 1 ) . the average duration of success was approximately 21 months , according to the kaplan - meier estimates ( table 1 ) . the cox regression analysis showed no significant overall difference between the treatments ( p = 0.146 , figure 1 , table 2 ) , although a marginally significant difference was detected in the survival rates between the frc and tdw retainers . a hazard ratio of 2.3 indicated that the risk of failure may be two times higher in the case of frc retainers compared to tdw retainers , with a non - statistical trend ( p = 0.057 , table 2 ) . the risk of failure was approximately 50% less for tdw retainers compared to fsw retainers , though it was not statistically significant ( p = 0.317 ) . fsw retainer , and tdw retainers , 15 ( 35.7% ) , 11 ( 26.8% ) , and 8 ( 17.8% ) failed , respectively . three frc retainers , one fsw retainer , and one tw retainer broke during the stud period , and the rest of the failures were caused by detachment . the chi - squared test failed to detect a significant difference in the failure rates between the groups ( p = 0.167 , table 1 ) . the average duration of success was approximately 21 months , according to the kaplan - meier estimates ( table 1 ) . the cox regression analysis showed no significant overall difference between the treatments ( p = 0.146 , figure 1 , table 2 ) , although a marginally significant difference was detected in the survival rates between the frc and tdw retainers . a hazard ratio of 2.3 indicated that the risk of failure may be two times higher in the case of frc retainers compared to tdw retainers , with a non - statistical trend ( p = 0.057 , table 2 ) . the risk of failure was approximately 50% less for tdw retainers compared to fsw retainers , though it was not statistically significant ( p = 0.317 ) . we did not observe an overall statistically significant difference between the success rates of the three retainer types . nonetheless , the experimental retainers showed better survival results compared to the frc retainers . similar to metallic alloys , fibers might offer superior mechanical properties , while composite resins provide esthetic benefits.6222526 considering the increasing number of adult patients seeking orthodontic treatment , esthetics is now a major factor contributing to patient satisfaction.2227 frc retainers have advantages , including biocom patibility ( no metal content ) , making them safe for patients who are allergic to metals or who are undergoing mri assessments , and frc retainers can be bonded to dental tissues.2228 frcs have been intro duced in dental practice in recent years . the possibility of reinforcing composite resins with fibers such as aramid , polyethylene , carbon , or glass has numerous clinical applications , including the replacement of missing teeth , repair of complete dentures , preparation of overdenture parts , splinting of periodontal teeth , use in maryland bridges , or the direct construction of posts and cores.132229 the flexural strength of frcs might be similar to that of gold and cr - co alloys and greater than that of stainless steel.222530 in orthodontic treatment , frcs have passive and active applications , such as post - orthodontic tooth retention and anchorage unit increases.62226 glass frcs are recommended for post - orthodontic fixed lingual retention in the anterior segment.6222526 the clinical efficacy of frc retainers may be based on the internal structure of the complex . the homogeneous structure of integrated resin matrix , adhesive , and fiber might absorb and dissipate mechanical stresses.622252630 conventional retainers use mechanical retention between non - bondable materials , such as metal and composite adhesives , creating a point of weakness at the junction between different materials.6222526 multistranded fsws are broadly accepted for routine use in modern orthodontics.6172226 both frcs and multi - stranded wires used for postorthodontic retention have shown 2-year success rates of approximately 50% to 90%.17222830 in this regard , our findings are consistent with studies reporting better results with multi - stranded wires compared to frc retainers , such as polyethylene ribbon - reinforced or glass fiber - reinforced ( gfr ) retainers , in vivo1317 or in vitro.16 for instance , the average success durations were approximately 24 and 12 months for multi - stranded and frc retainers , respectively.13 another study showed significantly higher success rates for multi - stranded retainers ( 88% ) compared to gfr retainers ( 49%).17 frc retainers might have a higher failure rate because of their lower flexibility , which results in higher strain in the inter - dental areas under loading.231 this can lead to a higher probability of microfracture or debonding , particularly in the case of teeth that have become more mobile after orthodontic treatment.213173132 other chemomechanical properties , such as water sorption and thermal expansion of polyethylene materials , might also contribute to the higher failure rates of frc retainers.213 capillary forces might cause water to enter non - polymerized voids along the woven fibers and alter the mechanical characteristics.213 moreover , the lingual placement of frc , which is necessary for esthetic reasons , is not the best option to reinforce a composite.17333435 furthermore , frc retainers are technique - sensitive , which may indicate higher rate of human error.17 multi - stranded retainers have suitable efficacy and reliability ; hence , they are considered a standard treatment option in modern orthodontics.245 despite the success of multi - stranded retainers , splinting the teeth with an frc is also a popular choice.212 on the other hand , the results of the present study are in contrast to some previous studies showing insignificant differences between multi - stranded metal wires and frc retainers.22228 the discrepancies might be attributable to the use of different materials and methodologies , including differences in the method used for light curing ( light - emitting diode versus quartz - tungsten halogen ) , which might affect the polymerization , or differences in the interval for follow - up ( 6-month follow - up visits versus monthly follow - up visits).22 this study was limited by several factors . in addition , if the sample size were increased , a p - value less than 0.05 might be obtained in the comparison between frc and tdw , which resulted in a p - value of 0.057 in this study . it would be preferable to also control for chewing habits , vertical skeletal patterns , and inclination of the lower incisors , which might influence the success rate . this study was advantageous in terms of the use of standardized materials for bonding,22 the well - balanced groups and homogenous randomization of the treatments,17 and the introduction of a successful retainer , which might be more flexible than the traditional spiral wires . however , the findings are only generalizable to the mandible and to the material brands used ( results achieved with a particular brand are less likely to be generalizable to other brands ) . within the limitations of this preliminary clinical trial , the three types of retainers , including the experimental retainer created by twisting two dead soft wires , appear to present similar success rates within a 2-year follow - up period . the survival rate of the experimental tw retainers might be better than that of the frc retainers , although we can not decisively conclude this from a statistical point of view , and future studies with larger samples are necessary to confirm or reject the success of this retainer and to assess the gingival response . although the superiority of the tdw retainer over the frc could not be confirmed , the results show that the tw retainer was at least equivalent to the frc .

in the early days of renal transplantation , preexisting dsa caused hyperacute rejections within minutes to hours with destruction of the graft 3 . histologic evaluation disclosed glomerulitis ( g ) and ptcitis ( ptc ) that involved predominantly polymorphonuclear leukocytes ( pmns ) . with the advent of the t - cell cross - match in 1969 , hyperacute rejections became rare , and the emphasis became the prevention and treatment for cell - mediated rejections ( cmr ) . in 1990 , halloran et al . 4 placed the focus back toward antibodies by describing seven patients with very early acute rejections ( ar ) in the presence of positive t - cell cytotoxic cross - matches . these rejections were viewed as very early aamr and ascribed to preformed class i dsa . the main inflammatory cell appeared to be the pmn , as in hyperacute rejection 4,5 . with the discovery of ptc c4d staining as a marker of alloantibody injury and its application in acute and chronic situations , it became evident that mononuclear cells were more prominent than pmns in biopsies diagnosed with aamr 6 . current banff criteria for diagnosing aamr include dsa , c4d , and the morphological alterations summarized in table1 that include mvi , g and/or ptc 2 . if only dsa or c4d are positive , mvi allows a diagnosis of presumptive aamr . hence , a biopsy with any degree of mvi ( theoretically one inflamed glomerulus ) with detectable dsa or c4d has presumptive aamr . with regard to g ( table2 ) 7 , current banff criteria do not detail the minimum number of cells / glomerulus required , but mononuclear cells and endothelial cell swelling are specified . regarding ptc ( table3 ) 8 , 10% of ptcs must be inflamed with at least three to three inflammatory cells . morphologic evidence of aamr ( type / grade ) aamr , acute antibody - mediated rejection . adapted from 2 . banff quantitative criteria for glomerulitis ( g ) score minimum number of cells required for consideration not specified . banff quantitative criteria for peritubular capillaritis ( ptc ) ptc , peritubular capillaries . note the composition ( mononuclear vs. neutrophils ) and extent : 50% ( focal ) vs. > 50% ( diffuse ) . adapted from 8 . as noted above , g in hyperacute rejections and very early aamr may involve pmns . although a criterion for the diagnosis of aamr , the specificity of g is not 100% . seven patients with ar and positive ptc c4d staining ( c4d - pos ) had significantly higher glomerular monocytes ( mo ) and pmns , as compared to 10 with negative staining ( c4d - neg ) ar and 10 with no ar . similarly , magil and tinckham compared 23 early biopsies ( less than six months ) with diffuse c4d - pos ar with acute tubular injury to 28 with c4d - neg cmr 10 . pmns were detectable in the glomeruli of 96% and 93% of both groups , as well as 100% of ptc of both groups . distinguishing features included presence of g ( 57% in c4d - pos vs. 11% in c4d - neg ) and the presence of glomerular mo ( 96% vs. 43% ) . subsequently , magil studied 42 patients with g and ar in the first six months 11 . in the 20 c4d - pos patients , mo predominated , and in the 22 c4d - neg cases , t cells ( tc ) did . the mean glomerular mo / tc ratio was > 1.0 in 75% of c4d - pos vs. 14% of c4d - neg rejections ( p < 0.0001 ) . studying 96 patients with ar in the first 12 months , this group again noted this correlation : 83% of biopsies with ( mean ) 1 mo / glomerulus ( deemed high ) were c4d - pos vs. 11% of those with a ratio < 1 , and 78% vs. 18% had banff criteria for g , respectively 12 . a high mo count was predictive of one- and three - yr egfr by multivariable analysis , and this obtained whether c4d - pos or c4d - neg . these data indicate that using c4d as a marker of humoral activity , a strong but not absolute correlation exists between antibodies and g , especially g involving mo in early ( less than one yr ) biopsies . cell - mediated mechanisms as evidenced by cd3-positive staining , however , result in g in c4d - neg cases and may contribute in c4d - pos ones . in a study of 240 biopsies obtained after one yr or more , papadimitriou et al . found those in the upper quartile of maximum number of mo in the most involved glomerulus ( > 12 ) were more likely to have dsa , c4d - pos , tg , and the worst allograft survival ( gs ) . indeed , the maximum mo count was the only factor significant for gs on multivariable analysis 13 . acute cmr in this late cohort did not affect the composition of cells in g. unlike previous data from earlier biopsies 11 , the mo / tc ratio was not different in c4d - pos vs. c4d - neg cases . in a later study of 1101 biopsies , pure cmr did not have more g ( or ptc ) than dsa negative ( dsa - neg)/c4d - neg biopsies without any rejection ( combined g + ptc > 0 in 6% vs. 0.6% ) 14 . 15 studied 111 biopsies with at least interstitial inflammation obtained at a mean of 504 d. using strict criteria ( minimum 5 leukocytes / glomerulus to be considered positive ) , 44 were graded g0 by banff . even with this stricter criterion than banff s , 47% of dsa - neg , c4d - neg cmrs had g>0 , and 62% of dsa - neg , c4d - neg borderline cmrs did as well . of the 36 with g>0 , 22 had c4d - neg cmr and 10 had cmr that was suspicious for aamr ( c4d - pos ) . these data reinforce those of magil 11 that cmr in the absence of detectable antibodies can produce g. gibson et al . overall , most cases were moderate ( ptc 2 ) , focal ( involving 1050% of ptc ) and mononuclear predominant . there was a strong correlation of ptc with g ( if g > 0 , then ptc > 0 in over 80% ) and c4d ( 75% had ptc > 0 if c4d - pos vs. 24% if c4d - neg ) . interestingly , 68% of 76 biopsies with 1a cmr had ptc > 0 as did 45% of 105 with borderline rejections . furthermore , 14% of biopsies without any rejection ( aamr or cmr ) had ptc 1 . 18 studied 42 biopsies , comparing 10 with pure cmr in the absence of g with 32 having g : 13 c4d - neg and 19 c4d - pos . no significant difference in ptc total cell count , mononuclear count ( mo + tc ) , or pmn count existed between the three groups . the ptc mo / tc ratio was significantly higher in c4d - pos g cases ( 2.3 ) than c4d - neg g cases ( 1 , p = 0.0008 ) and pure cmr ones ( 0.9 , p = 0.0014 ) , results mirroring the glomerular findings of magil 11 , but unlike those of papadimitriou 13 . these data support the concept that cell - mediated mechanisms produce mvi without the help of antibodies . several groups have studied the prognostic significance of mvi . among 878 transplants , cosio et al . 19 found by multivariable analysis that c4d - neg ar ( presumably cmr ) was only associated with reduced gs if g or ptc was present . in the absence of mvi 20 studied 58 conventional risk transplants with development of de novo dsa ( dndsa ) who had indication biopsies . despite dndsa and an indication for the biopsy six ( 23% ) of the 26 were c4d - pos and none had cmr . as the degree of mvi increased ( g + ptc = 1 or 2 , or g + ptc 3 ) , c4d - pos increased ( 29% and 64% respectively ) , as well as cmr ( 33% and 73% ) , suggesting significant contributions from both arms of adaptive immunity in the development of mvi . using mvi as a predictor of gs , the area under the curve in roc analysis was 0.84 , better than that for c4d ( p = 0.006 ) . mvi ( g + ptc > 0 ) in early biopsies ( less than one yr ) was non - specific , being found in aamr , cmr , and atn . only in the presence of c4d - pos was mvi likely to indicate dsa at this early time . in later biopsies , this group has also demonstrated a role for nk cells in antibody - mediated cases 22 , an observation supported by experimental studies 23 . in summary , mvi is found in aamr . in hyperacute rejections and some that occur very early , most commonly the inflammatory infiltrate , however , is mononuclear , especially mo and possibly nk cells . such mvi can be ascribed to amr in the presence of both c4d - pos and dsa - pos . acute cmr results in mvi as well , both g and/or ptc . in the presence of cmr and non - alloimmune mechanisms such as atn may result in mvi in the absence of any rejection . in the future , identification of pathogenesis - based transcripts ( pbts ) specific for the type of injury may determine the predominant cause 24,25 , along with cell type ( mo vs. tc , perhaps nk - cell ) . in any case , these chronic changes are considered synonymous with antibody causation and either is considered sufficient tissue injury required for diagnosis of caamr by banff . transplant glomerulopathy is diagnosed by light microscopy when 10% of capillary loops in the most affected glomerulus have double contours ( table4 ) 7 . by em , at least three capillary loops should show gbm reduplication 13 , and probably over 90% have associated ptcbmml 26 . although it may be clinically silent , clinical manifestations include declining gfr , proteinuria , and hypertension 27 . in a large series of conventional transplants undergoing protocol biopsies , tg was detected in 4% at one yr , increasing to 20% by five yr 28 . in higher - risk , live donor transplants performed against a positive cross - match , the one - yr incidence was 22% 29 . banff quantitative criteria for transplant glomerulopathy ( cg ) number of glomeruli and percentage sclerotic . adapted from 7 . evidence indicates that chronic antibody - mediated injury results in tg . preformed hla antibodies , especially if dsa and class ii , correlate with future development of tg . among 582 transplants , the presence of class ii antibodies increased the hazard ratio ( hr ) for tg by 5 , as compared to those without any class ii antibodies . the frequency of their occurrence , however , has varied considerably in the published series . these include 0% of 22 biopsies 30 , 15% of 36 31 , 24% of 68 32 , 36% of 53 26 , 48% of 25 33 , 55% of 58 34 , and 67% of 59 6 . where assessed , these c4d - pos cases had detectable dsa , as did some of the c4d - neg ones . overall , 2560% of these tg cases , however , lacked any evidence of antibody mediation ( both hla / dsa - neg and c4d - neg ) 26,30,31 . other potential causes of tg include cmr , thrombotic microangiopathy ( tma ) , and chronic hepatitis c virus ( hcv ) infection . as noted above , cmr results in g and ptc . such mvi precedes or coexists with tg . whereas both aamr and subclinical amr 35 can significantly increase the risk of tg , most cases are not preceded by such events 32 . in one series of 73 patients , furthermore , acute cmr more commonly precedes tg than does aamr 26,28 , although the exact significance of this remains uncertain ( see below ) . for example , in the series of 582 transplants noted above , prior cmr predominated ( 15 , including nine borderline ) compared with aamr ( three cases ) 28 . in the series of sis et al . 26 , prior ar occurred in 20 of 37 patients with rejection history . among 35 biopsies in these 20 , cmr existed in 74% ( only one of which was borderline ) , as compared to 26% with aamr . 36 compared six biopsies with tg to 17 with chronic allograft nephropathy ( can , now referred to as interstitial fibrosis / tubular atrophy , or if / ta ) . all tg biopsies had glomerular staining for the costimulatory molecule icos and the chemokine receptor cxcr3 with its ligand mig , findings that indicate the presence of activated t cells . 37 found increased mrna expression for interferon - gamma ( infg ) , the th1 transcription factor t - bet , and the cytotoxic t - lymphocyte cytokine granzyme - b in the glomeruli of 22 patients with tg as compared to 18 with can alone . similarly , sun et al . 38 demonstrated significantly increased t - bet expression by immunohistochemistry in glomeruli and ptc of 32 patients with tg , as compared to 23 with if / ta and 15 stable grafts . 39 compared intragraft gene expression in 22 positive cross - match transplants with tg to 10 conventional dsa - neg controls . despite the antibody involvement , gene expression profiles of pbts associated with cell - mediated immunity were significantly enriched , including those for cytotoxic t lymphocytes and infg . finally , we retrospectively studied 50 consecutive biopsies with tg at thomas jefferson university hospital . only 45% had detectable dsa by luminex solid phase assay ( spa ) , and only 14% were c4d - pos . surprisingly , 49 of 50 had lymphocytic infiltration of fibrous intimal thickening , thereby satisfying banff criteria for chronic active cmr ( filippone et al . double contours are found by light microscopy ( lm ) , and the ultrastructural changes are also similar to tg 33,40,41 . pancreas ( spk ) patients who developed tg to those of eight spk patients that did not . within one yr , swelling and vacuolization of endothelial cells with loss of their fenestrations was observed in those destined to develop tg . they also had a widened subendothelial space with accumulation of flocculent electron - lucent material . haas and mirocha 43 noted similar early changes ( endothelial cell swelling , subendothelial widening , and gbm duplication ) in three - month indication biopsies , many of which demonstrated tg on subsequent biopsies . these early and later changes of tg are also those of acute and chronic tma . post - transplantation tma may represent recurrent disease , for example inherited complement regulatory protein abnormalities , or arise de novo 40 . these latter cases may develop very early ( even within one wk ) and have been ascribed to calcineurin - inhibitor ( cni ) toxicity 44 . post - transplantation tma is often graft limited , but about 25% of cases have systemic features , including those of a microangiopathic hemolytic anemia 41 . 41 studied 59 patients with de novo tma and found diffuse c4d - pos in 55% . of note , 13% of c4d - pos biopsies evidenced tma no significant difference in the severity of tg between c4d - pos and c4d - neg groups was noted , although greater mvi was observed in the former . in 37 tma cases ( of 1101 biopsies ) , only six were c4d - pos . the incidence of tma was the same in c4d - pos and c4d - neg biopsies overall ( 3.4% of each ) 46 . if the biopsy occurred at 90 d , however , the probability of observing tma in c4d - pos biopsies was significantly greater than in c4d - neg ones . by contrast , after 90 d , tma was less frequent in c4d - pos biopsies 46 . they found 48% to be c4d - pos ; 32% had tma , and 36% were hcv positive . significant overlap existed between the presence of tma and hcv : five had both tma and hcv , as compared to three with just tma and four with just hcv . a significant association of hcv and tg was found by others 28,47 , although not all 48 . 47 found positive serology for hcv in 29% of 41 patients with g and 33% of 27 patients with tg , as compared to 1.8% of 105 patients with neither ( p = 0.0004 for both comparisons ) . 28 noted a significant association by multivariable analysis between hcv and development of tg in 582 conventional transplants with a hr of 2.9 ( p = 0.01 ) . ptcbmml alone represents sufficient tissue injury for a diagnosis of caamr per banff if both dsa - pos and c4d - pos . the minimum requirements to diagnose ptcbmml , however , are not well specified by banff . in an important paper , this required circumferential involvement ( > 75% of the circumference of a ptc ) with the most involved capillary having 7 layers or the three most involved capillaries having 56 layers . such a lesion had a sensitivity for chronic rejection ( defined as arterial fibrous intimal thickening or tg ) of 59% , a number similar to the 57% reported in an earlier series 50 . notably , lm evidence of acute cmr was found in 78% of cases , which suggested that cpc is attributable to slowly progressive , smoldering cmr . severe ptcbmml ( equivalent to cpc ) was rarely found in native kidneys with the exception of late tma . in transplants , this lesion lacked specificity , as it was found in 20% of c4d - neg acute cmr , 36% of cases felt to have cni toxicity , and 67% of c4d - neg chronic active cmr . finally , cpc was found in approximately half of antibody - mediated cases ( aamr / caamr ) . hence , although ptcbmml is usually found together with tg , neither of these lesions is specific for antibody causation . the immunologic destruction of a renal allograft involves a complex interplay between innate and adaptive immune systems ( recent review 52 ) . the methodology for antibody detection is constantly evolving , and consensus guidelines have recently been published 53 . here , we have detailed the lack of specificity of histologic and ultrastructural features elicited by humoral injury , although part of the discrepancies noted relate to variability between laboratories in the methodology and proficiency in determining dsa and c4d . in addition , interobserver discrepancy in the diagnosis of cell - mediated rejection is significant . for example , with three pathologists evaluating the same 245 biopsies , agreement between any two was only 45% with regard to a diagnosis of tcmr or mixed t - cell- or antibody - mediated rejection 25 . these issues reduce both the sensitivity and specificity of associated morphologic features of any diagnostic category . alloantibodies are considered the prime mediators of chronic rejection . both cell - mediated immunity and antibodies , however , are capable of producing the acute and chronic lesions discussed above ( table5 ) . treatment of cell - mediated and antibody - mediated rejections differ . in the former , steroids with the possible addition of thymoglobulin would be in order , possibly with increased chronic therapy . in the latter , and despite a dearth of high - level evidence 54 , plasmapheresis , ivig , and steroids are used , with consideration of rituximab , bortezomib , thymoglobulin , eculizumab , and possibly splenectomy depending on the circumstances . more difficult is the case with features of both , such as a c4d - neg 1a cmr with significant mvi and dsa . manuscript in preparation . includes borderline acmr . at the present time , no gold standard exists to identify the predominant pathophysiologic mechanism of allograft injury . such antibodies may simply indicate a more aggressive alloimmune diathesis and are not causally related to the injury in a particular case . dsa frequently exist , however , without c4d , and the reverse may occur . among late ( seven yr ) biopsies for allograft dysfunction , 40 were double positive , 74 cases double negative , but 28 had c4d alone , and 31 had dsa alone . the presence of c4d - pos correlated with reduced gs , whereas dsa alone did not 56 . they may have been present earlier as initiators of disease but not required to sustain the injury . such a situation exists in caamr for antibodies against agrin 57 , glutathione - s - transferase t1 58 , and peroxisomal - trans-2-enoyl - coa - reductase 59 . dsa can occur in the absence of c4d for several reasons and still be pathogenic . c4d staining may be fleeting , with cases changing from positive to negative in days to weeks 60 . experimental and clinical evidence indicates that antibodies via fc receptors recruit nk cells and macrophages that injure tissue 23 . for example , hla antibodies can induce endothelial cell exocytosis of p - selectin and induce inflammation in the absence of complement 61 . more importantly , in the absence of c4d , dsa may be present but not pathogenic . the current trend is to label such cases as c4d - neg antibody - mediated rejection 63 , although by banff they would be presumptive amr . given the non - specificity of mvi and tg / ptcbmml , it is critical to determine whether dsa are pathogenic in a particular patient . preformed dsa , even at low levels detectable only by spa , are associated with adverse outcomes 64 . this is not always the case , however , as they can frequently wane or disappear over time , either spontaneously 65 or after desensitization 66 . 65 prospectively followed 69 non - desensitized flow cross - match ( fcxm)-positive patients with spas . within one yr , two - thirds showed elimination of fcxm positivity and had no dsa by spa . within one yr , 52% lost dsa , and another 30% had reductions in the number or strength of their dsas . when persistent , dsa may not always cause detectable harm 67 . 67 compared 34 non - desensitized patients with excellent graft function after one yr to 130 patients with decreased gfr and/or proteinuria . all five maintained excellent graft function ( egfr > 60 ) for at least five more yr , including one with persistingly positive fcxms ( both t- and b - cell ) . at present , the best evidence for the pathogenicity of a given dsa , aside from its ability to fix complement ( c4d+ ) , is a rising and high titer : prior to transplantation a high titer predicts a positive cross - match 68 ; in the early post - transplant period , rising titers predict aamr 69 ; and later , they predict graft failure 70 . as well as being preformed , dsa may develop de novo ( dndsa ) in up to 25% of cases 71 . this may occur late ( after one yr ) 7173 , although earlier appearance has been reported 7476 . some of the very early cases may represent activation of memory b - cells detectable at the time of transplantation by hla - specific tetramer staining in the absence of circulating antibody 78 . a significant number of patients tolerate their presence , however , without obvious adverse outcomes . as with tg , dndsa may follow cmr 73,74 . in one study with protocol screening , dndsa rarely preceded ar ( 3 of 19 with ar ) , but usually occurred concurrently or following the rejection 74 . furthermore , detection of dndsa had a positive predictive value of only 6% for subsequent ar . do these antibodies detectable following cmr then assume prime importance for further graft injury ? or are they merely epiphenomena with smoldering cmr the real culprit ? dndsa are most troublesome when found in the setting of late graft dysfunction in a non - adherent patient , where aamr and cmr may coexist 63,73 . the ability to detect the complement fixation capability of dsa in vitro by spa ( e.g. , c1q or c4d positivity on luminex beads ) may help predict development of aamr or more chronic issues such as tg and late graft failure 7981 . most promisingly , the ability to differentiate the specific cause of issue injury by evaluation of pbts may allow determination that antibodies are the proximate cause 22,24,25 . unfortunately , this kind of analysis , as spearheaded by the edmonton group and others , is not generally available and still requires confirmation . in conclusion , both acute mvi ( g and ptc ) and chronic microvascular changes ( tg and ptcbmml ) can clearly be caused by alloantibodies . both cell - mediated and non - alloimmune mechanisms , however , similarly result in these acute and chronic lesions , either together with antibodies or as the primary process . the pathogenicity of these alternatives to amr needs to be considered for appropriate stratification in the design of therapeutic clinical trials and obviously for consideration of treatment in the individual patient .

the diagnosis of an antibody - mediated rejection ( amr ) is made when there is evident histologic injury in the presence of detectable donor - specific alloantibodies ( dsa ) and diffuse peritubular capillary c4d staining ( c4d - pos ) . in the presence of only detectable dsa or c4d - pos , the tissue injury is currently considered presumptive for antibody causation . in acute antibody - mediated rejection ( aamr ) , diagnostic morphologic features include microvascular inflammation ( mvi ) , specifically glomerulitis and peritubular capillaritis . in the case of chronic active amr ( caamr ) , these inflammatory lesions have progressed to chronic microvascular injury , transplant glomerulopathy ( tg ) and peritubular capillary basement membrane multilayering ( ptcbmml ) . either tg or ptcbmml is sufficient morphological evidence for a diagnosis of caamr . unfortunately , these lesions are not specific . mvi , tg , and ptcbmml are found in the setting of cell - mediated immunity , as well as in association with non - alloimmune mechanisms . the available treatments for amr and cmr are different , and it is important to ascertain the dominant mechanism when approaching an individual patient . at present , no gold standard exists to establish the specific pathogenesis in the more ambiguous cases . we detail here the differential diagnosis of mvi , tg , and ptcbmml .

Acute humoral rejection and microvascular inflammation (MVI) Chronic microvascular injury: TG and PTCBMML Discussion

in the early days of renal transplantation , preexisting dsa caused hyperacute rejections within minutes to hours with destruction of the graft 3 . histologic evaluation disclosed glomerulitis ( g ) and ptcitis ( ptc ) that involved predominantly polymorphonuclear leukocytes ( pmns ) . with the advent of the t - cell cross - match in 1969 , hyperacute rejections became rare , and the emphasis became the prevention and treatment for cell - mediated rejections ( cmr ) . 4 placed the focus back toward antibodies by describing seven patients with very early acute rejections ( ar ) in the presence of positive t - cell cytotoxic cross - matches . the main inflammatory cell appeared to be the pmn , as in hyperacute rejection 4,5 . with the discovery of ptc c4d staining as a marker of alloantibody injury and its application in acute and chronic situations , it became evident that mononuclear cells were more prominent than pmns in biopsies diagnosed with aamr 6 . current banff criteria for diagnosing aamr include dsa , c4d , and the morphological alterations summarized in table1 that include mvi , g and/or ptc 2 . if only dsa or c4d are positive , mvi allows a diagnosis of presumptive aamr . hence , a biopsy with any degree of mvi ( theoretically one inflamed glomerulus ) with detectable dsa or c4d has presumptive aamr . morphologic evidence of aamr ( type / grade ) aamr , acute antibody - mediated rejection . banff quantitative criteria for peritubular capillaritis ( ptc ) ptc , peritubular capillaries . although a criterion for the diagnosis of aamr , the specificity of g is not 100% . seven patients with ar and positive ptc c4d staining ( c4d - pos ) had significantly higher glomerular monocytes ( mo ) and pmns , as compared to 10 with negative staining ( c4d - neg ) ar and 10 with no ar . similarly , magil and tinckham compared 23 early biopsies ( less than six months ) with diffuse c4d - pos ar with acute tubular injury to 28 with c4d - neg cmr 10 . pmns were detectable in the glomeruli of 96% and 93% of both groups , as well as 100% of ptc of both groups . distinguishing features included presence of g ( 57% in c4d - pos vs. 11% in c4d - neg ) and the presence of glomerular mo ( 96% vs. 43% ) . in the 20 c4d - pos patients , mo predominated , and in the 22 c4d - neg cases , t cells ( tc ) did . the mean glomerular mo / tc ratio was > 1.0 in 75% of c4d - pos vs. 14% of c4d - neg rejections ( p < 0.0001 ) . studying 96 patients with ar in the first 12 months , this group again noted this correlation : 83% of biopsies with ( mean ) 1 mo / glomerulus ( deemed high ) were c4d - pos vs. 11% of those with a ratio < 1 , and 78% vs. 18% had banff criteria for g , respectively 12 . a high mo count was predictive of one- and three - yr egfr by multivariable analysis , and this obtained whether c4d - pos or c4d - neg . cell - mediated mechanisms as evidenced by cd3-positive staining , however , result in g in c4d - neg cases and may contribute in c4d - pos ones . found those in the upper quartile of maximum number of mo in the most involved glomerulus ( > 12 ) were more likely to have dsa , c4d - pos , tg , and the worst allograft survival ( gs ) . acute cmr in this late cohort did not affect the composition of cells in g. unlike previous data from earlier biopsies 11 , the mo / tc ratio was not different in c4d - pos vs. c4d - neg cases . in a later study of 1101 biopsies , pure cmr did not have more g ( or ptc ) than dsa negative ( dsa - neg)/c4d - neg biopsies without any rejection ( combined g + ptc > 0 in 6% vs. 0.6% ) 14 . even with this stricter criterion than banff s , 47% of dsa - neg , c4d - neg cmrs had g>0 , and 62% of dsa - neg , c4d - neg borderline cmrs did as well . of the 36 with g>0 , 22 had c4d - neg cmr and 10 had cmr that was suspicious for aamr ( c4d - pos ) . these data reinforce those of magil 11 that cmr in the absence of detectable antibodies can produce g. gibson et al . overall , most cases were moderate ( ptc 2 ) , focal ( involving 1050% of ptc ) and mononuclear predominant . there was a strong correlation of ptc with g ( if g > 0 , then ptc > 0 in over 80% ) and c4d ( 75% had ptc > 0 if c4d - pos vs. 24% if c4d - neg ) . furthermore , 14% of biopsies without any rejection ( aamr or cmr ) had ptc 1 . 18 studied 42 biopsies , comparing 10 with pure cmr in the absence of g with 32 having g : 13 c4d - neg and 19 c4d - pos . the ptc mo / tc ratio was significantly higher in c4d - pos g cases ( 2.3 ) than c4d - neg g cases ( 1 , p = 0.0008 ) and pure cmr ones ( 0.9 , p = 0.0014 ) , results mirroring the glomerular findings of magil 11 , but unlike those of papadimitriou 13 . these data support the concept that cell - mediated mechanisms produce mvi without the help of antibodies . several groups have studied the prognostic significance of mvi . 19 found by multivariable analysis that c4d - neg ar ( presumably cmr ) was only associated with reduced gs if g or ptc was present . in the absence of mvi 20 studied 58 conventional risk transplants with development of de novo dsa ( dndsa ) who had indication biopsies . despite dndsa and an indication for the biopsy six ( 23% ) of the 26 were c4d - pos and none had cmr . as the degree of mvi increased ( g + ptc = 1 or 2 , or g + ptc 3 ) , c4d - pos increased ( 29% and 64% respectively ) , as well as cmr ( 33% and 73% ) , suggesting significant contributions from both arms of adaptive immunity in the development of mvi . using mvi as a predictor of gs , the area under the curve in roc analysis was 0.84 , better than that for c4d ( p = 0.006 ) . mvi ( g + ptc > 0 ) in early biopsies ( less than one yr ) was non - specific , being found in aamr , cmr , and atn . only in the presence of c4d - pos was mvi likely to indicate dsa at this early time . in later biopsies , this group has also demonstrated a role for nk cells in antibody - mediated cases 22 , an observation supported by experimental studies 23 . in summary , mvi is found in aamr . such mvi can be ascribed to amr in the presence of both c4d - pos and dsa - pos . acute cmr results in mvi as well , both g and/or ptc . in the presence of cmr and non - alloimmune mechanisms such as atn may result in mvi in the absence of any rejection . in the future , identification of pathogenesis - based transcripts ( pbts ) specific for the type of injury may determine the predominant cause 24,25 , along with cell type ( mo vs. tc , perhaps nk - cell ) . in any case , these chronic changes are considered synonymous with antibody causation and either is considered sufficient tissue injury required for diagnosis of caamr by banff . transplant glomerulopathy is diagnosed by light microscopy when 10% of capillary loops in the most affected glomerulus have double contours ( table4 ) 7 . in higher - risk , live donor transplants performed against a positive cross - match , the one - yr incidence was 22% 29 . banff quantitative criteria for transplant glomerulopathy ( cg ) number of glomeruli and percentage sclerotic . evidence indicates that chronic antibody - mediated injury results in tg . among 582 transplants , the presence of class ii antibodies increased the hazard ratio ( hr ) for tg by 5 , as compared to those without any class ii antibodies . the frequency of their occurrence , however , has varied considerably in the published series . these include 0% of 22 biopsies 30 , 15% of 36 31 , 24% of 68 32 , 36% of 53 26 , 48% of 25 33 , 55% of 58 34 , and 67% of 59 6 . where assessed , these c4d - pos cases had detectable dsa , as did some of the c4d - neg ones . overall , 2560% of these tg cases , however , lacked any evidence of antibody mediation ( both hla / dsa - neg and c4d - neg ) 26,30,31 . other potential causes of tg include cmr , thrombotic microangiopathy ( tma ) , and chronic hepatitis c virus ( hcv ) infection . whereas both aamr and subclinical amr 35 can significantly increase the risk of tg , most cases are not preceded by such events 32 . in the series of sis et al . among 35 biopsies in these 20 , cmr existed in 74% ( only one of which was borderline ) , as compared to 26% with aamr . all tg biopsies had glomerular staining for the costimulatory molecule icos and the chemokine receptor cxcr3 with its ligand mig , findings that indicate the presence of activated t cells . 37 found increased mrna expression for interferon - gamma ( infg ) , the th1 transcription factor t - bet , and the cytotoxic t - lymphocyte cytokine granzyme - b in the glomeruli of 22 patients with tg as compared to 18 with can alone . 38 demonstrated significantly increased t - bet expression by immunohistochemistry in glomeruli and ptc of 32 patients with tg , as compared to 23 with if / ta and 15 stable grafts . despite the antibody involvement , gene expression profiles of pbts associated with cell - mediated immunity were significantly enriched , including those for cytotoxic t lymphocytes and infg . only 45% had detectable dsa by luminex solid phase assay ( spa ) , and only 14% were c4d - pos . surprisingly , 49 of 50 had lymphocytic infiltration of fibrous intimal thickening , thereby satisfying banff criteria for chronic active cmr ( filippone et al . double contours are found by light microscopy ( lm ) , and the ultrastructural changes are also similar to tg 33,40,41 . haas and mirocha 43 noted similar early changes ( endothelial cell swelling , subendothelial widening , and gbm duplication ) in three - month indication biopsies , many of which demonstrated tg on subsequent biopsies . 41 studied 59 patients with de novo tma and found diffuse c4d - pos in 55% . of note , 13% of c4d - pos biopsies evidenced tma no significant difference in the severity of tg between c4d - pos and c4d - neg groups was noted , although greater mvi was observed in the former . in 37 tma cases ( of 1101 biopsies ) , only six were c4d - pos . the incidence of tma was the same in c4d - pos and c4d - neg biopsies overall ( 3.4% of each ) 46 . if the biopsy occurred at 90 d , however , the probability of observing tma in c4d - pos biopsies was significantly greater than in c4d - neg ones . by contrast , after 90 d , tma was less frequent in c4d - pos biopsies 46 . they found 48% to be c4d - pos ; 32% had tma , and 36% were hcv positive . significant overlap existed between the presence of tma and hcv : five had both tma and hcv , as compared to three with just tma and four with just hcv . 47 found positive serology for hcv in 29% of 41 patients with g and 33% of 27 patients with tg , as compared to 1.8% of 105 patients with neither ( p = 0.0004 for both comparisons ) . ptcbmml alone represents sufficient tissue injury for a diagnosis of caamr per banff if both dsa - pos and c4d - pos . the minimum requirements to diagnose ptcbmml , however , are not well specified by banff . such a lesion had a sensitivity for chronic rejection ( defined as arterial fibrous intimal thickening or tg ) of 59% , a number similar to the 57% reported in an earlier series 50 . notably , lm evidence of acute cmr was found in 78% of cases , which suggested that cpc is attributable to slowly progressive , smoldering cmr . severe ptcbmml ( equivalent to cpc ) was rarely found in native kidneys with the exception of late tma . in transplants , this lesion lacked specificity , as it was found in 20% of c4d - neg acute cmr , 36% of cases felt to have cni toxicity , and 67% of c4d - neg chronic active cmr . finally , cpc was found in approximately half of antibody - mediated cases ( aamr / caamr ) . hence , although ptcbmml is usually found together with tg , neither of these lesions is specific for antibody causation . the methodology for antibody detection is constantly evolving , and consensus guidelines have recently been published 53 . here , we have detailed the lack of specificity of histologic and ultrastructural features elicited by humoral injury , although part of the discrepancies noted relate to variability between laboratories in the methodology and proficiency in determining dsa and c4d . in addition , interobserver discrepancy in the diagnosis of cell - mediated rejection is significant . for example , with three pathologists evaluating the same 245 biopsies , agreement between any two was only 45% with regard to a diagnosis of tcmr or mixed t - cell- or antibody - mediated rejection 25 . these issues reduce both the sensitivity and specificity of associated morphologic features of any diagnostic category . both cell - mediated immunity and antibodies , however , are capable of producing the acute and chronic lesions discussed above ( table5 ) . treatment of cell - mediated and antibody - mediated rejections differ . in the former , steroids with the possible addition of thymoglobulin would be in order , possibly with increased chronic therapy . in the latter , and despite a dearth of high - level evidence 54 , plasmapheresis , ivig , and steroids are used , with consideration of rituximab , bortezomib , thymoglobulin , eculizumab , and possibly splenectomy depending on the circumstances . more difficult is the case with features of both , such as a c4d - neg 1a cmr with significant mvi and dsa . at the present time , no gold standard exists to identify the predominant pathophysiologic mechanism of allograft injury . such antibodies may simply indicate a more aggressive alloimmune diathesis and are not causally related to the injury in a particular case . the presence of c4d - pos correlated with reduced gs , whereas dsa alone did not 56 . such a situation exists in caamr for antibodies against agrin 57 , glutathione - s - transferase t1 58 , and peroxisomal - trans-2-enoyl - coa - reductase 59 . more importantly , in the absence of c4d , dsa may be present but not pathogenic . the current trend is to label such cases as c4d - neg antibody - mediated rejection 63 , although by banff they would be presumptive amr . given the non - specificity of mvi and tg / ptcbmml , it is critical to determine whether dsa are pathogenic in a particular patient . this is not always the case , however , as they can frequently wane or disappear over time , either spontaneously 65 or after desensitization 66 . within one yr , 52% lost dsa , and another 30% had reductions in the number or strength of their dsas . 67 compared 34 non - desensitized patients with excellent graft function after one yr to 130 patients with decreased gfr and/or proteinuria . at present , the best evidence for the pathogenicity of a given dsa , aside from its ability to fix complement ( c4d+ ) , is a rising and high titer : prior to transplantation a high titer predicts a positive cross - match 68 ; in the early post - transplant period , rising titers predict aamr 69 ; and later , they predict graft failure 70 . as well as being preformed , dsa may develop de novo ( dndsa ) in up to 25% of cases 71 . some of the very early cases may represent activation of memory b - cells detectable at the time of transplantation by hla - specific tetramer staining in the absence of circulating antibody 78 . in one study with protocol screening , dndsa rarely preceded ar ( 3 of 19 with ar ) , but usually occurred concurrently or following the rejection 74 . furthermore , detection of dndsa had a positive predictive value of only 6% for subsequent ar . dndsa are most troublesome when found in the setting of late graft dysfunction in a non - adherent patient , where aamr and cmr may coexist 63,73 . , c1q or c4d positivity on luminex beads ) may help predict development of aamr or more chronic issues such as tg and late graft failure 7981 . most promisingly , the ability to differentiate the specific cause of issue injury by evaluation of pbts may allow determination that antibodies are the proximate cause 22,24,25 . unfortunately , this kind of analysis , as spearheaded by the edmonton group and others , is not generally available and still requires confirmation . in conclusion , both acute mvi ( g and ptc ) and chronic microvascular changes ( tg and ptcbmml ) can clearly be caused by alloantibodies . both cell - mediated and non - alloimmune mechanisms , however , similarly result in these acute and chronic lesions , either together with antibodies or as the primary process . the pathogenicity of these alternatives to amr needs to be considered for appropriate stratification in the design of therapeutic clinical trials and obviously for consideration of treatment in the individual patient .

a retrospective chart review was conducted of patients from september 2006 through september 2010 who presented with an acute central scotoma or defective vision secondary to smh from n - amd . inclusion criteria for the study included : symptoms of 7 days duration , thick ( causing visible elevation of the fovea by slit lamp biomicroscopy according to criteria used in previously published studies ) , 2 disc areas hemorrhage , hemorrhagic component 50% of total lesion size , and follow - up of 6 months . exclusion criteria included absence of blood beneath the fovea , indeterminate duration , smh secondary to any condition other than n - amd , pre - existing sub - foveal fibrosis , vision 20/400 prior to the occurrence of smh , co - existent vitreous hemorrhage , any other ocular condition responsible for decrease in vision , or recent ( 3 months ) thrombo - embolic event . a complete eye examination , including best - corrected snellen va , slit lamp examination , and fundus biomicroscopy were performed at presentation . fundus photos and intraveonus fluorescein angiography ( ivfa ) were performed to document an active cnvm . images were imported into ois winstation software ( ophthalmic imaging systems , sacramento , ca ) . the software was used to measure total lesion size and size of cnvm in mid - phase angiogram spectral domain optical coherence tomography ( sdoct ) was performed with either the optovue rtvue fd - oct system ( optovue corporation , fremont , ca ) using the emm5 ( 26 803 + 16 535 a - scans ) and raster ( 17 parallel scans , 17 1024 a - scans ) protocols or zeiss cirrus hd - oct system model 4000 ( carl zeiss meditec , inc . , dublin , ca ) with macular cube ( 512 128 line scans over a 6 mm 6 mm area ) and hd raster ( 4096 a scans per line , 5 parallel lines , 0.25 mm apart ) protocols . oct scans were manually checked to avoid quantitative errors . at presentation , retinal thickness ( distance between rpe to the ilm ) , location of hemorrhage ( subretinal or subrpe ) , and thickness of subfoveal hemorrhage were measured manually on a high resolution raster image . for subretinal hemorrhage , thickness was equal to the distance between the rpe and the first high reflectivity line within the retina representing the junction between inner and outer segment of photoreceptors ( is / os line ) . thickness of subrpe hemorrhage was the distance from the rpe to an imaginary line contiguous with the edge of rpe adjacent to hemorrhage . the same oct machine that was used for the initial evaluation was utilized for follow - up visits to allow for direct comparison between results . after obtaining informed consent , an intravitreal injection of 1.25 mg bevacizumab was administered under topical or subconjunctival anesthesia with sterile technique using a lid speculum , 10% povidone iodine scrub , and topical 5% povidone iodine solution . ranibizumab ( 0.5 mg ) was used in patient 9 who developed smh 4 weeks after bevacizumab injection . the patients were seen monthly and they underwent a complete ophthalmic examination , fundus photos , and sdoct . ivfa was performed every 6 - 12 months or when there was a worsening of the patient 's condition . monthly injections were continued until complete resolution of hemorrhage and submacular / intraretinal fluid based on clinical examination and sdoct . thereafter , interval between injections was increased by 1 - 2 weeks , but not more than 2 months to optimize the intravitreal therapy to keep the lesion dry . va results were analyzed at 3 and 6 month , at 1 year , and at final follow - up . va of counting fingers and hand motions were assigned logmar value of 1.6 and 2.0 , respectively . a two - tailed paired student 's t - test was applied for oct values and hemorrhage size using microsoft excel ( microsoft corporation , redmond , wa ) . wilcoxon signed rank test was applied to the visual acuity results using megastat plug - in for excel ( j.b.orris , butler university ) . p values less than 0.05 were considered to be statistically significant . at final follow - up , appearance of the macula ( presence of any scar , rpe tear , or geographic atrophy ) was recorded . fourteen eyes of 14 patients ( m = 2 , f = 12 ) , mean age sd 80.1 9.54 , range 60 - 91 years were included in the study [ table 1 ] . eight had previously been treated for n - amd , six with anti - vegf monotherapy alone , one with thermal laser for an extrafoveal cnvm , followed by three applications of photodynamic therapy for subfoveal recurrence followed by bevacizumab injections and 1 with foveal thermal laser . va prior to the onset of smh ranged from 20/20 to 20/400 ( median 20/40 ) . those with a history of bevacizumab injections ( n = 7 ) had received an average of 7.4 3.8 ( median 8 , range 2 - 12 ) injections prior to the onset of smh . of these , five had received their last injection less than 20 weeks prior to the onset of smh ( range 4 - 104 , median 20 ) . baseline characteristics patients presented after a median of 4 ( range 1 - 7 ) days after the onset of acute central scotoma or defective vision . ten were on systemic anticoagulation , seven on aspirin alone , two on aspirin and an antiplatelet agent , and one on warfarin . reason for anticoagulant use included ischemic heart disease in 6 patients and a history of deep vein thrombosis in 2 patients . two patients who were taking over - the - counter aspirin did not have any past history of systemic thrombo - embolic event . eleven eyes were pseudophakic and 3 phakic eyes remained phakic throughout the period of the study . mean lesion size was 27.9 24.5 mm ( range 5.47 - 100 , median 15 ) and mean smh size was 25.1 24.3 mm ( range 4.22 - 90 , median 13.1 ) [ table 2 ] . hemorrhage comprised a mean of 87.7 6.3% ( median 90.0% , range 77 - 98% ) of the lesion . at 3 months , the mean size of the hemorrhage decreased to 10.3 11.8 mm ( median 6.08 , range 0 - 36 , p = 0.0025 ) . at 6 months , the mean size of the hemorrhage decreased further to 4.3 9 mm ( median 0.37 , range 0 - 31 ) , which was statistically significant as compared to baseline ( p = 0.0037 ) and 3 months ( p = 0.019 ) . subfoveal hemorrhage resolved in all eyes in a mean of 4.8 1.6 ( range 2 - 8 , median 5 ) months . there was no statistically significant difference in time to resolution between predominantly subretinal hemorrhage and predominantly subrpe hemorrhages [ figs . 1a and b , 2 , 3a c , 4a c , 5 , 6a c ] . ( b ) ivfa at presentation showing blocked fluorescence and poorly defined leakage indicating a cnvm patient 6 at 1 month after presentation showing significant decrease in thickness of blood ( a ) patient 6 at 7 months follow - up . ( b ) ivfa of patient 6 at 7 months showing a large occult subfoveal choroidal neovascular membrane . ( c ) oct of patient 6 at 7 months showing a dry macula ( a ) patient 5 . oct at 4 months showing persistent ped , but no intra or subretinal fluid during the first 6 months , the patients received a mean of 5.4 ( median 5 , range 5 - 6 ) injections . those with 12 months follow - up ( n = 10 ) received a mean of 9.6 ( median 9 , range 9 - 12 ) injections during the first year . over a mean follow - up of 18.4 11 ( range 7 - 50 ) months , patients received a mean of 11.4 4.4 ( range 5 - 20 ) injections . presenting va ranged from 20/60 to hm ( mean 20/400 , median 20/200 ) [ table 3 ] . at 3 months , visual acuity ranged from 20/30 to counting fingers ( median logmar 1.15 ; snellen equivalent 20/280 ] ) . compared to baseline , change in va at 3 months was not statistically significant ( p = 0.104 ) . visual acuity results at 6 months ( n = 14 ) , va ranged from 20/25 - 20/400 with a mean and median of 20/100 . mean va gain at 6 months compared to baseline was 0.54 0.57 logmar ( range : 1.5 to 1 , p = 0.0037 ) . eleven patients experienced va gain of 0.2 logmar and 7 gained 0.6 logmar with 5 eyes having vision 20/50 . patient 11 , who presented with visual acuity of 20/60 , remained at the 20/60 level at 6 months despite resolution of subfoveal hemorrhage after 4 months . patient 12 , who had previously received foveal thermal laser , presented with a visual acuity of 20/400 that remained at that level throughout follow - up despite complete clearance of smh after 3 months . patient 2 suffered visual decline from 20/80 at presentation to 20/400 6 weeks later due to an rpe tear . she remained at 20/400 level throughout the duration of follow - up of 50 months . at 1 year ( n = 9 ) , va ranged from 20/30 - 20/400 with a mean and median of 20/100 and 20/50 , respectively . all 6 eyes that gained 0.2 logmar va at 6 months maintained that gain at 1 year . mean change in va from baseline was 0.43 0.6 logmar ( median 0.60 , range 1.42 to 1 ) . there was no significant difference between the va at 6 months and at 1 year ( p = 0.38 ) . at final follow - up , ( mean 18.4 , range 7 - 50 months ) , va ranged from 20/30 - 20/400 , mean 20/100 , median 20/60 for the 14 eyes . the mean change in va from baseline in logmar was 0.58 0.57 ( median 0.6 , range 1.6 to 1 , p = 0.0022 ) . no statistical significance was found between the va at 6 months and at final follow - up ( p = 0.23 ) . oct results were available at presentation in 8 patients and showed a mean foveal thickness of 506 218 ( range 371 - 978 , median 405 ) . mean thickness of hemorrhage was 322 ( range 127 - 637 , median 301 ) . oct results at 6 months showed a mean foveal thickness of 243.5 41 ( range 174 - 330 , median 231 , p = 0.023 as compared to baseline ) . at 1 year , the mean foveal thickness was 256.5 37 ( range 206 - 313 , median 263 , p = 0.89 as compared to foveal thickness at 6 months ) . at final follow - up , mean foveal thickness was 246.8 43 ( range 187 - 329 , median 233 , p = 0.81 as compared to foveal thickness at 6 months ) . fa after resolution of smh showed an occult cnvm in 10 eyes , peripapillary classic cnvm in 2 eyes , regressed classic cnvm in 1 eye , and an rpe tear in 1 eye [ table 2 ] . thin subretinal scar was noted in 2 eyes , laser scar in 1 eye , and no scar in the remaining 10 eyes . no ocular or systemic adverse effects secondary to the use of the anti - vegf medications were noted during the study . fourteen eyes of 14 patients ( m = 2 , f = 12 ) , mean age sd 80.1 9.54 , range 60 - 91 years were included in the study [ table 1 ] . eight had previously been treated for n - amd , six with anti - vegf monotherapy alone , one with thermal laser for an extrafoveal cnvm , followed by three applications of photodynamic therapy for subfoveal recurrence followed by bevacizumab injections and 1 with foveal thermal laser . va prior to the onset of smh ranged from 20/20 to 20/400 ( median 20/40 ) . those with a history of bevacizumab injections ( n = 7 ) had received an average of 7.4 3.8 ( median 8 , range 2 - 12 ) injections prior to the onset of smh . of these , five had received their last injection less than 20 weeks prior to the onset of smh ( range 4 - 104 , median 20 ) . baseline characteristics patients presented after a median of 4 ( range 1 - 7 ) days after the onset of acute central scotoma or defective vision . ten were on systemic anticoagulation , seven on aspirin alone , two on aspirin and an antiplatelet agent , and one on warfarin . reason for anticoagulant use included ischemic heart disease in 6 patients and a history of deep vein thrombosis in 2 patients . two patients who were taking over - the - counter aspirin did not have any past history of systemic thrombo - embolic event . eleven eyes were pseudophakic and 3 phakic eyes remained phakic throughout the period of the study . mean lesion size was 27.9 24.5 mm ( range 5.47 - 100 , median 15 ) and mean smh size was 25.1 24.3 mm ( range 4.22 - 90 , median 13.1 ) [ table 2 ] . hemorrhage comprised a mean of 87.7 6.3% ( median 90.0% , range 77 - 98% ) of the lesion . at 3 months , the mean size of the hemorrhage decreased to 10.3 11.8 mm ( median 6.08 , range 0 - 36 , p = 0.0025 ) . at 6 months , the mean size of the hemorrhage decreased further to 4.3 9 mm ( median 0.37 , range 0 - 31 ) , which was statistically significant as compared to baseline ( p = 0.0037 ) and 3 months ( p = 0.019 ) . subfoveal hemorrhage resolved in all eyes in a mean of 4.8 1.6 ( range 2 - 8 , median 5 ) months . there was no statistically significant difference in time to resolution between predominantly subretinal hemorrhage and predominantly subrpe hemorrhages [ figs . 1a and b , 2 , 3a c , 4a c , 5 , 6a c ] . ( b ) ivfa at presentation showing blocked fluorescence and poorly defined leakage indicating a cnvm patient 6 at 1 month after presentation showing significant decrease in thickness of blood ( a ) patient 6 at 7 months follow - up . note resolution of subfoveal hemorrhage . ( b ) ivfa of patient 6 at 7 months showing a large occult subfoveal choroidal neovascular membrane . ( c ) oct of patient 6 at 7 months showing a dry macula ( a ) patient 5 . oct at 4 months showing persistent ped , but no intra or subretinal fluid during the first 6 months , the patients received a mean of 5.4 ( median 5 , range 5 - 6 ) injections . those with 12 months follow - up ( n = 10 ) received a mean of 9.6 ( median 9 , range 9 - 12 ) injections during the first year . over a mean follow - up of 18.4 11 ( range 7 - 50 ) months , patients received a mean of 11.4 4.4 ( range 5 - 20 ) injections . presenting va ranged from 20/60 to hm ( mean 20/400 , median 20/200 ) [ table 3 ] . at 3 months , visual acuity ranged from 20/30 to counting fingers ( median logmar 1.15 ; snellen equivalent 20/280 ] ) . compared to baseline , change in va at 3 months was not statistically significant ( p = 0.104 ) . visual acuity results at 6 months ( n = 14 ) , va ranged from 20/25 - 20/400 with a mean and median of 20/100 . mean va gain at 6 months compared to baseline was 0.54 0.57 logmar ( range : 1.5 to 1 , p = 0.0037 ) . eleven patients experienced va gain of 0.2 logmar and 7 gained 0.6 logmar with 5 eyes having vision 20/50 . patient 11 , who presented with visual acuity of 20/60 , remained at the 20/60 level at 6 months despite resolution of subfoveal hemorrhage after 4 months . patient 12 , who had previously received foveal thermal laser , presented with a visual acuity of 20/400 that remained at that level throughout follow - up despite complete clearance of smh after 3 months . patient 2 suffered visual decline from 20/80 at presentation to 20/400 6 weeks later due to an rpe tear . she remained at 20/400 level throughout the duration of follow - up of 50 months . at 1 year ( n = 9 ) , va ranged from 20/30 - 20/400 with a mean and median of 20/100 and 20/50 , respectively . all 6 eyes that gained 0.2 logmar va at 6 months maintained that gain at 1 year . mean change in va from baseline was 0.43 0.6 logmar ( median 0.60 , range 1.42 to 1 ) . there was no significant difference between the va at 6 months and at 1 year ( p = 0.38 ) . at final follow - up , ( mean 18.4 , range 7 - 50 months ) , va ranged from 20/30 - 20/400 , mean 20/100 , median 20/60 for the 14 eyes . the mean change in va from baseline in logmar was 0.58 0.57 ( median 0.6 , range 1.6 to 1 , p = 0.0022 ) . no statistical significance was found between the va at 6 months and at final follow - up ( p = 0.23 ) . oct results were available at presentation in 8 patients and showed a mean foveal thickness of 506 218 ( range 371 - 978 , median 405 ) . mean thickness of hemorrhage was 322 ( range 127 - 637 , median 301 ) . oct results at 6 months showed a mean foveal thickness of 243.5 41 ( range 174 - 330 , median 231 , p = 0.023 as compared to baseline ) . at 1 year , the mean foveal thickness was 256.5 37 ( range 206 - 313 , median 263 , p = 0.89 as compared to foveal thickness at 6 months ) . at final follow - up , mean foveal thickness was 246.8 43 ( range 187 - 329 , median 233 , p = 0.81 as compared to foveal thickness at 6 months ) . fa after resolution of smh showed an occult cnvm in 10 eyes , peripapillary classic cnvm in 2 eyes , regressed classic cnvm in 1 eye , and an rpe tear in 1 eye [ table 2 ] . thin subretinal scar was noted in 2 eyes , laser scar in 1 eye , and no scar in the remaining 10 eyes . no ocular or systemic adverse effects secondary to the use of the anti - vegf medications were noted during the study . our study showed excellent anatomical and visual results with anti - vegf monotherapy alone in patients with thick smh secondary to n - amd who presented in 7 days . visual gain was usually observed between 3 - 6 months . at 6 months , 11/14 ( 79% ) experienced va gain equivalent of 2 lines and 7/14 ( 50% ) gained equivalent of 6 lines on an etdrs chart . of the remaining 3 patients , 1 presented with good va ( 20/60 ) , and 1 had limited potential for improvement due to prior foveal thermal laser . only 1 patient suffered visual decline due to an rpe tear . visual gain was accompanied by resolution of subfoveal hemorrhage in all eyes in a mean of 4.8 months with monthly anti - vegf injections . anatomical and visual improvement was maintained for 1 year and beyond with ongoing anti - vegf injections . although numbers were small , there was no apparent difference in outcome between subrpe and subretinal hemorrhages . demonstrated that , in 21 eyes with hemorrhagic n - amd treated with intravitreal bevacizumab , 48% had improvement in va by > 1 letter , 9.5% demonstrated no change , while 43% lost <3 lines at 4 months , although median va remained unchanged . however , only 9 ( 43% ) eyes had subfoveal hemorrhage , thickness of hemorrhage was not determined , 7 ( 33% ) had symptoms for > 30 days , presenting va was relatively better ( mean 20/80 versus 20/400 in our study ) , patients received fewer injections ( median 3 compared to 9 in our study ) over a 1-year period , visual gain was modest , and only 25% gaining approximately 2 lines of vision compared to 67% in our study at 1 year . mckibbin et al . reported outcomes with intravitreal ranibizumab injections in 11 eyes with smh secondary to n - amd . at 6 months , 3 ( 28% ) gained > 15 etdrs letters , 4 ( 36% ) gained 1 - 14 letters , and 4 ( 36% ) lost 1 - 14 letters . patients were treated with 3 monthly loading injections , followed by prn dosing ; 7 eyes followed for 1 year received a mean of 6 injections . patients were included in the study regardless of the size or thickness of hemorrhage ( range : 0.5 - 45.4 mm ) . they found no difference in outcome for duration 4 weeks compared to > 4 weeks , but duration of symptoms exceeded 7 days in all eyes . sacu et al . found that eyes treated with pneumatic displacement with intravitreal tpa and sf6 gas and anti - vegf had superior outcomes as compared to eyes treated with anti - vegf agents alone . most notably , the mean duration of symptoms in tpa / gas / anti - vegf group was 7 days as compared to 12.9 days in the anti - vegf group . pneumatic displacement has potential advantage of faster visual rehabilitation and shorter exposure time of the rpe and photoreceptors to toxic degradation products of blood clot , but it may result in shearing injury to photoreceptors , may not be effective in subrpe hemorrhages , has lower success rate for hemorrhages larger than 5 mm diameter , and has potential of displacing the blood into the fovea if significant amount of blood is located superior to fovea . combination of pneumatic displacement with an anti - vegf agent has been evaluated in a few small case series . although this approach seems logical , it is currently not known whether it is superior to anti - vegf injections alone . a recent publication found no evidence that addition of gas injection to displace smh provided any benefit over anti - vegf injections alone . chang et al . reported a prospective study of 7 eyes treated with ranibizumab for predominantly hemorrhagic n - amd lesions . patients were treated with 3 monthly injections followed by prn dosing , receiving a median of 7 injections in 1 year . va gain of > 2 lines was noted in 43% , and the median va improved from 20/320 to 20/250 . however , duration and thickness of smh were not specified , and 5/7 eyes had subfoveal fibrosis prior to the onset of smh . although our patients had large and thick lesions and had poor presenting va , we believe our study demonstrated outcomes better than any of the previously reported studies for three reasons . first , treatment was initiated within 1 week . earlier treatment in our patients compared to previous studies might have played a significant beneficial role in improving outcome . second , patients with pre - existing subfoveal fibrosis secondary to n - amd were not included in this review . third , our patients received more intravitreal injections during the first and second year than in previously reported studies . we attempted to achieve a completely fluid - free macula guided by spectral domain oct , similar to the criteria used in catt . although we utilized treat and extend protocol , we did not extend treatment - free interval beyond 8 weeks . about half of our patients had previously received anti - vegf injections for n - amd and presented with acute decrease in central vision due to thick smh after a median of 20 weeks from their last anti - vegf injection , similar to that in levine et al . , while we are aware of potential side effects , expense , and inconvenience associated with multiple intravitreal injections , we were hesitant to extend the treatment - free interval too much and risk a re - bleed . we noted that mean injections of 11.4 over a mean follow - up of 18.4 months are not much different from the mean of 14.1 injections of bevacizumab over 2 years in the prn arm of catt . in a retrospective study , dadgostar et al . showed that patients who received more frequent injections had superior visual outcome . less frequent dosing might have contributed to worse visual results in other studies . a major goal of therapy for neovascular amd is prevention of disciform scar . however , no disciform scar forms in patients where etiology of smh is other than a cnvm . thus , vascular proliferation is needed for scar formation , and its inhibition by early anti - vegf monotherapy may prevent scarring . snellen , rather than standardized etdrs va , was used , leading to lack of precise va measurements below 20/400 . definition of thick hemorrhage was based on clinical criteria and not on sdoct , which was not available in 6 ( 43% ) at baseline . other limitations of our study include retrospective and noncomparative nature and a small sample size . however , it seems that disciform scar formation can be prevented and good anatomical and visual outcome can be accomplished in patients treated with anti - vegf monotherapy within 1 week from the onset of smh secondary to n - amd .

aim : to investigate the role of anti - vegf monotherapy in patients with thick submacular hemorrhage ( smh ) of 1 week duration secondary to neovascular age - related macular degeneration ( n - amd).materials and methods : a retrospective chart review of 14 eyes of 14 patients presenting with acute decrease in central vision of 1 week duration secondary to a thick smh measuring 2 mps disk areas from n - amd was performed . intravitreal injections of bevacizumab 1.25 mg ( 13 eyes ) or ranibizumab 0.5 mg ( 1 eye ) were given monthly until resolution of smh and less frequently thereafter , based on treat - and - extend approach utilizing spectral domain optical coherence tomography ( sdoct ) . patients with follow - up of 6 months were included.results:patients presented after a median of 4 ( range 1 - 7 ) days from the onset of smh . mean lesion size was 27.9 mm2 ( range 5.47 - 100 , median 15 ) , with blood comprising 77 - 98% of the lesion . presenting visual acuity ( va ) ranged from 20/60 to hand motions ( median 20/200 ) . patients received a mean of 11.4 ( range 5 - 20 ) injections over 18.4 ( range 7 - 50 ) months . smh resolved in all eyes in a mean of 4.8 ( range 2 - 8 ) months . at 6 months follow - up , mean va gain was 0.54 logmar ( range : 1.5 to + 1 , snellen range 20/25 - 20/400 , median 20/100 , p = 0.0037 ) , with 11 gaining 0.2 logmar . mean change in va from baseline at final follow - up was 0.58 logmar ( range 1.6 to + 1 , snellen range 20/30 - 20/400 , median 20/60 ; p = 0.0022).conclusion : a good anatomical and visual outcome can be accomplished in patients with thick smh secondary to n - amd treated with anti - vegf monotherapy within 1 week .

Materials and Methods Results Baseline characteristics Lesion and treatment details VA results SDOCT results Fundoscopic and angiographic findings Discussion

a retrospective chart review was conducted of patients from september 2006 through september 2010 who presented with an acute central scotoma or defective vision secondary to smh from n - amd . inclusion criteria for the study included : symptoms of 7 days duration , thick ( causing visible elevation of the fovea by slit lamp biomicroscopy according to criteria used in previously published studies ) , 2 disc areas hemorrhage , hemorrhagic component 50% of total lesion size , and follow - up of 6 months . exclusion criteria included absence of blood beneath the fovea , indeterminate duration , smh secondary to any condition other than n - amd , pre - existing sub - foveal fibrosis , vision 20/400 prior to the occurrence of smh , co - existent vitreous hemorrhage , any other ocular condition responsible for decrease in vision , or recent ( 3 months ) thrombo - embolic event . the software was used to measure total lesion size and size of cnvm in mid - phase angiogram spectral domain optical coherence tomography ( sdoct ) was performed with either the optovue rtvue fd - oct system ( optovue corporation , fremont , ca ) using the emm5 ( 26 803 + 16 535 a - scans ) and raster ( 17 parallel scans , 17 1024 a - scans ) protocols or zeiss cirrus hd - oct system model 4000 ( carl zeiss meditec , inc . after obtaining informed consent , an intravitreal injection of 1.25 mg bevacizumab was administered under topical or subconjunctival anesthesia with sterile technique using a lid speculum , 10% povidone iodine scrub , and topical 5% povidone iodine solution . ranibizumab ( 0.5 mg ) was used in patient 9 who developed smh 4 weeks after bevacizumab injection . monthly injections were continued until complete resolution of hemorrhage and submacular / intraretinal fluid based on clinical examination and sdoct . thereafter , interval between injections was increased by 1 - 2 weeks , but not more than 2 months to optimize the intravitreal therapy to keep the lesion dry . va results were analyzed at 3 and 6 month , at 1 year , and at final follow - up . va of counting fingers and hand motions were assigned logmar value of 1.6 and 2.0 , respectively . at final follow - up , appearance of the macula ( presence of any scar , rpe tear , or geographic atrophy ) was recorded . fourteen eyes of 14 patients ( m = 2 , f = 12 ) , mean age sd 80.1 9.54 , range 60 - 91 years were included in the study [ table 1 ] . eight had previously been treated for n - amd , six with anti - vegf monotherapy alone , one with thermal laser for an extrafoveal cnvm , followed by three applications of photodynamic therapy for subfoveal recurrence followed by bevacizumab injections and 1 with foveal thermal laser . va prior to the onset of smh ranged from 20/20 to 20/400 ( median 20/40 ) . those with a history of bevacizumab injections ( n = 7 ) had received an average of 7.4 3.8 ( median 8 , range 2 - 12 ) injections prior to the onset of smh . of these , five had received their last injection less than 20 weeks prior to the onset of smh ( range 4 - 104 , median 20 ) . baseline characteristics patients presented after a median of 4 ( range 1 - 7 ) days after the onset of acute central scotoma or defective vision . mean lesion size was 27.9 24.5 mm ( range 5.47 - 100 , median 15 ) and mean smh size was 25.1 24.3 mm ( range 4.22 - 90 , median 13.1 ) [ table 2 ] . hemorrhage comprised a mean of 87.7 6.3% ( median 90.0% , range 77 - 98% ) of the lesion . at 3 months , the mean size of the hemorrhage decreased to 10.3 11.8 mm ( median 6.08 , range 0 - 36 , p = 0.0025 ) . at 6 months , the mean size of the hemorrhage decreased further to 4.3 9 mm ( median 0.37 , range 0 - 31 ) , which was statistically significant as compared to baseline ( p = 0.0037 ) and 3 months ( p = 0.019 ) . subfoveal hemorrhage resolved in all eyes in a mean of 4.8 1.6 ( range 2 - 8 , median 5 ) months . ( b ) ivfa at presentation showing blocked fluorescence and poorly defined leakage indicating a cnvm patient 6 at 1 month after presentation showing significant decrease in thickness of blood ( a ) patient 6 at 7 months follow - up . oct at 4 months showing persistent ped , but no intra or subretinal fluid during the first 6 months , the patients received a mean of 5.4 ( median 5 , range 5 - 6 ) injections . those with 12 months follow - up ( n = 10 ) received a mean of 9.6 ( median 9 , range 9 - 12 ) injections during the first year . over a mean follow - up of 18.4 11 ( range 7 - 50 ) months , patients received a mean of 11.4 4.4 ( range 5 - 20 ) injections . presenting va ranged from 20/60 to hm ( mean 20/400 , median 20/200 ) [ table 3 ] . at 3 months , visual acuity ranged from 20/30 to counting fingers ( median logmar 1.15 ; snellen equivalent 20/280 ] ) . compared to baseline , change in va at 3 months was not statistically significant ( p = 0.104 ) . visual acuity results at 6 months ( n = 14 ) , va ranged from 20/25 - 20/400 with a mean and median of 20/100 . mean va gain at 6 months compared to baseline was 0.54 0.57 logmar ( range : 1.5 to 1 , p = 0.0037 ) . eleven patients experienced va gain of 0.2 logmar and 7 gained 0.6 logmar with 5 eyes having vision 20/50 . patient 11 , who presented with visual acuity of 20/60 , remained at the 20/60 level at 6 months despite resolution of subfoveal hemorrhage after 4 months . patient 12 , who had previously received foveal thermal laser , presented with a visual acuity of 20/400 that remained at that level throughout follow - up despite complete clearance of smh after 3 months . she remained at 20/400 level throughout the duration of follow - up of 50 months . at 1 year ( n = 9 ) , va ranged from 20/30 - 20/400 with a mean and median of 20/100 and 20/50 , respectively . all 6 eyes that gained 0.2 logmar va at 6 months maintained that gain at 1 year . mean change in va from baseline was 0.43 0.6 logmar ( median 0.60 , range 1.42 to 1 ) . there was no significant difference between the va at 6 months and at 1 year ( p = 0.38 ) . at final follow - up , ( mean 18.4 , range 7 - 50 months ) , va ranged from 20/30 - 20/400 , mean 20/100 , median 20/60 for the 14 eyes . the mean change in va from baseline in logmar was 0.58 0.57 ( median 0.6 , range 1.6 to 1 , p = 0.0022 ) . no statistical significance was found between the va at 6 months and at final follow - up ( p = 0.23 ) . oct results were available at presentation in 8 patients and showed a mean foveal thickness of 506 218 ( range 371 - 978 , median 405 ) . oct results at 6 months showed a mean foveal thickness of 243.5 41 ( range 174 - 330 , median 231 , p = 0.023 as compared to baseline ) . at 1 year , the mean foveal thickness was 256.5 37 ( range 206 - 313 , median 263 , p = 0.89 as compared to foveal thickness at 6 months ) . at final follow - up , mean foveal thickness was 246.8 43 ( range 187 - 329 , median 233 , p = 0.81 as compared to foveal thickness at 6 months ) . fa after resolution of smh showed an occult cnvm in 10 eyes , peripapillary classic cnvm in 2 eyes , regressed classic cnvm in 1 eye , and an rpe tear in 1 eye [ table 2 ] . no ocular or systemic adverse effects secondary to the use of the anti - vegf medications were noted during the study . fourteen eyes of 14 patients ( m = 2 , f = 12 ) , mean age sd 80.1 9.54 , range 60 - 91 years were included in the study [ table 1 ] . eight had previously been treated for n - amd , six with anti - vegf monotherapy alone , one with thermal laser for an extrafoveal cnvm , followed by three applications of photodynamic therapy for subfoveal recurrence followed by bevacizumab injections and 1 with foveal thermal laser . va prior to the onset of smh ranged from 20/20 to 20/400 ( median 20/40 ) . those with a history of bevacizumab injections ( n = 7 ) had received an average of 7.4 3.8 ( median 8 , range 2 - 12 ) injections prior to the onset of smh . of these , five had received their last injection less than 20 weeks prior to the onset of smh ( range 4 - 104 , median 20 ) . baseline characteristics patients presented after a median of 4 ( range 1 - 7 ) days after the onset of acute central scotoma or defective vision . mean lesion size was 27.9 24.5 mm ( range 5.47 - 100 , median 15 ) and mean smh size was 25.1 24.3 mm ( range 4.22 - 90 , median 13.1 ) [ table 2 ] . hemorrhage comprised a mean of 87.7 6.3% ( median 90.0% , range 77 - 98% ) of the lesion . at 3 months , the mean size of the hemorrhage decreased to 10.3 11.8 mm ( median 6.08 , range 0 - 36 , p = 0.0025 ) . at 6 months , the mean size of the hemorrhage decreased further to 4.3 9 mm ( median 0.37 , range 0 - 31 ) , which was statistically significant as compared to baseline ( p = 0.0037 ) and 3 months ( p = 0.019 ) . subfoveal hemorrhage resolved in all eyes in a mean of 4.8 1.6 ( range 2 - 8 , median 5 ) months . ( b ) ivfa at presentation showing blocked fluorescence and poorly defined leakage indicating a cnvm patient 6 at 1 month after presentation showing significant decrease in thickness of blood ( a ) patient 6 at 7 months follow - up . oct at 4 months showing persistent ped , but no intra or subretinal fluid during the first 6 months , the patients received a mean of 5.4 ( median 5 , range 5 - 6 ) injections . those with 12 months follow - up ( n = 10 ) received a mean of 9.6 ( median 9 , range 9 - 12 ) injections during the first year . over a mean follow - up of 18.4 11 ( range 7 - 50 ) months , patients received a mean of 11.4 4.4 ( range 5 - 20 ) injections . presenting va ranged from 20/60 to hm ( mean 20/400 , median 20/200 ) [ table 3 ] . at 3 months , visual acuity ranged from 20/30 to counting fingers ( median logmar 1.15 ; snellen equivalent 20/280 ] ) . compared to baseline , change in va at 3 months was not statistically significant ( p = 0.104 ) . visual acuity results at 6 months ( n = 14 ) , va ranged from 20/25 - 20/400 with a mean and median of 20/100 . mean va gain at 6 months compared to baseline was 0.54 0.57 logmar ( range : 1.5 to 1 , p = 0.0037 ) . eleven patients experienced va gain of 0.2 logmar and 7 gained 0.6 logmar with 5 eyes having vision 20/50 . patient 11 , who presented with visual acuity of 20/60 , remained at the 20/60 level at 6 months despite resolution of subfoveal hemorrhage after 4 months . patient 12 , who had previously received foveal thermal laser , presented with a visual acuity of 20/400 that remained at that level throughout follow - up despite complete clearance of smh after 3 months . she remained at 20/400 level throughout the duration of follow - up of 50 months . at 1 year ( n = 9 ) , va ranged from 20/30 - 20/400 with a mean and median of 20/100 and 20/50 , respectively . all 6 eyes that gained 0.2 logmar va at 6 months maintained that gain at 1 year . mean change in va from baseline was 0.43 0.6 logmar ( median 0.60 , range 1.42 to 1 ) . there was no significant difference between the va at 6 months and at 1 year ( p = 0.38 ) . at final follow - up , ( mean 18.4 , range 7 - 50 months ) , va ranged from 20/30 - 20/400 , mean 20/100 , median 20/60 for the 14 eyes . the mean change in va from baseline in logmar was 0.58 0.57 ( median 0.6 , range 1.6 to 1 , p = 0.0022 ) . no statistical significance was found between the va at 6 months and at final follow - up ( p = 0.23 ) . oct results were available at presentation in 8 patients and showed a mean foveal thickness of 506 218 ( range 371 - 978 , median 405 ) . oct results at 6 months showed a mean foveal thickness of 243.5 41 ( range 174 - 330 , median 231 , p = 0.023 as compared to baseline ) . at 1 year , the mean foveal thickness was 256.5 37 ( range 206 - 313 , median 263 , p = 0.89 as compared to foveal thickness at 6 months ) . at final follow - up , mean foveal thickness was 246.8 43 ( range 187 - 329 , median 233 , p = 0.81 as compared to foveal thickness at 6 months ) . fa after resolution of smh showed an occult cnvm in 10 eyes , peripapillary classic cnvm in 2 eyes , regressed classic cnvm in 1 eye , and an rpe tear in 1 eye [ table 2 ] . no ocular or systemic adverse effects secondary to the use of the anti - vegf medications were noted during the study . our study showed excellent anatomical and visual results with anti - vegf monotherapy alone in patients with thick smh secondary to n - amd who presented in 7 days . visual gain was usually observed between 3 - 6 months . at 6 months , 11/14 ( 79% ) experienced va gain equivalent of 2 lines and 7/14 ( 50% ) gained equivalent of 6 lines on an etdrs chart . of the remaining 3 patients , 1 presented with good va ( 20/60 ) , and 1 had limited potential for improvement due to prior foveal thermal laser . visual gain was accompanied by resolution of subfoveal hemorrhage in all eyes in a mean of 4.8 months with monthly anti - vegf injections . anatomical and visual improvement was maintained for 1 year and beyond with ongoing anti - vegf injections . demonstrated that , in 21 eyes with hemorrhagic n - amd treated with intravitreal bevacizumab , 48% had improvement in va by > 1 letter , 9.5% demonstrated no change , while 43% lost <3 lines at 4 months , although median va remained unchanged . however , only 9 ( 43% ) eyes had subfoveal hemorrhage , thickness of hemorrhage was not determined , 7 ( 33% ) had symptoms for > 30 days , presenting va was relatively better ( mean 20/80 versus 20/400 in our study ) , patients received fewer injections ( median 3 compared to 9 in our study ) over a 1-year period , visual gain was modest , and only 25% gaining approximately 2 lines of vision compared to 67% in our study at 1 year . reported outcomes with intravitreal ranibizumab injections in 11 eyes with smh secondary to n - amd . at 6 months , 3 ( 28% ) gained > 15 etdrs letters , 4 ( 36% ) gained 1 - 14 letters , and 4 ( 36% ) lost 1 - 14 letters . patients were treated with 3 monthly loading injections , followed by prn dosing ; 7 eyes followed for 1 year received a mean of 6 injections . patients were included in the study regardless of the size or thickness of hemorrhage ( range : 0.5 - 45.4 mm ) . found that eyes treated with pneumatic displacement with intravitreal tpa and sf6 gas and anti - vegf had superior outcomes as compared to eyes treated with anti - vegf agents alone . combination of pneumatic displacement with an anti - vegf agent has been evaluated in a few small case series . a recent publication found no evidence that addition of gas injection to displace smh provided any benefit over anti - vegf injections alone . reported a prospective study of 7 eyes treated with ranibizumab for predominantly hemorrhagic n - amd lesions . patients were treated with 3 monthly injections followed by prn dosing , receiving a median of 7 injections in 1 year . however , duration and thickness of smh were not specified , and 5/7 eyes had subfoveal fibrosis prior to the onset of smh . second , patients with pre - existing subfoveal fibrosis secondary to n - amd were not included in this review . about half of our patients had previously received anti - vegf injections for n - amd and presented with acute decrease in central vision due to thick smh after a median of 20 weeks from their last anti - vegf injection , similar to that in levine et al . we noted that mean injections of 11.4 over a mean follow - up of 18.4 months are not much different from the mean of 14.1 injections of bevacizumab over 2 years in the prn arm of catt . thus , vascular proliferation is needed for scar formation , and its inhibition by early anti - vegf monotherapy may prevent scarring . however , it seems that disciform scar formation can be prevented and good anatomical and visual outcome can be accomplished in patients treated with anti - vegf monotherapy within 1 week from the onset of smh secondary to n - amd .

the competition for solar photons among aquatic photosynthetic organisms striving to maintain viability at various depths in the water column is fierce , often requiring adaptation of the species for survival . a prime example of the development of adaptive traits is found in the purple photosynthetic bacterium , rhodoblastus ( rbl . ) acidophilus ( formerly rhodopseudomonas acidophila ) strain 7050 . this bacterium is able to alter its number and size of photosynthetic units as well as its pigment composition and light absorption properties of the major light harvesting ii ( lh2 ) antenna pigment protein complex in response to changes in illumination conditions . if the bacterium is grown under high light , the well - characterized b800 - 850 lh2 complex is formed having bacteriochlorophyll ( bchl ) absorption bands near 800 and 850 nm , and rhodopin and rhodopin glucoside are the primary carotenoid pigments . under low - light conditions , genes that code for a variant lh2 denoted b800 - 820 ( also sometimes referred to as lh3 in the literature ) are activated , the bchl qy absorption band near 850 nm shifts to 820 nm , and the organism accumulates rhodopinal and rhodopinal glucoside in the lh2 complex as primary carotenoid pigments ( figures 1a and 1b ) . this response to changes in ambient light is controlled by a combination of a classical two component regulatory system and bacteriophytochromes that regulate the synthesis of the photosynthetic apparatus . structures of ( a ) all - trans - rhodopin glucoside and 13-cis - rhodopinal glucoside and ( b ) one - third portion of the lh2 b800 - 820 ring complex from rbl . acidophilus strain 7050 ( pdb 1ijd ) showing the protein - bound bchls ( green ) and carotenoids ( purple ) . the shift of the bchl qy band from 850 nm to 820 nm that occurs at low light is due to alterations in the amino acid sequence of the apoproteins that are assembled in the variant lh2 pigment protein complex . results from x - ray crystallography ( figure 1b ) , site - directed mutagenesis , and resonance raman spectroscopy indicate that h - bonding residues 44 ( tyr ) and 45 ( trp ) in the b800 - 850 lh2 prevent rotation of the c3-acetyl group of the b850 bchl and fix the functional group so that its c = o -electron bond resides in a planar orientation relative to the porphyrin macrocycle . the conversion of these h - bonding residues to non - h - bonding 44 ( phe ) and 45 ( leu ) in the b800 - 820 lh2 leads to a rotation of the c3-acetyl group out of the plane of the porphyrin ring , thereby inhibiting delocalization of the -electron conjugation to the acetyl carbonyl , resulting in more restricted -electron delocalization and consequently a blue shift of the qy band from 850 nm to 820 nm . accompanying the shift of the bchl qy absorption band in the lh2 complex is a change in the absorption spectrum of the carotenoid . under low - light growth conditions , rhodopin and rhodopin glucoside are enzymatically converted to rhodopinal and rhodopinal glucoside as an aldehyde group replaces the methyl group at carbon c20 in the carotenoid structures ( figure 1a ) . the spectral origin ( 00 ) vibronic band of rhodopin glucoside in methanol appears at 500 nm , whereas for rhodopinal glucoside , the band is less resolved spectrally , and it is located at 540 nm in the same solvent ( figure 2 ) . previous workers compared the carotenoid - to - bchl energy transfer properties of lh2 complexes isolated from cells of rbl . acidophilus strain 7050 grown under different illumination conditions and found that there was an increase in the energy transfer efficiency from between 50 and 55% for the b800 - 850 complex to between 70 and 75% for the b800 - 820 complex . however , the previous investigation did not address the specific reasons for the increase , i.e. , whether changes in the bchl absorption spectra , or the conversion of rhodopin to rhodopinal in the protein complex , or both factors , were responsible for the enhanced ability of the lh2 complex to effectively harvest photons in the region of carotenoid absorption . moreover , the previous work and subsequent ultrafast spectroscopic experiments carried out on the b800 - 820 lh2 complex from rbl . acidophilus strain 7050 did not assign specific values to the energy transfer efficiencies of the individual carotenoids bound in the complexes , nor has there been any direct comparison of the spectra and dynamics of the excited states of rhodopin and rhodopinal either in solution or in the lh2 complexes . these data are important for addressing the specific mechanism of how these alterations in bchl and carotenoid structures and spectra increase the carotenoid - to - bchl energy transfer efficiency and , as a consequence , enhance the viability of the photosynthetic bacterial organism . normalized steady - state absorption spectra of ( a ) rhodopin glucoside and ( b ) rhodopinal glucoside in carbon disulfide , benzyl alcohol , methanol , and acetonitrile recorded in 2 mm path length cuvettes at room temperature . energy transfer from carotenoids involves at least two excited singlet states that can act as donors of absorbed light energy to bchl . these are the s1 ( 2ag ) and s2 ( 1bu ) states whose properties are strikingly distinct . a one - photon transition from the ground s0 ( 1ag ) state to the s1 ( 2ag ) state is forbidden by symmetry , whereas a transition to the s2 ( 1bu ) state is strongly allowed . the s0 ( 1ag ) s2 ( 1bu ) transition is responsible for the vibrant coloration of carotenoids in nature . motivated by the landmark report of the x - ray crystal structure of the lh2 complex from rbl . acidophilus strain 10050 , several investigators sought to understand the role of the s1 ( 2ag ) and s2 ( 1bu ) excited singlet states in the mechanism of energy transfer to bchl in this pigment used ultrafast time - resolved optical spectroscopy applied to the lh2 complex prepared from rbl . acidophilus strain 10050 and reported that the s2 ( 1bu ) state of rhodopin glucoside dominated the pathway for energy transfer to bchl despite its extremely short intrinsic lifetime of 120 fs in solution . the s1 ( 2ag ) state of rhodopin glucoside , which has a much longer lifetime of 4 ps in solution , was reported to make only a minor contribution to the overall energy transfer efficiency . in another study using steady - state and ultrafast time - resolved spectroscopy to elucidate the carotenoid - to - bchl energy transfer mechanism in the lh2 complex from rbl . acidophilus 10050 , cong et al . reported the partitioning of energy transfer to be 23 7% from the s1 ( 2ag ) state and 63 10% from the s2 ( 1bu ) state . a recent broadband 2d electronic spectroscopic investigation of lh2 complexes from rbl . acidophilus 10050 and rhodobacter sphaeroides strain 2.4.1 provided convincing evidence for the additional involvement of a dark intermediate state in the carotenoid - to - bchl energy transfer pathway . the present work provides a detailed experimental and computational comparison of the excited state energy levels , spectra , and dynamics of rhodopin , rhodopinal , and their associated glucosides in various solvents and in their respective lh2 complexes isolated from rbl . the use of steady - state absorption , fluorescence and fluorescence excitation spectroscopy and ultrafast time - resolved transient absorption spectroscopy in the visible spectral region have revealed the rates and efficiencies of carotenoid - to - bchl energy transfer for the individual carotenoids . the data address the questions of how and why photosynthetic organisms alter their pigment composition and light - harvesting characteristics to ensure survival under the challenging , light - deprived environmental conditions in which they are sometimes found . acidophilus 10050 and 7050 cultures were grown anaerobically in the light using pfennig s medium . normal growth conditions ( hereafter referred to as high - light ( hl ) conditions ) used continuous illumination at an intensity of 30 mol s m. the rbl . acidophilus 7050 culture was also grown at a lower light intensity ( hereafter denoted low - light ( ll ) ) ranging from 3.7 to 5 mol s m. cells were harvested by centrifugation at 4000 g in a beckman model j-6b centrifuge . the resulting pellets were resuspended in 1 l of 20 mm mes ph 6.8 buffer , containing 100 mm kcl , and centrifuged again to remove any residual media . extraction of the carotenoids from whole cells of the bacteria was accomplished by mixing 24 g of thawed cells with 30 ml of methanol at room temperature and stirring in the dark for 15 min . the mixture was then centrifuged at 3000 g using an ss-34 rotor at 4 c in a sorvall rc-5b centrifuge . the pellet was then mixed with 30 ml of fresh methanol and centrifuged repeatedly until the cells appeared gray , signifying that all the pigments had been extracted . the supernatant from the second and subsequent extractions contained primarily carotenoids as evidenced by absorption spectra recorded using a varian cary 50 uv / visible spectrometer . the dried extracts were dissolved in acetonitrile / methanol ( 6:4 , v / v ) and analyzed using a waters 600e/600s hplc system equipped with a waters atlantis t3 obd preparative column having dimensions of 19 100 mm . the mobile phase consisted of acetonitrile / methanol ( 6:4 , v / v ) delivered isocratically at a flow rate of 7.0 ml / min . individual peaks were collected and identified by mass spectrometry using a fisons quattro ii instrument employing atmospheric pressure chemical ionization ( apci ) in negative mode with the following conditions : corona voltage , 2.5 v ; cone voltage , 25 v ; source temperature , 120 c ; probe temperature , 300 c , mobile phase , acetonitrile . all solvents were hplc - grade and were purchased from sigma - aldrich corp . briefly , 5 g of pelleted whole cells of the bacteria were suspended in 30 ml of 20 mm tris buffer adjusted to ph 8.0 using 6 m hcl ( hereafter referred to as tris buffer ) , and 2050 mg of dnase and a few grains of mgcl2 were added to degrade the released dna during cellular disruption . the sample was briefly homogenized using a glass tissue homogenizer to ensure smooth passage through the french press operating at 15,000 psi . cells were passed through the press three times to ensure complete disruption , and the resulting suspension was centrifuged for 2 h at 4 c in a type 70 ti rotor spinning at 180000 g in a beckman l8 - 55 m ultracentrifuge . the resultant pellet containing membrane fragments was diluted using tris buffer to an od of 50 measured in a 1 cm cuvette at the bchl qy absorption band maximum ( 800 nm for ll grown rbl . the membranes were then solubilized by adding 30% lauryldimethylamine oxide ( ldao ) dropwise to a final concentration of 1.0% and allowing the sample to incubate for 60 min while being stirred at room temperature in the dark . the sample was then centrifuged for 30 min at 27000 g in the sorvall rc-5b centrifuge to remove denatured protein and any nonsolubilized material . the supernatant containing the solubilized proteins was then collected for further treatment using sucrose density gradient ultracentrifugation . solutions of 0.2 , 0.4 , 0.6 , and 0.8 m sucrose were prepared using tris buffer containing 0.1% ldao and were carefully layered to form discontinuous gradients in the tubes by using the following amounts : 0.8 m , 5.0 ml ; 0.6 m , 6.5 ml ; 0.4 m , 6.5 ml ; and 0.2 m , 5.0 ml . the tubes were then placed in a ti70 rotor and spun at 160000 g for 12 h at 4 c in the beckman l8 - 55 m ultracentrifuge . the procedure effectively separates any free pigments present in the 0.2 m sucrose layer , from the lh2 complex appearing in the 0.4 m sucrose layer , from the lh1-rc core complexes that appear at the interface of the 0.6 and 0.8 m sucrose solutions . the 0.4 m sucrose solution layer containing the lh2 complex was carefully removed from each tube and pooled . the complex was then purified further by column chromatography using de52 anion exchange resin ( 15 g , whatman scientific ) packed into a 5 cm diameter 30 cm long solid phase , sintered glass column pre - equilibrated with several bed volumes of tris buffer . after loading the sample onto the column , several bed volumes of tl buffer ( 0.1% ldao , 20 mm tris ph 8.0 ) were applied to remove the sucrose . the lh2 complex was then eluted using tl buffer containing increasing concentrations of nacl in 30 mm increments starting with 10 mm . acidophilus 10050 and 7050 cells eluted between 10 and 20 mm nacl , whereas that isolated from ll rbl . the purity of the eluting fractions was monitored by absorption spectroscopy using a shimadzu uv-1700 pharmaspec spectrometer . fractions from hl samples exhibiting spectra with an 850 nm to 280 nm ratio of > 3.0 were pooled for further purification . for the ll sample , fractions exhibiting spectra with an 800 nm to 280 nm absorbance ratio of > 2.5 were pooled for further purification . the pooled sample was reduced to a volume of < 1 ml by centrifugation using vivaspin 4 50 k mw cutoff concentrators ( sartorius stedim biotech ) placed in an 11390 rotor and spun at 3250 g in a sigma 3k30 centrifuge . the concentrated lh2 was further purified using an akta primeplus ( ge healthcare ) automated chromatography system equipped with an xk-16 long gel filtration column filled with superdex-200 . 0.5 ml fractions were collected of the lh2 band and subsequently assayed using the ratio of the absorbance maximum of the bchl qy band to the protein absorbance at 280 nm . fractions having an 800 nm to 280 nm ( from the ll - grown rbl . acidophilus 7050 ) ratio of > 2.7 or an 850 nm to 280 nm ( from the hl - grown rbl . acidophilus cells ) ratio of 3.3 were pooled and concentrated using the vivaspin 4 50 k mw cutoff concentrators to an od of 100 measured at the absorbance maximum of the bchl qy band in a 1 cm cuvette . the sample was then divided into 30 l aliquots , placed in pcr tubes , flash frozen in liquid nitrogen , and stored in a 80 c freezer . a 10 l aliquot of the frozen rbl . acidophilus 7050 lh2 complex was thawed on ice , and the liquid was evaporated to dryness using nitrogen gas . the remaining residue was then redissolved in 2 ml of methanol to release the pigments . this extract was then centrifuged at 13600 g for 2 min at room temperature in a fisher scientific 235c benchtop microcentrifuge . the supernatant was then analyzed using a waters 600e/600s hplc system equipped with a waters atlantis t3 5 m analytical column having dimensions of 4.6 250 mm . the mobile phase consisted of an isocratic delivery of acetonitrile / methanol ( 6:4 , v / v ) at a rate of 2.0 ml / min . acidophilus 10050 lh2 complex was dried under nitrogen gas and denatured with 2 ml of acetone . following centrifugation , the supernatant was removed and 2 ml of fresh acetone was added to the remaining pellet , and the mixture was centrifuged again , this time resulting in a colorless pellet . the sample was taken up in 1 ml of acetonitrile / methanol ( 6:4 , v / v ) and analyzed on the same hplc system with a waters atlantis t3 obd 5 m preparative column . the mobile phase was the same as described above , but with the flow rate increased to 7.0 ml / min . the molar percentages of the carotenoids were calculated using the area of each hplc peak detected at the wavelength of maximum absorption divided by the extinction coefficient of the carotenoid , and then determining the percentage of each pigment relative to the total carotenoid content . peaks that could not be conclusively identified by mass spectrometry remain unidentified , but are presumed to be isomers formed during the extraction procedure . all steady - state absorption and fluorescence emission and excitation spectroscopic measurements were carried out in 1 cm square cuvettes at room temperature unless otherwise stated . rhodopin glucoside and rhodopinal glucoside were dissolved in spectroscopic grade carbon disulfide ( acros organics ) , benzyl alcohol ( sigma - aldrich ) , methanol ( sigma - aldrich ) , or acetonitrile ( sigma - aldrich ) . steady - state absorption spectra of the carotenoids in various solvents and in the lh2 complexes were obtained using either a varian cary 50 or a cary 5000 uv visible spectrophotometer . fluorescence emission and excitation spectra were recorded using a jobin - yvon horiba fl3 - 22 fluorimeter equipped with double excitation and emission monochromators having 1200 grooves / mm gratings , a 450 w osram xbo xenon arc lamp , and a hamamatsu r928p photomultiplier tube detector . emission spectra of the lh2 complexes were recorded using samples having an od between 0.025 and 0.1 in a 1 cm path cuvette at the bchl qx band at 591 nm , which was also the excitation wavelength . the excitation and emission slit widths corresponded to bandpasses of 6 and 3 nm , respectively for experiments on the lh2 complex from rbl . acidophilus 7050 ( hl and ll ) lh2 complexes were obtained using excitation and emission slit widths corresponding to bandpasses of 12 and 6 nm , respectively . all emission spectra were corrected using an emission correction factor file generated by taking the ratio of the spectral response of a calibrated 200 w quartz tungsten - halogen filament lamp and the instrument detection system . fluorescence excitation spectra were recorded by monitoring the bchl emission at its maximum wavelength ( 870 nm for the b800 - 850 complexes and 860 nm for the b800 - 820 complex ) using samples with an od of 0.025 in a 1 cm path cuvette at the bchl qx band . emission was detected at a right angle relative to the excitation with bandpasses corresponding to 6 nm ( 10050 lh2 ) , 7 nm ( 7050 hl ) , and 4 nm ( 7050 ll ) for the excitation monochromator , and 12 nm ( 10050 lh2 ) and 14 nm ( 7050 hl and ll ) for the emission monochromator . an excitation correction factor file was used to correct for the wavelength variability of the source lamp and excitation monochromator . this file was generated using a photodiode calibration kit consisting of a photodiode assembly and a dm303-p module that was rented from horiba . probe ultrafast transient absorption spectroscopy was carried out using a helios femtosecond transient absorption spectrometer ( ultrafast systems llc , sarasota , fl , usa ) coupled to a laser setup that has been previously described . surface xplorer pro 1.2.2.26 ( ultrafast systems llc , sarasota , fl , usa ) was used to correct for the dispersion in the transient absorption spectra . samples having an od between 0.2 and 0.5 in a 2 mm path cuvette at the carotenoid spectral origin ( 00 ) band were mixed continuously using a magnetic microstirrer to avoid photodegradation . the pump laser had an energy of 1 j / pulse focused on a 1 mm diameter spot , which corresponds to a laser intensity between 3.2 and 3.9 10 photons / cm . the integrity of the samples was assayed by taking steady - state absorption spectra before and after laser excitation . global fitting of the transient absorption data sets was performed in asufit 3.0 provided by dr . evaldas katilius at arizona state university and carried out according to a sequential excited state decay model which yielded evolution associated difference spectra ( eads ) . reconstruction of the 1-t and fluorescence excitation spectra was done using origin software version 9 . ground state geometries were generated using b3lyp/6 - 31g(d ) methods as implemented within gaussian 09 . excited state geometries were generated using single - configuration interaction ( cis ) methods and full single ci . the effect of the solvent environment was simulated by using the polarizable continuum model ( pcm ) . spectroscopic properties were calculated using mndo - psdci and equation of motion coupled - cluster singles and doubles ( eom - ccsd ) methods . the mndo - psdci methods are semiempirical and have been used successfully to study long - chain polyenes and carotenoids . the eom - ccsd calculations were carried out using a d95 double - zeta basis set , but the size of the target systems limited the use of this method to only rhodopin . the steady - state absorption spectra of rhodopin glucoside and rhodopinal glucoside recorded in carbon disulfide , benzyl alcohol , methanol , and acetonitrile are shown in figure 2 . the primary bands in these spectra represent an electronic transition from the ground s0 ( 1ag ) state to the s2 ( 1bu ) state . in any particular solvent , the spectrum of rhodopinal glucoside is shifted to longer wavelength by 3040 nm compared to that of rhodopin glucoside . the spectrum of rhodopin glucoside displays well - resolved vibronic bands in all of the solvents , whereas the spectrum of rhodopinal glucoside is much less structured , except in carbon disulfide , where a shoulder on the long - wavelength side of the primary absorption band is observed . also , the high polarizability of carbon disulfide ( p( ) = 0.354 ) results in a 3040 nm red shift of the spectra of rhodopin glucoside and rhodopinal glucoside compared to their spectra recorded in the less polarizable solvents , acetonitrile ( p( ) = 0.210 ) and methanol ( p( ) = 0.202 ) . benzyl alcohol , which has a polarizability value ( p( ) = 0.314 ) between those of carbon disulfide and methanol or acetonitrile , red shifts the spectrum of rhodopinal glucoside more than for rhodopin glucoside ( figure 2 ) . this is undoubtedly due to the presence of the aldehyde group on rhodopinal glucoside , which interacts more strongly with this solvent than the methyl group in the same position on rhodopin glucoside . except for a small change in relative intensities of the vibronic bands of rhodopin glucoside , the spectra are not significantly affected by changing the solvent from a protic ( methanol ) to a nonprotic ( acetonitrile ) polar solvent . it should be mentioned that the glucoside moiety has no effect on the positions and intensities of the absorption bands . the spectra of rhodopin glucoside and rhodopinal glucoside are indistinguishable from the corresponding spectra of rhodopin and rhodopinal . acidophilus 10050 , 7050 hl , and 7050 ll recorded at room temperature are shown in figure 3 . all spectra show the strong s0 ( 1ag ) s2 ( 1bu ) transition characteristic of carotenoids in the 400550 nm region . also , the positions of the bchl soret band at 375 nm and the bchl qx band at 590 nm are nearly identical for all of the complexes . however , one bchl qy band of the lh2 complex is located at 800 nm , and the other is at 859 nm for strain 10050 , at 855 nm for 7050 hl , and at 823 nm for 7050 ll . in addition , compared to the spectrum of the lh2 complex from strain 10050 , the spectra of the lh2 complexes from 7050 hl and 7050 ll show more absorption in the region between 550 and 600 nm where the spectrum of the carotenoid partially overlaps with the bchl qx band at 590 nm . this is due to the presence of rhodopinal and rhodopinal glucoside in the lh2 complexes from strain 7050 . acidophilus 7050 will always result in a very small amount of the b800 - 820 lh2 complex in hl cells due to the effect of light shading in the culture media . likewise , a small amount of b800 - 850 lh2 complex will be present in the ll cells due to the fact that there is a limit to how low the light intensity can be adjusted to ensure a realistic amount of bacterial growth . a spectral analysis of the bchl absorption bands in the qy region ( figure s1 in the supporting information ) shows that this amount is less than 20% . moreover , it should be noted that the regulatory pathways for the switch from rhodopin glucoside to rhodopinal glucoside and from b800 - 850 to b800 - 820 are independent of each other . during growth at progressively decreasing light intensity , the carotenoid pathway switch is activated before , i.e. , at a higher light intensity than the switch that controls the type of complex present . therefore , it is possible to obtain a b800 - 850 complex that contains a significant amount of rhodopinal glucoside as is the case for lh2 complex isolated from 7050 hl cells ( table 1 ) . the pronounced similarity in the structures of the b800 - 850 and b800 - 820 protein complexes precludes complete separation by chromatographic techniques . however , by a judicious choice of excitation and detection wavelengths in the steady - state and transient absorption spectroscopic experiments , the properties of the individual pigment acidophilus 10050 , 7050 ll , and 7050 hl recorded in 2 mm path length cuvettes at room temperature . uncertainties in the values , based on standard deviations from the mean , were equal to or less than two percentage points . the carotenoid composition of the different lh2 complexes was determined by hplc analyses carried out as illustrated in figure 4 . the molar percentages of the carotenoids in the different lh2 complexes are given in table 1 . acidophilus 7050 cells display a significant increase in total rhodopinal ( defined in this context as rhodopinal plus rhodopinal glucoside ) concurrent with a decrease in total rhodopin ( defined here as rhodopin plus rhodopin glucoside ) compared to that found in the bacterium grown under hl conditions . total rhodopinal in the lh2 complexes increased from 32% in the 7050 hl sample to 65% ( 58% + 7% ) in the 7050 ll sample ( table 1 ) . concurrently , total rhodopin in the lh2 complexes decreased from 56% ( 28% + 28% ) to 15% ( 5% + 10% ) when cells were grown using ll ( table 1 ) . as previously reported and confirmed by the present work , the lh2 complex from rbl . acidophilus 7050 lh2 complexes prepared from cells grown under ll ( top trace ) and hl ( bottom trace ) conditions . the major pigments were identified as follows : 1 , rhodopinal glucoside ; 2 , rhodopinal ; 3 , rhodopin glucoside ; 4 , bchl a ; 5 , rhodopin ; and 6 , lycopene . the fluorescence spectra of the lh2 complexes recorded using 591 nm excitation , which excites the bchl qx band , are shown in figure 5 ( blue traces ) . the maximum wavelength of emission occurs at 870 nm for the lh2 complexes from 10050 and 7050 hl , and at 860 nm for the lh2 complex from 7050 ll . fluorescence spectra were also recorded using excitation wavelengths of 560 , 570 , 580 , and 590 nm , but no changes in the position or shape of the resulting emission spectra were observed . fluorescence excitation spectra of the lh2 complexes are also shown in figure 5 ( red traces ) . these spectra were recorded by detecting the fluorescence from the samples at the maximum wavelength of bchl emission , but identical lineshapes were observed using any detection wavelength between 820 and 880 nm ( figure s2 in the supporting information ) . emission ( blue ) , excitation ( red ) , and 1-t ( black ) spectra of lh2 complexes obtained from rbl . the green line shows the ratio of the normalized excitation and 1-t spectra and in the region of carotenoid absorption gives a quantitative measurement of the carotenoid - to - bchl energy transfer efficiency . it is somewhat surprising that the fluorescence maximum of the b800 - 820 complex occurs at 860 nm rather than closer to the 820 nm qy band . however , as mentioned above , previous work detailing the effect of light intensity and temperature on cell growth and the formation of the b800 - 820 complex indicated that the conversion of rhodopin to rhodopinal occurs prior to the conversion of b800 - 850 to b800 - 820 . therefore , it is likely that the fraction of b800 - 850 complex in our ll sample ( see figure s1 in the supporting information ) contains rhodopinal and represents the main emission component . this interpretation rationalizes the appearance of the fluorescence maximum at 860 nm and the fluorescence excitation spectra not being affected by changing the detection wavelength . t is transmittance ( black traces ) , and the excitation spectra ( red traces ) were normalized at the bchl qx and qy bands , and the ratio of the excitation and 1-t spectral amplitudes were determined in the region from 300 to 650 nm ( green traces ) . these traces represent the efficiency of excitation energy transfer ( eet ) to bchl , which was found in the carotenoid absorption region between 425 and 550 nm to be in the range of 55% to 61% for the 10050 lh2 complex consistent with previously published results . the lh2 complexes from 7050 hl and 7050 ll displayed higher carotenoid - to - bchl eet efficiencies and were in the range of 63% to 71% and 76% to 86% , respectively . hplc analysis ( table 1 ) revealed that the lh2 samples from the 7050 samples contain both rhodopin and rhodopinal ( and their associated glucosides ) in different amounts . therefore , in order to examine the contributions of these individual chromophores to the spectra of the pigment protein complexes , reconstructions of the 1-t ( left panel , figure 6 ) and fluorescence excitation ( right panel , figure 6 ) spectra were carried out based on the absorption spectra of the hplc - purified carotenoids recorded in benzyl alcohol . the spectra of the carotenoids in the lh2 protein ( figure 3 ) are well - reproduced by their spectra in benzyl alcohol ( figure 2 ) due to the fact that this solvent has a polarity and polarizability similar to the average of those of the protein in the binding environment of the carotenoid . the spectral reconstructions yielded the specific carotenoid - to - bchl eet efficiencies for rhodopin and rhodopinal from the ratios of the individual fluorescence excitation and 1-t bands in the profiles . ( because rhodopin and rhodopinal have identical absorption ( and 1-t ) spectra as their corresponding glucoside derivatives , in this context and unless explicitly noted otherwise , any statements about the spectra of rhodopin or rhodopinal should be taken to mean the combined contribution from the chromophores associated with both the glucoside and non - glucoside molecules . ) the eet efficiency for rhodopin in the lh2 complex from the 10050 sample becomes evident from a side - by - side , horizontal comparison of the amplitudes of the 1-t and fluorescence excitation spectra ( figures 6a and 6b ) in the region of carotenoid absorption , and was found to be 56 1% , consistent with previous reports . a similar analysis was carried out to obtain the values for the individual carotenoid - to - bchl eet efficiencies in the lh2 complex from the 7050 ll sample ( figures 6c and 6d ) , which were found from the spectral reconstruction to be 54 5% for rhodopin ( orange traces ) and 97 2% for rhodopinal ( purple traces ) . the computed individual carotenoid - to - bchl eet efficiencies for the lh2 complex prepared from 7050 hl cells ( figures 6e and 6f ) were 50 3% for rhodopin ( orange traces ) and 98 2% for rhodopinal ( purple traces ) , consistent with the values determined for the lh2 complex from 7050 ll . these results are summarized in table 2 and reveal the remarkable finding that rhodopinal in the lh2 complex from rbl . acidophilus 7050 transfers essentially all of its excited state energy to bchl with minimal loss through internal conversion to the ground state . this stands in striking contrast to rhodopin in the lh2 complexes from all rbl . reconstruction of the ( a , c , e ) 1-t and ( b , d , f ) fluorescence excitation spectra ( black traces ) of the lh2 complexes from rbl . the 1-t spectra of purified rhodopin glucoside ( orange traces ) and rhodopinal glucoside ( purple traces ) were recorded in benzyl alcohol and summed to generate the reconstructed spectra ( red traces ) . the bchl bands in the soret region between 300 and 400 nm and in the qx region near 600 nm were modeled using gaussian functions ( green lines ) for simplicity . the uncertainties in the amplitudes were obtained by comparing the reconstructed spectra with those experimentally recorded . those values figure 7 shows ultrafast time - resolved transient absorption spectra of rhodopin glucoside ( left panels ) and rhodopinal glucoside ( right panels ) recorded in carbon disulfide , benzyl alcohol , methanol , or acetonitrile at various delay times after laser excitation . upon excitation , an immediate onset of bleaching of the strongly allowed s0 ( 1ag ) s2 ( 1bu ) transition occurs , resulting in a negative signal in the 450550 nm region . in addition , a strong positive signal in the 520700 nm region appears that can be attributed to excited state absorption ( esa ) associated with the s1 ( 2ag ) sn transition . this suggests that the decay of the s2 state via internal conversion to populate the s1 state is occurring on the same time scale as the instrument response time of 100 fs . similar to the steady - state absorption spectra of the carotenoids , the excited state absorption bands shift to longer wavelength and are broader as the polarizability of the solvent increases . the spectra are broadest and most red - shifted in the highly polarizable solvent , carbon disulfide , compared to the spectra recorded in the other solvents . a similar effect is evident in the spectrum of rhodopinal glucoside in carbon disulfide and benzyl alcohol ( figure 7 , right panels ) , where it is also clear that the spectral bands are broader overall than the spectra of rhodopin glucoside in the same solvents ( figure 7 , left panels ) . also , an additional peak at 740 nm , which is not found in the spectra of rhodopin glucoside , is seen in the transient absorption spectra from rhodopinal glucoside . unlike the main s1 ( 2ag ) sn transient absorption band , this feature is insensitive to the polarizability of the solvent , and therefore appears more separated from the main esa feature in methanol and acetonitrile compared to in carbon disulfide . transient absorption spectra of rhodopin glucoside and rhodopinal glucoside in carbon disulfide , benzyl alcohol , methanol , and acetonitrile recorded at room temperature using the indicated excitation wavelengths . in order to obtain detailed information regarding the dynamics of the excited states of the carotenoids , global fitting according to a sequential decay model , resulting in evolution associated difference spectra ( eads ) , was carried out on the transient absorption data sets . for both rhodopin glucoside and rhodopinal glucoside in all four solvents , the first eads has a very short lifetime ranging from < 100 to 120 fs , and the profile of this component shows bleaching of the ground state s0 ( 1ag ) s2 ( 1bu ) absorption and stimulated emission from the s2 ( 1bu ) state . it is important to point out here that although the best global fitting results are achieved in some cases using time constants smaller than 100 fs , these very small values can not be taken factually because the instrument response time of the laser spectrometer is on the order of 100 fs . hence , these values are specified as < 100 fs . more precise values in this time domain were reported by macpherson et al . who used fluorescence upconversion spectroscopy and found the s2 ( 1bu ) lifetime of rhodopin glucoside in benzyl alcohol to be 124 8 fs . the second eads component for both molecules has a time constant that ranges from 330 to 530 fs in the different solvents . this component can be assigned to a transition from a vibronically hot s1 ( 2ag ) sn excited singlet state due to the fact that its broad esa peak narrows and shifts to shorter wavelength upon decaying to form the third eads component . the second and third eads for rhodopinal glucoside in all solvents also show the additional peak at 740 nm alluded to above , which is not found in the spectra of rhodopin glucoside . the fact that this feature does not depend on solvent polarity indicates that it is not associated with the formation of an intramolecular charge transfer ( ict ) state in rhodopinal glucoside . evolution associated difference spectra ( eads ) obtained from globally fitting the transient absorption data sets from rhodopin glucoside and rhodopinal glucoside in carbon disulfide , benzyl alcohol , methanol , and acetonitrile given in figure 7 . the band profile of the third eads component for both molecules is very strong and well - known to be associated with esa from the relaxed s1 ( 2ag ) state making a transition to a higher excited singlet state . the lifetime of this component for rhodopin glucoside in all solvents was found to be in the narrow range of 3.8 to 4.3 ps with no obvious effect of solvent polarity on the value . however , the s1 ( 2ag ) lifetime for rhodopinal glucoside in the polar solvents methanol and acetonitrile was 3.7 and 3.9 ps respectively , which are slightly shorter than the 4.4 and 4.7 ps values found for the molecule in the less polar solvents , benzyl alcohol and carbon disulfide , respectively . the fact that the lifetime of this component is reasonably similar for both rhodopin glucoside and rhodopinal glucoside indicates that neither the configuration , which is 13-cis for rhodopinal glucoside in solution compared to all - trans for rhodopin glucoside ( figure 1a ) , nor the presence of the aldehyde group at carbon c20 on rhodopinal glucoside compared to the methyl group at the same position on rhodopin glucoside results in any significant change in the dynamics of the s1 state that would impact its role in light - harvesting . the fourth eads component of both molecules in all solvents has a lifetime that ranges from 5.9 to 15.3 ps . this component is rather weak , but displays negative features associated with ground state bleaching as well as a positive feature on the short wavelength side of the main s1 sn absorption profile . the negative features indicate that some fraction of the carotenoid population is still in an excited state . the short wavelength band is reminiscent of the s * state initially proposed to be an intermediate state between the s1 ( 2ag ) and s2 ( 1bu ) excited singlet states that is involved in both s2 ( 1bu ) depopulation and carotenoid triplet state formation in light harvesting complexes . subsequent work on several open - chain carotenoids has shown that the s * yield is larger for molecules with longer -electron conjugation suggestive of it being associated with a twisted molecular conformation of the carotenoid in the s1 ( 2ag ) state . finally , a very weak , infinitely long - lived component was necessary to obtain a completely satisfactory fit to the data sets . the lh2 complexes were excited in the region of carotenoid absorption at either 525 or 570 nm , and then transient absorption spectra were recorded at various delay times after the pump laser pulse ( figure 9 ) . acidophilus 10050 displayed transient absorption profiles ( top panel of figure 9 ) that were similar to previously reported spectra and include the instantaneous onset of bleaching of the carotenoid ground state spectrum and broad positive esa at intermediate times , followed by the appearance of a strong narrow s1 sn transition at 580 nm at later times . the hplc pigment analysis of the lh2 complex from strain 10050 ( table 1 ) revealed that the carotenoid composition consists of roughly equal amounts of rhodopin and rhodopin glucoside with a small amount of lycopene , all of which have identical absorption spectra . using this information regarding the spectra of the carotenoids in the lh2 complex from rbl . acidophilus 10050 as a control , the features attributable to rhodopin and rhodopinal can be distinguished in the transient spectra of the lh2 complexes prepared from 7050 ll and 7050 hl cells , which contain a mixture of these carotenoids ( table 1 ) . ( recall that any reference to rhodopin or rhodopinal in this context should be taken to mean the combined spectral properties of the molecules and their respective glucosides , which are indistinguishable . ) laser excitation at 525 nm of the 7050 ll or 7050 hl lh2 complexes results in a combination of transient absorption signals from rhodopin and rhodopinal because both molecules absorb at this wavelength . selective excitation of rhodopinal can be achieved by tuning the pump laser to 570 nm , which is a wavelength where rhodopin does not absorb . acidophilus 10050 , 7050 ll , and 7050 hl recorded at room temperature using the indicated excitation wavelengths . transient absorption spectra from the 7050 ll and hl lh2 complexes excited at 525 nm ( combined rhodopin plus rhodopinal excitation ) or at 570 nm ( selective rhodopinal excitation ) are shown in figure 9 . similar to the transient absorption spectra from strain 10050 , the transient spectra recorded for these lh2 complexes at a 50 fs delay time ( black traces in figure 9 ) have negative features corresponding to the bleaching of the carotenoid s0 ( 1ag ) s2 ( 1bu ) ground state absorption bands . in addition , there is a positive signal in the 540700 nm region which represents an s1 ( 2ag ) sn transition indicating very fast decay of the s2 ( 1bu ) state to populate the s1 ( 2ag ) state of the carotenoid . in the 200 fs delay time spectra ( red traces ) , the ground state bleaching has partially recovered , indicative of energy transfer to bchl , but the strong positive s1 ( 2ag ) sn absorption band has gained amplitude and is broader compared to the subsequent 1 ps delay time spectrum ( green traces ) . the gain in amplitude of the feature associated with the s1 ( 2ag ) sn transition indicates that internal conversion from s2 to populate the s1 state is competing effectively with eet from s2 to bchl . a close examination of the 1 ps time delay ( green trace ) spectrum recorded for the 7050 ll lh2 excited at 525 nm ( second panel in figure 9 ) shows a narrow positive feature at 580 nm and a broader one at 630 nm . comparison of this spectrum with those taken at the same time delay from the 10050 lh2 excited at 525 nm ( selective rhodopin excitation , upper panel of figure 9 ) and the 7050 ll lh2 sample excited at 570 nm ( selective rhodopinal excitation , third panel of figure 9 ) reveals that the narrow peak at 580 nm in the spectrum from the 7050 ll lh2 excited at 525 nm is due to the s1 sn transition of rhodopin whereas the 630 nm peak is from rhodopinal . this is expected because excitation of the 7050 ll sample using 525 nm light excites both carotenoids which are present in this lh2 sample ( table 1 ) . this interpretation is supported by the transient absorption spectra resulting from 525 nm excitation of the 7050 hl lh2 complex ( fourth panel of figure 9 ) . note that the narrow positive feature at 580 nm in the 1 ps time delay spectrum ( green trace ) attributable to the s1 sn transition of rhodopin is much more pronounced relative to the broader band at 630 nm associated with rhodopinal . this is because the lh2 complex from 7050 hl contains 56% total rhodopin compared to the 7050 ll lh2 sample , which has only 15% rhodopin ( table 1 ) . as expected , selective excitation of rhodopinal at 570 nm in the 7050 hl lh2 sample ( bottom panel of figure 9 ) shows no sign of the narrow s1 sn feature belonging to rhodopin . the major positive band in the 1 ps time delay spectrum ( green trace ) is that of the s1 sn esa of rhodopinal . the 5 ps delay time spectra of the 10050 and 7050 ll lh2 samples ( blue traces in figure 9 ) show significantly diminished carotenoid ground state bleaching and reduced s1 sn esa indicating that a substantial amount of the carotenoid s1 state excited state population either has been transferred to bchl or has decayed via internal conversion back to the ground state . also in this time frame , an additional peak appears at 560 nm on the short wavelength side of the main esa peak and persists longer than 100 ps ( magenta trace ) . the eads components of the lh2 complexes obtained from a global fitting of the transient absorption data sets are shown in figure 10 . the fitting of the transient absorption spectra required five components , the first of which has a lifetime < 100 fs for all the lh2 complexes and has features associated with the bleaching of the ground state absorption as well as stimulated emission from s2 ( 1bu ) . this is consistent with the value of 57 fs reported by macpherson et al . the second eads component ( red traces in figure 10 ) has a lifetime ranging from 250 to 490 fs and has broad features in the carotenoid absorption region characteristic of vibronically hot s1 ( 2ag ) sn excited singlet state transition . this second eads has weaker negative bands in the carotenoid ground state absorption region than the first eads , indicating that some of the carotenoid molecules have returned to the ground state via eet from the s2 state to bchl . also evident in the second eads is a small negative dip at 590 nm appearing on the broad positive esa spectrum . this is due to the bleaching of the bchl qx band brought about by eet to bchl from the carotenoids . as the second eads component decays into a third ( green lines in figure 10 ) , the line shape narrows considerably for the 10050 lh2 excited at 525 nm and a strong band associated with the s1 ( 2ag ) sn transition is evident at 580 nm . this line narrowing is accompanied by a decrease in the extent of ground state bleaching indicating that more of the carotenoid molecules have returned to the ground state , perhaps by eet from the vibronically hot s1 state . this third component decays in 3.0 ps for the 10050 lh2 and represents the s1 lifetime of rhodopin in the lh2 pigment protein complex . the peak at 580 nm has much less amplitude in the third eads component from the 7050 ll lh2 sample obtained from data also using 525 nm excitation ( green trace in the second panel of figure 10 ) . this is due to the fact that this sample has much less total rhodopin ( rhodopin plus rhodopin glucoside ) than the 10050 lh2 . in fact , the peak at 580 nm is completely absent in the third eads obtained from data using 570 nm excitation ( green trace in the third panel of figure 10 ) . instead , a broad line shape peaking at 620 nm and belonging to rhodopinal is observed . note that the lifetime of this component is 1.2 ps in the 7050 ll lh2 and 1.3 ps in the 7050 hl lh2 , which is significantly shorter than the average value of 4.1 ps found for rhodopinal in the different solvents and also shorter than the value of 3.0 ps found for rhodopin in the lh2 complex from strain 10050 . this is suggestive of significantly faster eet to bchl from the s1 state of rhodopinal compared to rhodopin . evolution associated difference spectra ( eads ) obtained from globally fitting the transient absorption data sets of lh2 complexes from rbl . the uncertainties in the values were obtained by exploring the region of solution for each parameter according to the goodness of fit and minimization of the residuals . an infinitely long ( on the time scale of the experiment ) component the uncertainties were obtained by exploring the region of solution for each parameter according to the goodness of fit and minimization of the residuals . an infinitely long ( on the time scale of the experiment ) component was required for a good fit in all cases . the fourth eads component has a lifetime in the range 13 to 27 ps for all the data sets and a significantly diminished overall amplitude relative to the preceding eads profiles . the lifetime of this component , along with the clearly evident positive band on the short wavelength side of the feature attributable to the main s1 sn absorption band , and the wavy features in the region of the carotenoid ground state absorption are all characteristics of the s * state in accord with previous reports . the fifth and final eads component has an infinitely long lifetime on the time scale of the experiment and was necessary to obtain a satisfactory fit in all cases . the primary spectral feature in this last eads component is the negative amplitude at 590 nm , which corresponds to the bleaching of the bchl qx band that is expected to persist on the order of nanoseconds until the s1 state of bchl relaxes back to the ground state . quantum computations were carried out to augment the experimental results , but in order to make them tractable , the majority were carried out on the model chromophores shown in figures 11c and 11d . the model chromophores include the entire central polyene portion of rhodopin and rhodopinal , but replace the glucoside and aliphatic end groups with methyl groups . the resulting model polyenes have cs or c2h symmetry , and this symmetry , in combination with the smaller size , allows higher quality calculations to be carried out than would otherwise be possible . test calculations on the full and model chromophores indicate that the end groups do not have a significant impact on the atomic charges other than small changes in the charges on the carbon atoms at the ends of the polyene chains . single ci calculations on the full system ( figures 11a and 11b ) and the model systems ( figures 11c and 11d ) demonstrate that the end groups create less than a 0.02 ev shift in the transition energies . this conclusion is consistent with the observation that the absorption spectra of rhodopin and rhodopin glucoside are indistinguishable . b3lyp/6 - 31g(d ) calculated structures of ( a ) trans - rhodopin glucoside , ( b ) trans - rhodopinal glucoside , ( c ) trans - rhodopin , ( d ) trans - rhodopinal , ( e ) the s1 relaxed excited state of trans - rhodopinal , ( f ) 13-cis - rhodopinal , and 13-cis - rhodopinal model . the structures given in panels c , d , e , and g are simplified , higher - symmetry analogues used in the mndo - psdci , eom - ccsd and cas - scf theoretical calculations , which retain the full -system . the calculated vacuum dipole moments ( in debyes ( d ) ) and the dipole moment vectors ( the length is not relevant ) are shown underneath those structures that have a dipole moment . the dashed ellipse in panel d shows the primary repulsive atom atom interaction responsible for making the cis configuration more stable than the trans configuration by 8 kj / mol in nonpolar solvent ( n - hexane ) and 8.5 kj / mol in polar solvent ( acetonitrile ) . although a primary goal of this work is to understand the photophysical properties of rhodopinal in lh2 complexes , the process is started by identifying the geometry of rhodopinal in solution . previous studies have proposed that rhodopinal acquires a cis configuration in solution . based on density functional theory and an analysis of the absorption spectra , it is demonstrated here that rhodopinal takes on a cis configuration in both nonpolar and polar solvent . the all - trans rhodopinal chromophore exhibits just one significant intramolecular repulsion involving the interaction of the aldehyde hydrogen with the nearest hydrogen atom on the main polyene chain . the repulsion is both electrostatic ( both atoms have positive charge ) and steric ( the atoms are separated by 2 ) . . the aldehyde group can rotate out of plane , or the double bond adjacent to the aldehyde group can rotate 180 to create a cis linkage near the center of the polyene chain . based on b3lyp/6 - 31g(d)/pcm calculations the resulting 13-cis configuration is more stable than the all - trans configuration by 8 kj / mol in nonpolar solvent ( n - hexane ) and 8.5 kj / mol in polar solvent ( acetonitrile ) . this number is invariant to whether the calculation is done on the smaller model chromophore ( figure 11d ) or the full chromophore ( figure 11b ) . the value in n - hexane increases to 9.3 kj / mol when a much larger basis set [ 6 - 311+g(2d , p ) ] is used . experimental support for the 13-cis configuration of rhodopinal in solution is provided by comparing the excited state manifolds calculated for both the cis and the trans configurations with the experimental spectra . note that there is a relatively strong vibronically resolved band at 28 kk ( 28,000 cm ) observed in the experimental spectra ( solid yellow ) . the mndo - psdci calculations indicate that this band is associated with a ag excited state ( top right - hand panel of figure 12 ) , resulting in transition from the ground state that is often called the cis - band . it should be mentioned that the viability of the mndo - psdci methods was explored by carrying out an eom - ccsd calculation on rhodopin . the level ordering of the first eight excited singlet states was identical with the exception that the eom - ccsd methods predicted that the bu state is higher in energy than the bu state . the high relative intensity of the cis - band band provides spectroscopic evidence for a cis linkage near the center of the polyene chain . the combination of theory and experiment provide strong support for rhodopinal having a cis configuration in both polar and nonpolar solvent . analysis of the excited state manifold responsible for the electronic absorption spectrum of rhodopinal in n - hexane ( solid yellow spectra ) based on mndo - psdci theory . four configurations ( figure 11 ) were investigated relative to experimental observations : trans - rhodopinal ( upper left ) , 13-cis - rhodopinal ( upper right ) , trans - rhodopinal in a corkscrew conformation ( lower left ) , and 13-cis - rhodopinal in a corkscrew conformation ( lower right ) . the corkscrew conformation was generated by adjusting the dihedral angles of the single bonds 10 from planar and the double bonds 5 from planar , with all dihedral distortions in the same direction . the cis configuration involves rotation of the double bond directly connected to the aldehyde group , identified using the torsional arrow in figure 11d . the heights of the bars are proportional to the calculated oscillator strengths of the transitions , and the color reflects the ionic versus covalent character ( figure 13 ) . the approximate symmetry is indicated for selected states , and 1 kilokayser ( kk ) = 1000 cm . the computations suggest , however , that 13-cis - rhodopinal may form a corkscrew conformation to help stabilize the molecule in solution . a corkscrew conformation involves clockwise or counterclockwise dihedral distortion involving both single and double bonds , with a majority of the distortion in the single bonds . the change in geometry costs very little in terms of torsional distortion energy but provides intramolecular electrostatic stabilization in nonpolar solvent and solute solvent stabilization in polar solvent . a modest corkscrew rotation of 10 in the single bonds and 5 in the double bonds generates improved agreement between experiment and theory ( bottom panels in figure 12 ) . it should be emphasized that the same improvement in simulating the observed spectra is obtained by generating modest , random dihedral distortions , such as would be generated via thermal motions and occupation of low - frequency torsional modes . the crystal structure of the b800 - 820 lh2 complex did not fully resolve the rhodopinal structure , but provided ample evidence that the structure is in an all - trans configuration . given the above results that demonstrate that 13-cis - rhodopinal is more stable than trans - rhodopinal in solution , the question arises as to why an all - trans configuration for rhodopinal exists in the lh2 complex . first , the binding site for rhodopinal glucoside , which , as mentioned previously , is formed independently of , and prior to , the conversion of b800 - 850 to b800 - 820 , must also serve as the binding site for rhodopin glucoside , whose most stable configuration is undisputedly all - trans . second , the formation of rhodopinal requires enzymatic activity to attach the aldehyde group . the enzyme would likely release trans - rhodopinal glucoside into solution for assembly into the pigment protein complex , and formation of the 13-cis - rhodopinal glucoside would require either an isomerase or > 30 min for thermal isomerization to generate the equilibrium structure . as shown in figure 7 , there are significant differences in the transient absorption spectra of rhodopinal compared to rhodopin . in particular , the transient absorption maximum is red - shifted and an additional feature at 740 nm is observed for rhodopinal . to explore these differences theoretically , mndo - psdci theory was used to calculate the energies and oscillator strengths for excitations from the ground state and the relaxed first excited state ( figure 13 ) . the computations also simulated the transient absorption spectra based on the assumption that the origin states are the fully relaxed lowest - excited s1 ( 2ag ) states generated using full single ci and acetonitrile as the solvent . however , the mndo - psdci calculations are for vacuum conditions , and the effect of solvent is limited to the geometry of the relaxed origin state . the simulated spectra ( figure 14 ) reproduce the key features shown in figure 7 by predicting both the red shift of the transient maximum and the farther red - shifted weaker band observed only for rhodopinal . the calculations of the s1 sn transitions for rhodopin predict a maximum absorbance at 500 nm , which corresponds to the experimental band observed at 600 nm . this band is associated with a transition from the relaxed s1 ( 2ag ) excited state to the 11th excited ( 5bu ) singlet state ( figure 13 ) . this state also gives rise to a weakly allowed transition from the ground state calculated to be at 340 nm . this ensuing discussion of the transient absorption spectrum of rhodopinal in solution is limited to an analysis of the 13-cis configuration because it was demonstrated above that the 13-cis - isomer dominates in that environment ( figure 12 ) . the strong band observed spectroscopically for rhodopinal at 630 nm ( calculated to be at 520 nm ) is associated with a transition from the relaxed s1 ( 2ag ) excited state to the 11th excited ( 5bu ) singlet state ( figure 13 ) , which is an assignment identical to that for the intense band of rhodopin . the major difference is a lower energy transition calculated to be at 680 nm and experimentally observed at 740 nm . this more red - shifted band is associated with a transition from the 2ag excited state to the eighth excited ( 5ag ) singlet state ( figure 13 ) . reported similar red - shifted transient absorption bands from 8-apo--carotenal and also interpreted the results in terms of a transition from the s1 ( 2ag ) state to a higher ag state . moreover , they proposed that the allowedness for the transition arises due to the carbonyl group introducing asymmetry into the conjugated polyene system . the presence of asymmetry in the molecule would have the effect of relaxing the selection rules that render the transition forbidden in more symmetric , linear carotenoids , e.g. , rhodopin . excited state * level ordering for trans - rhodopin , trans - rhodopinal , and 13-cis - rhodopinal for excitations from the ground state for the equilibrium ground state conformation ( s0 geom ) and the relaxed first excited singlet conformation ( s1 rlxd ) . the energies and oscillator strengths were calculated based on mndo - psdci theory using a ci basis set of the 10 highest energy filled orbitals and the 10 lowest energy unfilled orbitals . the stationary states are represented by rectangles where the height is proportional to the oscillator strength , and the color reflects the ionic versus covalent character of the state ( see inset ) . the values in parentheses index electronic states 8 and 11 discussed in the text . simulation of the s1 to sn spectra of trans - rhodopin ( top ) , trans - rhodopinal ( middle ) , and 13-cis - rhodopinal ( bottom ) based on mndo - psdci theory ( full single and double configuration interaction involving the 11 highest energy filled and 11 lowest energy unfilled orbitals ) . the horizontal axis is linear in energy , where 1 kilokayser ( kk ) = 1000 cm , and the corresponding wavelength is marked as an inset in green . although there are higher energy bands calculated to be present only in 13-cis - rhodopinal ( compare the middle and bottom spectra in figure 14 ) , the above - mentioned red - shifted band calculated to be at 680 nm appears to be present in both cis- and trans - rhodopinal . this observation can be traced to the fact that , whereas a transition from the ground s0 ( 1ag ) state to the 5ag state only has intensity in molecules with cis configurations , hence its designation as the cis - band , a transition from the s1 ( 2ag ) excited state is strong for both trans- and cis - rhodopinal . therefore , the additional red - shifted band seen only in the transient absorption spectrum of rhodopinal in solution is due to the presence of the aldehyde group on the molecule , and not to the configuration of the carotenoid . reconstruction and comparison of the 1-t and fluorescence excitation spectra from the lh2 complexes ( figures 5 and 6 ) show clearly that rhodopinal is much more efficient than rhodopin at transferring excited state energy to bchl . the precise factors responsible for this effect can be obtained from a consideration of the rate constants for de - excitation of the carotenoid excited states derived from the global fitting analysis . the s1 and s2 states of carotenoids represent important donor excited states for eet to bchl . these routes are represented in figure 15 by the rate constants ket1 and ket2 , respectively . deactivation of these two excited states may also occur via internal conversion , and these processes are indicated in the figure by the rate constants , k10 and k21 . it is important to mention here that broadband 2d electronic spectroscopic results provide compelling evidence for the participation in eet of a dark intermediate state of the carotenoid having an energy in the vicinity of the state associated with the qx transition of bchl . this dark state was reported to be populated in 21 fs and depopulated in 62 fs . both of these times are shorter than the 100 fs time resolution of the laser spectrometer used in the present work . hence , the rate constant ket2 illustrated in figure 15 should be interpreted as including the kinetics of this dark state in addition to those of s2 . the total eet efficiency , et , resulting from both the s1 and s2 pathways is given by the expression1where the rate constants correspond to the processes illustrated in figure 15 . the rate constants can be computed from the lifetimes of the s1 and s2 excited states measured in solution , sisoln , and in the lh2 complexes , silh2 . the relevant expressions are2and3 tables 3 , 4 , and 5 summarize the lifetimes obtained from the global fits and the rate constants and efficiencies obtained either from the data presented here and using eqs 13 or from previous work . rate constants for the lh2 complexes were obtained using laser excitation at 525 nm ( rhodopin excitation ) for the sample prepared from rbl . acidophilus strain 10050 ( which contains only the rhodopin chromophore ) and at 570 nm ( selective rhodopinal excitation ) for the sample prepared from rbl . acidophilus strain 7050 ll . determined from the dynamics ( dyn ) of the excited states according to eq 1 . determined from steady - state fluorescence excitation spectroscopy ( fl ) as shown in figures 5 and 6 . computed from eq 2 using the values of 3.0 ps ( 3 in table 4 ) and 4.1 ps measured here for the lifetime of the s1 state of rhodopin in the lh2 complex and as an average value in solution ( 3 in table 3 ) , respectively . computed from eq 2 using the values of 57 and 95 fs for the lifetime of the s2 state in the lh2 complex and in solution , respectively . the 57 fs value was obtained from fluorescence upconversion spectroscopic experiments reported in ref ( 33 ) . the lifetime of the s2 state in solution was treated as an adjustable parameter to achieve agreement between the energy transfer efficiency of 56% computed from the dynamics data and that measured by fluorescence excitation spectroscopy . computed from eq 2 using the values of 1.2 ps ( 3 in table 4 ) and 4.1 ps measured here for the lifetime of the s1 state of rhodopinal in the lh2 complex and as an average value in solution ( 3 in table 3 ) , respectively . computed from eq 2 using the values of 10 and 100 fs for the lifetime of the s2 state in the lh2 complex and in solution , respectively . in fact , the s2 lifetime obtained for rhodopinal glucoside in solution was found here to be < 100 fs , which means that the k21 value of 10 ps based on this lifetime is a lower limit to what the value could be . the lifetime of the s2 state in the complex was treated as an adjustable parameter to achieve agreement between the energy transfer efficiency of 97% computed from the dynamics data and that measured by fluorescence excitation spectroscopy . the key result for rhodopin ( and rhodopin glucoside ) in the lh2 complex from rbl . acidophilus 10050 is the extent of partitioning between the s1 and s2 donor states of the efficiency of energy transfer to bchl . the data in table 5 show that et1 = 27% and et2 = 40% , which according to eq 1 leads to an overall energy transfer efficiency of 56% . however , it is important to point out that , in the current analysis , the lifetime of the s2 state of rhodopin in solution was treated as an adjustable parameter and set to 95 fs to achieve agreement between the energy transfer efficiency of 56% computed from the dynamics data , and that measured by fluorescence excitation spectroscopy ( figures 5 and 6 ) . the value of 95 fs is in very good agreement with the s2 lifetime of 105 fs reported by macpherson et al . for rhodopin glucoside in ethanol , and is consistent with the present findings that the value is < 100 fs . previous workers concur that the s2 pathway is most important in the carotenoid - to - bchl energy transfer mechanism in this lh2 complex ( table 5 ) . when rhodopinal ( and rhodopinal glucoside ) is present in the lh2 complex , as is the case for rbl . acidophilus 7050 ll and hl , the overall carotenoid - to - bchl energy transfer efficiency becomes much greater . as shown clearly from the analysis of the fluorescence excitation spectra given in figure 6 , this can be traced directly to the fact that rhodopinal is much more efficient at transferring energy to bchl than rhodopin . the data presented in table 5 reveal that this enhancement is due to a significant increase in both ket1 and ket2 for rhodopinal compared to those obtained for rhodopin . note from the data in table 5 that ket1 increases from 0.089 ps to 0.59 ps and ket2 increases from 7.0 ps to 90 ps in going from rhodopin to rhodopinal as the energy donor to bchl in the lh2 complex from 7050 ll . these combined increases result in a substantial enhancement in the carotenoid - to - bchl eet efficiency from 56% for rhodopin to 97% for rhodopinal . however , as was the case for the s2 lifetime of rhodopin discussed above , owing to the fact that lifetimes of the s2 state of rhodopinal in solution and in the lh2 complex are faster than the time resolution of the laser spectrometer , these values were treated as adjustable parameters to achieve agreement between the energy transfer efficiency computed from the dynamics data and that measured by fluorescence excitation spectroscopy . a consideration of the fundamental quantum mechanical expression for the rate constant for eet4where t is the electronic coupling term and j is the normalized spectral overlap5and fd( ) is the emission spectrum of the donor carotenoid and a( ) is the absorption spectrum of the bchl acceptor , leads to the conclusion that the increase in the values of the rate constants in going from rhodopin to rhodopinal ( table 5 ) can not be attributed solely to the different positions of the energy levels of the carotenoids , and consequently neither to differences in spectral overlap . this is particularly evident for the route involving the s2 ( 1bu ) state of the carotenoid and the state corresponding to the qx band of bchl . taking the s0 ( 1ag ) s2 ( 1bu ) absorption spectrum of rhodopin and rhodopinal reflected about their spectral origins as approximations to their ( very weak ) emission profiles , and overlaying these lineshapes with the qx absorption band of bchl in the lh2 complex ( figure 16 ) , one obtains a rhodopinal - to - rhodopin spectral overlap ratio of 1.2 . this value is significantly different from the rhodopinal - to - rhodopin ratio of ket2 rate constants given in table 5 which indicate a value of ( 90/7.0 ) = 13 . although it is impossible to compute the spectral overlap integrals associated with the s1 ( 2ag ) state of the carotenoid due to the lack of either detectable s0 ( 1ag ) s1 ( 2ag ) absorption or fluorescence from the s1 ( 2ag ) state , the small difference in the s1 lifetimes of rhodopin and rhodopinal in solution ( table 3 ) suggests very similar s1 excited state energies for the two molecules . therefore , a minimal effect of the position of the energy levels , and consequently of spectral overlap , is expected for the ket1 rate constant in the lh2 complexes . however , a significant change in ket1 is evidenced by comparing the s1 lifetime of rhodopin in the lh2 complex from rbl . acidophilus 10050 ( 3.0 ps ) with that of rhodopinal ( selectively excited at 570 nm ) in the lh2 complex from rbl . acidophilus 7050 ll ( 1.2 ps ) . using eq 2 and an average lifetime of 4.1 ps for the carotenoids in the four different solvents , the value of ket1 is shown in table 5 to increase by a factor of ( 0.59/0.089 ) = 6.6 in going from rhodopin to rhodopinal . because only small differences in excited state energies and spectral overlap with the bchl qx and qy absorption bands are evident for both the s1 and s2 states of these carotenoids , stronger electronic coupling between the donor and acceptor electronic states induced by the presence of the aldehyde group on the -electron polyene chain in rhodopinal must be a major factor determining why rhodopinal is much more efficient at carotenoid - to - bchl eet than rhodopin in the lh2 pigment protein complex . spectral overlap of the hypothetical fluorescence of the carotenoid donor ( orange and purple traces ) and absorption of the bchl acceptor ( black trace ) . the absorption spectra of the carotenoids were reflected about their spectral origins to obtain approximations to the s2 ( 1bu ) s0 fluorescence spectra . although a rigorous analysis of the coulomb coupling terms describing eet from s1 and s2 is beyond the scope of this paper , this issue is addressed for s1 by the level ordering analysis shown in figure 13 . the lowest excited singlet state of both cis- and trans - configurations is calculated to be a forbidden 2ag state that gains oscillator strength due to dipole - induced coupling with the higher energy , strongly allowed 1bu state . the above kinetics analysis has revealed that energy is transferred from the s1 ( 2ag ) state to the bchl acceptor 6.6 times faster than the corresponding state in rhodopin . the computations predict that the enhanced energy transfer rate is associated in part with a significant increase in the oscillator strength of the s1 state in rhodopinal relative to rhodopin . moreover , the increase in oscillator strength in trans - rhodopinal is 35% larger than observed in 13-cis - rhodopinal ( figure 13 ) . because the frster coupling efficiency is proportional to the allowedness of the donor excited state , the all - trans configuration of rhodopinal provides added value to its light - harvesting function . acidophilus 7050 is grown under ll conditions , two major changes in the spectrum of the lh2 complex occur : a blue shift of the bchl qy band from 850 nm to 820 nm , which is caused by a change in the orientation of the c3-acetyl group of the b850 bchl ; and a red shift and broadening of the spectrum of the carotenoid , which is a consequence of the enzymatic substitution of an aldehyde group for a methyl group on rhodopin resulting in its conversion to rhodopinal . an important question is then , which of these factors is most important for enhancing the rate and efficiency of carotenoid - to - bchl eet ? the answer is that the conversion of rhodopin to rhodopinal is more important than the shift of the bchl qy band . data suggestive of this conclusion was provided previously by deinum et al . , who investigated the b800 - 820 lh2 complex from rbl . cells of this strain grown either at low temperatures or at ll do not contain rhodopinal ( or rhodopinal glucoside ) , and the carotenoid - to - bchl eet efficiency of its associated lh2 complex , which contains primarily rhodopin and rhodopin glucoside , was found to be 45 5% . if the change in the position of the bchl qy band from 850 nm to 820 nm was important for enhancing the efficiency of eet from the carotenoid to bchl , one would expect that the efficiency for rbl . acidophilus strain 7750 would have a value above 45% or even higher than 56% , which is the value found for rhodopin in the b800 - 850 lh2 complex from strain 10050 ( table 2 ) . the fact that it is not larger than 56% , and in fact is smaller than this value , i.e. , 45 5% , indicates that the change in the position of the qy band of bchl is not a factor in determining the efficiency of carotenoid - to - bchl eet . instead , the conversion of rhodopin to rhodopinal , which incidentally occurs prior to the conversion of b800 - 850 to b800 - 820 , is the primary controlling factor leading to more efficient eet in the lh2 complex of rbl . acidophilus 7050 grown under ll conditions results in a significant enhancement in the ability of the organism to transfer absorbed light energy to the reaction center . this is due almost entirely to the fact that rhodopinal , and its corresponding glucoside , are nearly 100% efficient at transferring excited state energy to bchl in the lh2 complex . also , the spectrum of rhodopinal occurs at a longer wavelength compared to that of rhodopin , which allows the bacterium containing this carotenoid to capture light energy in a spectral region where its photosynthetic bacterial competitors do not absorb light . this provides an important advantage for this organism as it competes for life - sustaining photons in the water column . moreover , based on the ket1 and ket2 rate constants given in table 5 , the energy absorbed by rhodopinal is transferred to bchl in the lh2 complex 6.6 times faster from the s1 state and > 10 times faster from the s2 state than energy absorbed by rhodopin . more effective electronic coupling between the electronic states of the carbonyl - containing rhodopinal and bchl is the primary reason for the increase in these rates . even though the blue shift of the bchl qy band from 850 nm to 820 nm induced by growing rbl . acidophilus 7050 under ll conditions does not have an effect on the rate and efficiency of carotenoid - to - bchl eet , this spectral change has another consequence . singlet annihilation measurements that , compared to the b800 - 850 complex , the b800 - 820 complex provides a more effective energy barrier for preventing back transfer of excitation energy out of the reaction center core complex and into the photosynthetic unit . thus , the combined effects of alterations in carotenoid and bchl structures and spectral properties resulting from the adaptation of the bacterial photosynthetic organism to the challenging environmental condition of limited light availability contribute in a very tangible way to ensure survival of this species .

rhodopin , rhodopinal , and their glucoside derivatives are carotenoids that accumulate in different amounts in the photosynthetic bacterium , rhodoblastus ( rbl . ) acidophilus strain 7050 , depending on the intensity of the light under which the organism is grown . the different growth conditions also have a profound effect on the spectra of the bacteriochlorophyll ( bchl ) pigments that assemble in the major lh2 light - harvesting pigment protein complex . under high - light conditions the well - characterized b800 - 850 lh2 complex is formed and accumulates rhodopin and rhodopin glucoside as the primary carotenoids . under low - light conditions , a variant lh2 , denoted b800 - 820 , is formed , and rhodopinal and rhodopinal glucoside are the most abundant carotenoids . the present investigation compares and contrasts the spectral properties and dynamics of the excited states of rhodopin and rhodopinal in solution . in addition , the systematic differences in pigment composition and structure of the chromophores in the lh2 complexes provide an opportunity to explore the effect of these factors on the rate and efficiency of carotenoid - to - bchl energy transfer . it is found that the enzymatic conversion of rhodopin to rhodopinal by rbl . acidophilus 7050 grown under low - light conditions results in nearly 100% carotenoid - to - bchl energy transfer efficiency in the lh2 complex . this comparative analysis provides insight into how photosynthetic systems are able to adapt and survive under challenging environmental conditions .

Introduction Materials and Methods Results Discussion Conclusions

a prime example of the development of adaptive traits is found in the purple photosynthetic bacterium , rhodoblastus ( rbl . ) this bacterium is able to alter its number and size of photosynthetic units as well as its pigment composition and light absorption properties of the major light harvesting ii ( lh2 ) antenna pigment protein complex in response to changes in illumination conditions . if the bacterium is grown under high light , the well - characterized b800 - 850 lh2 complex is formed having bacteriochlorophyll ( bchl ) absorption bands near 800 and 850 nm , and rhodopin and rhodopin glucoside are the primary carotenoid pigments . under low - light conditions , genes that code for a variant lh2 denoted b800 - 820 ( also sometimes referred to as lh3 in the literature ) are activated , the bchl qy absorption band near 850 nm shifts to 820 nm , and the organism accumulates rhodopinal and rhodopinal glucoside in the lh2 complex as primary carotenoid pigments ( figures 1a and 1b ) . the conversion of these h - bonding residues to non - h - bonding 44 ( phe ) and 45 ( leu ) in the b800 - 820 lh2 leads to a rotation of the c3-acetyl group out of the plane of the porphyrin ring , thereby inhibiting delocalization of the -electron conjugation to the acetyl carbonyl , resulting in more restricted -electron delocalization and consequently a blue shift of the qy band from 850 nm to 820 nm . under low - light growth conditions , rhodopin and rhodopin glucoside are enzymatically converted to rhodopinal and rhodopinal glucoside as an aldehyde group replaces the methyl group at carbon c20 in the carotenoid structures ( figure 1a ) . the spectral origin ( 00 ) vibronic band of rhodopin glucoside in methanol appears at 500 nm , whereas for rhodopinal glucoside , the band is less resolved spectrally , and it is located at 540 nm in the same solvent ( figure 2 ) . previous workers compared the carotenoid - to - bchl energy transfer properties of lh2 complexes isolated from cells of rbl . acidophilus strain 7050 grown under different illumination conditions and found that there was an increase in the energy transfer efficiency from between 50 and 55% for the b800 - 850 complex to between 70 and 75% for the b800 - 820 complex . , whether changes in the bchl absorption spectra , or the conversion of rhodopin to rhodopinal in the protein complex , or both factors , were responsible for the enhanced ability of the lh2 complex to effectively harvest photons in the region of carotenoid absorption . acidophilus strain 7050 did not assign specific values to the energy transfer efficiencies of the individual carotenoids bound in the complexes , nor has there been any direct comparison of the spectra and dynamics of the excited states of rhodopin and rhodopinal either in solution or in the lh2 complexes . these data are important for addressing the specific mechanism of how these alterations in bchl and carotenoid structures and spectra increase the carotenoid - to - bchl energy transfer efficiency and , as a consequence , enhance the viability of the photosynthetic bacterial organism . in another study using steady - state and ultrafast time - resolved spectroscopy to elucidate the carotenoid - to - bchl energy transfer mechanism in the lh2 complex from rbl . acidophilus 10050 and rhodobacter sphaeroides strain 2.4.1 provided convincing evidence for the additional involvement of a dark intermediate state in the carotenoid - to - bchl energy transfer pathway . the present work provides a detailed experimental and computational comparison of the excited state energy levels , spectra , and dynamics of rhodopin , rhodopinal , and their associated glucosides in various solvents and in their respective lh2 complexes isolated from rbl . the use of steady - state absorption , fluorescence and fluorescence excitation spectroscopy and ultrafast time - resolved transient absorption spectroscopy in the visible spectral region have revealed the rates and efficiencies of carotenoid - to - bchl energy transfer for the individual carotenoids . the spectrum of rhodopin glucoside displays well - resolved vibronic bands in all of the solvents , whereas the spectrum of rhodopinal glucoside is much less structured , except in carbon disulfide , where a shoulder on the long - wavelength side of the primary absorption band is observed . also , the high polarizability of carbon disulfide ( p( ) = 0.354 ) results in a 3040 nm red shift of the spectra of rhodopin glucoside and rhodopinal glucoside compared to their spectra recorded in the less polarizable solvents , acetonitrile ( p( ) = 0.210 ) and methanol ( p( ) = 0.202 ) . the spectra of rhodopin glucoside and rhodopinal glucoside are indistinguishable from the corresponding spectra of rhodopin and rhodopinal . in addition , compared to the spectrum of the lh2 complex from strain 10050 , the spectra of the lh2 complexes from 7050 hl and 7050 ll show more absorption in the region between 550 and 600 nm where the spectrum of the carotenoid partially overlaps with the bchl qx band at 590 nm . this is due to the presence of rhodopinal and rhodopinal glucoside in the lh2 complexes from strain 7050 . acidophilus 7050 will always result in a very small amount of the b800 - 820 lh2 complex in hl cells due to the effect of light shading in the culture media . likewise , a small amount of b800 - 850 lh2 complex will be present in the ll cells due to the fact that there is a limit to how low the light intensity can be adjusted to ensure a realistic amount of bacterial growth . moreover , it should be noted that the regulatory pathways for the switch from rhodopin glucoside to rhodopinal glucoside and from b800 - 850 to b800 - 820 are independent of each other . the green line shows the ratio of the normalized excitation and 1-t spectra and in the region of carotenoid absorption gives a quantitative measurement of the carotenoid - to - bchl energy transfer efficiency . however , as mentioned above , previous work detailing the effect of light intensity and temperature on cell growth and the formation of the b800 - 820 complex indicated that the conversion of rhodopin to rhodopinal occurs prior to the conversion of b800 - 850 to b800 - 820 . the lh2 complexes from 7050 hl and 7050 ll displayed higher carotenoid - to - bchl eet efficiencies and were in the range of 63% to 71% and 76% to 86% , respectively . hplc analysis ( table 1 ) revealed that the lh2 samples from the 7050 samples contain both rhodopin and rhodopinal ( and their associated glucosides ) in different amounts . the spectral reconstructions yielded the specific carotenoid - to - bchl eet efficiencies for rhodopin and rhodopinal from the ratios of the individual fluorescence excitation and 1-t bands in the profiles . ( because rhodopin and rhodopinal have identical absorption ( and 1-t ) spectra as their corresponding glucoside derivatives , in this context and unless explicitly noted otherwise , any statements about the spectra of rhodopin or rhodopinal should be taken to mean the combined contribution from the chromophores associated with both the glucoside and non - glucoside molecules . ) a similar analysis was carried out to obtain the values for the individual carotenoid - to - bchl eet efficiencies in the lh2 complex from the 7050 ll sample ( figures 6c and 6d ) , which were found from the spectral reconstruction to be 54 5% for rhodopin ( orange traces ) and 97 2% for rhodopinal ( purple traces ) . a similar effect is evident in the spectrum of rhodopinal glucoside in carbon disulfide and benzyl alcohol ( figure 7 , right panels ) , where it is also clear that the spectral bands are broader overall than the spectra of rhodopin glucoside in the same solvents ( figure 7 , left panels ) . also , an additional peak at 740 nm , which is not found in the spectra of rhodopin glucoside , is seen in the transient absorption spectra from rhodopinal glucoside . the fact that the lifetime of this component is reasonably similar for both rhodopin glucoside and rhodopinal glucoside indicates that neither the configuration , which is 13-cis for rhodopinal glucoside in solution compared to all - trans for rhodopin glucoside ( figure 1a ) , nor the presence of the aldehyde group at carbon c20 on rhodopinal glucoside compared to the methyl group at the same position on rhodopin glucoside results in any significant change in the dynamics of the s1 state that would impact its role in light - harvesting . the hplc pigment analysis of the lh2 complex from strain 10050 ( table 1 ) revealed that the carotenoid composition consists of roughly equal amounts of rhodopin and rhodopin glucoside with a small amount of lycopene , all of which have identical absorption spectra . using this information regarding the spectra of the carotenoids in the lh2 complex from rbl . acidophilus 10050 as a control , the features attributable to rhodopin and rhodopinal can be distinguished in the transient spectra of the lh2 complexes prepared from 7050 ll and 7050 hl cells , which contain a mixture of these carotenoids ( table 1 ) . note that the lifetime of this component is 1.2 ps in the 7050 ll lh2 and 1.3 ps in the 7050 hl lh2 , which is significantly shorter than the average value of 4.1 ps found for rhodopinal in the different solvents and also shorter than the value of 3.0 ps found for rhodopin in the lh2 complex from strain 10050 . this conclusion is consistent with the observation that the absorption spectra of rhodopin and rhodopin glucoside are indistinguishable . first , the binding site for rhodopinal glucoside , which , as mentioned previously , is formed independently of , and prior to , the conversion of b800 - 850 to b800 - 820 , must also serve as the binding site for rhodopin glucoside , whose most stable configuration is undisputedly all - trans . therefore , the additional red - shifted band seen only in the transient absorption spectrum of rhodopinal in solution is due to the presence of the aldehyde group on the molecule , and not to the configuration of the carotenoid . the rate constants can be computed from the lifetimes of the s1 and s2 excited states measured in solution , sisoln , and in the lh2 complexes , silh2 . however , it is important to point out that , in the current analysis , the lifetime of the s2 state of rhodopin in solution was treated as an adjustable parameter and set to 95 fs to achieve agreement between the energy transfer efficiency of 56% computed from the dynamics data , and that measured by fluorescence excitation spectroscopy ( figures 5 and 6 ) . previous workers concur that the s2 pathway is most important in the carotenoid - to - bchl energy transfer mechanism in this lh2 complex ( table 5 ) . acidophilus 7050 ll and hl , the overall carotenoid - to - bchl energy transfer efficiency becomes much greater . these combined increases result in a substantial enhancement in the carotenoid - to - bchl eet efficiency from 56% for rhodopin to 97% for rhodopinal . however , as was the case for the s2 lifetime of rhodopin discussed above , owing to the fact that lifetimes of the s2 state of rhodopinal in solution and in the lh2 complex are faster than the time resolution of the laser spectrometer , these values were treated as adjustable parameters to achieve agreement between the energy transfer efficiency computed from the dynamics data and that measured by fluorescence excitation spectroscopy . a consideration of the fundamental quantum mechanical expression for the rate constant for eet4where t is the electronic coupling term and j is the normalized spectral overlap5and fd( ) is the emission spectrum of the donor carotenoid and a( ) is the absorption spectrum of the bchl acceptor , leads to the conclusion that the increase in the values of the rate constants in going from rhodopin to rhodopinal ( table 5 ) can not be attributed solely to the different positions of the energy levels of the carotenoids , and consequently neither to differences in spectral overlap . taking the s0 ( 1ag ) s2 ( 1bu ) absorption spectrum of rhodopin and rhodopinal reflected about their spectral origins as approximations to their ( very weak ) emission profiles , and overlaying these lineshapes with the qx absorption band of bchl in the lh2 complex ( figure 16 ) , one obtains a rhodopinal - to - rhodopin spectral overlap ratio of 1.2 . although it is impossible to compute the spectral overlap integrals associated with the s1 ( 2ag ) state of the carotenoid due to the lack of either detectable s0 ( 1ag ) s1 ( 2ag ) absorption or fluorescence from the s1 ( 2ag ) state , the small difference in the s1 lifetimes of rhodopin and rhodopinal in solution ( table 3 ) suggests very similar s1 excited state energies for the two molecules . therefore , a minimal effect of the position of the energy levels , and consequently of spectral overlap , is expected for the ket1 rate constant in the lh2 complexes . because only small differences in excited state energies and spectral overlap with the bchl qx and qy absorption bands are evident for both the s1 and s2 states of these carotenoids , stronger electronic coupling between the donor and acceptor electronic states induced by the presence of the aldehyde group on the -electron polyene chain in rhodopinal must be a major factor determining why rhodopinal is much more efficient at carotenoid - to - bchl eet than rhodopin in the lh2 pigment protein complex . acidophilus 7050 is grown under ll conditions , two major changes in the spectrum of the lh2 complex occur : a blue shift of the bchl qy band from 850 nm to 820 nm , which is caused by a change in the orientation of the c3-acetyl group of the b850 bchl ; and a red shift and broadening of the spectrum of the carotenoid , which is a consequence of the enzymatic substitution of an aldehyde group for a methyl group on rhodopin resulting in its conversion to rhodopinal . an important question is then , which of these factors is most important for enhancing the rate and efficiency of carotenoid - to - bchl eet ? the answer is that the conversion of rhodopin to rhodopinal is more important than the shift of the bchl qy band . cells of this strain grown either at low temperatures or at ll do not contain rhodopinal ( or rhodopinal glucoside ) , and the carotenoid - to - bchl eet efficiency of its associated lh2 complex , which contains primarily rhodopin and rhodopin glucoside , was found to be 45 5% . acidophilus strain 7750 would have a value above 45% or even higher than 56% , which is the value found for rhodopin in the b800 - 850 lh2 complex from strain 10050 ( table 2 ) . , 45 5% , indicates that the change in the position of the qy band of bchl is not a factor in determining the efficiency of carotenoid - to - bchl eet . instead , the conversion of rhodopin to rhodopinal , which incidentally occurs prior to the conversion of b800 - 850 to b800 - 820 , is the primary controlling factor leading to more efficient eet in the lh2 complex of rbl . acidophilus 7050 grown under ll conditions results in a significant enhancement in the ability of the organism to transfer absorbed light energy to the reaction center . acidophilus 7050 under ll conditions does not have an effect on the rate and efficiency of carotenoid - to - bchl eet , this spectral change has another consequence .

percutaneous coronary intervention ( pci ) has become the most widely used method to treat patients with coronary artery disease . the introduction of drug - eluting stents ( dess ) led the interventional cardiology to a new era , in which the pci became an alternative to coronary artery bypass surgery even in complex lesions . paclitaxel , sirolimus , and its derivatives were the most widely used anti - proliferative drugs of des and their efficacy in inhibiting smooth muscle cell proliferation had been verified in several clinical trials . however , several studies found that both sirolimus and paclitaxel lead to delayed arterial healing and endothelial dysfunction , which could be the major reason of the increasing late and very late stent thrombosis ( vlst ) events . a new des platform combining the approved 316l stainless stent platform with a novel anti - proliferative agent , arsenic trioxide , and a biodegradable poly - l - lactic acid ( plla ) polymer has been developed . arsenic trioxide , a chemotherapy drug that has been widely used in the treatment of acute promyelocytic leukemia ( apl ) , was proved to inhibit cell growth and induce cell apoptosis . our previous study had proved this stent system significantly suppressed in - stent restenosis by reducing proliferation and inducing apoptosis of vascular smooth muscle cells ( vsmcs ) compared to uncoated stents in a rabbit iliac artery injury model . in addition , it also inhibits local inflammatory reactions , which suppresses the endothelial healing . the purpose of this study was to investigate whether the aes ( amsinomed medical company , beijing , china ) is effective and safe as an alternative to current des in human . the firebird ses ( microport corporation , shanghai , china ) , a widely used des in china , is selected as the comparison . the trial was a multicenter , perspective , randomized controlled trial designed to compare clinical and angiographic outcomes between arsenic trioxide - eluting stents ( aess ) and sirolimus - eluting stents ( ses ) [ figure 1 ] . the study protocol was approved by the ethics committee at each participating center and was conducted according to the principles of the declaration of helsinki regarding investigations in humans . patient flow chart . aes : arsenic trioxide - eluting stent(s ) ; ses : sirolimus - eluting stent(s ) ; tvf : target vessel failure ; lll : late luminal loss . patients 18 and < 80 years of age were eligible for enrollment if they showed symptoms of angina pectoris and/or signs of ischemia or angina and met the main inclusion criteria of coronary angiography : ( 1 ) lesion length 37 mm ; ( 2 ) reference vessel diameter ( rvd ) 2.04.0 mm ; ( 3 ) a diameter stenosis 70% or total occlusion lesion . no restriction was applied to the total number of treated lesions , treated vessels , or number of stents implanted . exclusion criteria included cardiac shock , congenital heart disease , severe valvular disease , left ventricular ejection fraction < 30% , allergy to antiplatelet drugs , heparin , stainless steel , contrast agents , surgery in 6 weeks , acute myocardial infarction ( mi ) within 2 weeks , transient ischemic attack or stroke within 7 days , wbc < 4.0 10/l , pbc < 100 10/l , 1.5 times higher than normal value of alanine aminotransferases , aspartate aminotransferase , or alkaline phosphate , using thrombolytic agents or gpiib / iiia antagonists in 1 week , gastrointestinal and fundus bleeding within 6 months , true bifurcation lesion with side branch diameter > 2.00 mm , life expectancy < 1 year , stented target lesion , inability or unwillingness to comply with all protocol - required procedure , and a positive pregnancy test within 24 h before enrollment . patients were assigned a number from a randomization sequence generated by sas 9.1 ( sas institute , north carolina , usa ) and maintained by a statistician who was blinded to the treatments . aes were available in diameters of 2.0 , 2.5 , 2.75 , 3.0 , 3.5 , and 4.0 mm and in lengths of 8 , 13 , 17 , 22 , 27 , 33 , and 37 mm . both the aes and ses were expanded to achieve < 20% residual stenosis by visual estimate in the treated segment , with a combination of the stent deployment balloon and at the operators discretion , subsequent postdilatation balloons . patients who were not on long - term antiplatelet or aspirin therapy were required to receive a loading dose of aspirin ( 300 mg / d ) and clopidogrel bisulfate ( 300 mg ) at least 24 h before the procedure , followed by 75 mg / d of clopidogrel after procedure . at discharge , patients were maintained on at least 100 mg / d of aspirin and 75 mg / d of clopidogrel for at least 6 months . heparin was administered throughout the procedure to maintain an activated clotting time 230 s. all patients were scheduled to undergo a repeated angiography at 9 months . clinical follow - up was performed at 1 , 3 , 6 , 9 , 18 , 24 months by telephone . at follow - up , patients were specifically questioned regarding any adverse events or angina symptoms or other interventional therapy . the primary end point was target vessel failure ( tvf ) , a composite of cardiac death , target vessel mi , clinically driven target vessel revascularization ( defined as a repeated revascularization due to either a > 50% restenosis in the treated lesion with symptoms of ischemia or for a > 70% restenosis with or without symptoms within the entire major coronary vessel proximal and distal to a target lesion , including upstream and downstream branches and the target lesion itself ) . the secondary endpoints included late luminal loss ( lll ) , procedure success rate , in - stent restenosis rate , target lesion revascularization ( tlr ) , all - cause death , cardiac death or mi . the lll was defined as the difference between postprocedural and 9-month angiography follow - up minimal lumen diameter . the quantitative coronary angiography measurements were obtained for both the stented segment ( in - stent ) and a segment covering the stented length as well as 5-mm distal and proximal margins ( in - segment ) . tlr was defined as any ischemia - driven repeat pci of the target lesion or bypass surgery of the target vessel . cardiac death was defined as death due to any of the following : acute mi ; cardiac perforation / pericardial tamponade ; arrhythmia or conduction abnormality . the trial was designed to test the efficacy of aes compared with ses with respect to the primary endpoint of tvf . all primary and secondary endpoints were adjudicated by the independent and blinded clinical event committee . the objective of the study was to assess efficacy of aes compared with the ses with regard to the primary endpoint , tvf . based on the previous studies , we expect a mean in - stent lll of 0.40 mm firebird ses . the standard deviation ( sd ) for lll was presumed to be 0.45 mm , so the noninferiority margin was set at 0.15 mm ( equaling 35% of 0.45 mm ) . based on these assumptions , 102 patients per treatment group needed to be analyzed to provide a statistical power of 0.80 at an alpha level of 0.05 . continuous variables are presented as mean sd , and categorical variables are presented as counts and percentages . categorical variables were compared using chi - square and fisher 's exact tests while continuous variables were compared using student 's t - test . survival and event - free status were assessed using the methods of kaplan meier . all statistical analyses were performed by a physician with the use of r version 3.0.2 ( http://www.r-project.org ) . all reported p values were two - sided , and p < 0.05 were regarded as statistically significant . the trial was a multicenter , perspective , randomized controlled trial designed to compare clinical and angiographic outcomes between arsenic trioxide - eluting stents ( aess ) and sirolimus - eluting stents ( ses ) [ figure 1 ] . the study protocol was approved by the ethics committee at each participating center and was conducted according to the principles of the declaration of helsinki regarding investigations in humans . patient flow chart . aes : arsenic trioxide - eluting stent(s ) ; ses : sirolimus - eluting stent(s ) ; tvf : target vessel failure ; lll : late luminal loss . patients 18 and < 80 years of age were eligible for enrollment if they showed symptoms of angina pectoris and/or signs of ischemia or angina and met the main inclusion criteria of coronary angiography : ( 1 ) lesion length 37 mm ; ( 2 ) reference vessel diameter ( rvd ) 2.04.0 mm ; ( 3 ) a diameter stenosis 70% or total occlusion lesion . no restriction was applied to the total number of treated lesions , treated vessels , or number of stents implanted . exclusion criteria included cardiac shock , congenital heart disease , severe valvular disease , left ventricular ejection fraction < 30% , allergy to antiplatelet drugs , heparin , stainless steel , contrast agents , surgery in 6 weeks , acute myocardial infarction ( mi ) within 2 weeks , transient ischemic attack or stroke within 7 days , wbc < 4.0 10/l , pbc < 100 10/l , 1.5 times higher than normal value of alanine aminotransferases , aspartate aminotransferase , or alkaline phosphate , using thrombolytic agents or gpiib / iiia antagonists in 1 week , gastrointestinal and fundus bleeding within 6 months , true bifurcation lesion with side branch diameter > 2.00 mm , life expectancy < 1 year , stented target lesion , inability or unwillingness to comply with all protocol - required procedure , and a positive pregnancy test within 24 h before enrollment . patients were assigned a number from a randomization sequence generated by sas 9.1 ( sas institute , north carolina , usa ) and maintained by a statistician who was blinded to the treatments . aes were available in diameters of 2.0 , 2.5 , 2.75 , 3.0 , 3.5 , and 4.0 mm and in lengths of 8 , 13 , 17 , 22 , 27 , 33 , and 37 mm . both the aes and ses were expanded to achieve < 20% residual stenosis by visual estimate in the treated segment , with a combination of the stent deployment balloon and at the operators discretion , subsequent postdilatation balloons . patients who were not on long - term antiplatelet or aspirin therapy were required to receive a loading dose of aspirin ( 300 mg / d ) and clopidogrel bisulfate ( 300 mg ) at least 24 h before the procedure , followed by 75 mg / d of clopidogrel after procedure . at discharge , patients were maintained on at least 100 mg / d of aspirin and 75 mg / d of clopidogrel for at least 6 months . clinical follow - up was performed at 1 , 3 , 6 , 9 , 18 , 24 months by telephone . at follow - up , patients were specifically questioned regarding any adverse events or angina symptoms or other interventional therapy . the primary end point was target vessel failure ( tvf ) , a composite of cardiac death , target vessel mi , clinically driven target vessel revascularization ( defined as a repeated revascularization due to either a > 50% restenosis in the treated lesion with symptoms of ischemia or for a > 70% restenosis with or without symptoms within the entire major coronary vessel proximal and distal to a target lesion , including upstream and downstream branches and the target lesion itself ) . the secondary endpoints included late luminal loss ( lll ) , procedure success rate , in - stent restenosis rate , target lesion revascularization ( tlr ) , all - cause death , cardiac death or mi . the lll was defined as the difference between postprocedural and 9-month angiography follow - up minimal lumen diameter . the quantitative coronary angiography measurements were obtained for both the stented segment ( in - stent ) and a segment covering the stented length as well as 5-mm distal and proximal margins ( in - segment ) . tlr was defined as any ischemia - driven repeat pci of the target lesion or bypass surgery of the target vessel . cardiac death was defined as death due to any of the following : acute mi ; cardiac perforation / pericardial tamponade ; arrhythmia or conduction abnormality . the trial was designed to test the efficacy of aes compared with ses with respect to the primary endpoint of tvf . all primary and secondary endpoints were adjudicated by the independent and blinded clinical event committee . the objective of the study was to assess efficacy of aes compared with the ses with regard to the primary endpoint , tvf . based on the previous studies , we expect a mean in - stent lll of 0.40 mm firebird ses . the standard deviation ( sd ) for lll was presumed to be 0.45 mm , so the noninferiority margin was set at 0.15 mm ( equaling 35% of 0.45 mm ) . based on these assumptions , 102 patients per treatment group needed to be analyzed to provide a statistical power of 0.80 at an alpha level of 0.05 . continuous variables are presented as mean sd , and categorical variables are presented as counts and percentages . categorical variables were compared using chi - square and fisher 's exact tests while continuous variables were compared using student 's t - test . survival and event - free status were assessed using the methods of kaplan meier . all statistical analyses were performed by a physician with the use of r version 3.0.2 ( http://www.r-project.org ) . all reported p values were two - sided , and p < 0.05 were regarded as statistically significant . between july 2009 and january 2010 , 212 patients and 265 lesions were enrolled into this randomized study at eight sites across china . at 24 months , clinical follow - up was available for 105 patients in aes group and 103 patients in ses group . baseline , procedural , and lesion characteristics of the two randomized groups of patients were similar . the average age of the patients was 58.6 years , and 69.8% of enrolled patients were male . all of them suffered a history of angina pectoris and presented with an abnormal electrocardiogram and abnormal ultrasonic cardiogram [ table 1 ] . of a total of 265 treated lesions , approximately half of them are type b2/c lesions with mean rvds of 3.01 0.36 mm and lesion lengths of 19.97 8.20 mm ( aes ) and 19.52 8.13 mm ( ses ) . there was no significant difference between the baseline target lesions , but more severe diameter stenosis was observed in aes group ( 87.09 8.86% vs. 83.36 9.00% , p = 0.001 ) [ table 2 ] . baseline patient characteristics ecg : electrocardiogram ; ucg : ultrasound cardiogram ; aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . lesion and procedure characteristics ds : diameter stenosis ; rvd : reference vessel diameter ; aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent ; acc : american college of cardiology ; aha : american heart association . in both groups , a certain proportion of patients were treated with balloon predilatation ( 62.9% aes vs. 51.4% ses ) and postdilatation ( 25.7% aes vs. 32.7% ses ) [ table 2 ] . average stent diameter ( 3.01 0.35 mm aes vs. 3.00 0.43 mm ses ) and mean stent length ( 21.22 5.77 mm aes vs. 22.12 6.59 mm ses ) have been illustrated in table 3 . stent implantation data aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . the follow - up rate was more than 80% through the 24-month follow - up . the primary endpoint tvf in aes group was stable ( 6.67% ) , but an increasing trend was observed in ses group ( 1.87%5.83% ) . no significant difference between two groups was concluded . all cause death rate , as well as cardiac death or mi rate ( 0 vs. 3.88% , p = 0.058 ) at 24 months , was lower in aes group compared with ses group ( 0 vs. 4.85% , other adverse events including tlr , cerebral hemorrhage , noncardiac death , stent thrombosis , and angina pectoris were similar in both groups [ table 4 ] . 85 patients with 101 lesions in aes group ( 80.95% ) and 57 patients with 62 lesions in ses group ( 53.27% ) underwent angiographic follow - up at 9 months after stent implantation . in - segment late loss was 0.20 0.22 mm in aes group and 0.10 0.35 mm ses group . in - stent late loss was 0.29 0.52 mm in aes group and 0.10 0.25 mm in ses group . the difference in in - stent late loss between two groups was 0.18 mm ( 95% confidence interval : 0.0770.313 mm ) , which was larger than the noninferiority margin as 0.15 mm . binary in - stent and in - segment restenosis rate were not different between the two groups ; however , diameter stenosis was statistically greater in aes group than ses group for both in - stent ( 21.05% 18.70% vs. 12.01% 16.26% , p = 0.002 ) and in - segment ( 22.86% 11.34% vs. 17.83% 13.59% , p = 0.012 ) [ table 5 ] . clinical outcomes , n ( % ) tvf : target vessel failure ; mi : myocardial infarction ; tlr : target lesion revascularization ; st : stent thrombosis ; aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . nine - month angiographic follow - up results aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . between july 2009 and january 2010 , 212 patients and 265 lesions were enrolled into this randomized study at eight sites across china . at 24 months , clinical follow - up was available for 105 patients in aes group and 103 patients in ses group . baseline , procedural , and lesion characteristics of the two randomized groups of patients were similar . the average age of the patients was 58.6 years , and 69.8% of enrolled patients were male . all of them suffered a history of angina pectoris and presented with an abnormal electrocardiogram and abnormal ultrasonic cardiogram [ table 1 ] . of a total of 265 treated lesions , approximately half of them are type b2/c lesions with mean rvds of 3.01 0.36 mm and lesion lengths of 19.97 8.20 mm ( aes ) and 19.52 8.13 mm ( ses ) . there was no significant difference between the baseline target lesions , but more severe diameter stenosis was observed in aes group ( 87.09 8.86% vs. 83.36 9.00% , p = 0.001 ) [ table 2 ] . baseline patient characteristics ecg : electrocardiogram ; ucg : ultrasound cardiogram ; aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . lesion and procedure characteristics ds : diameter stenosis ; rvd : reference vessel diameter ; aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent ; acc : american college of cardiology ; aha : american heart association . in both groups , a certain proportion of patients were treated with balloon predilatation ( 62.9% aes vs. 51.4% ses ) and postdilatation ( 25.7% aes vs. 32.7% ses ) [ table 2 ] . average stent diameter ( 3.01 0.35 mm aes vs. 3.00 0.43 mm ses ) and mean stent length ( 21.22 5.77 mm aes vs. 22.12 6.59 mm ses ) have been illustrated in table 3 . stent implantation data aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . the follow - up rate was more than 80% through the 24-month follow - up . the primary endpoint tvf in aes group was stable ( 6.67% ) , but an increasing trend was observed in ses group ( 1.87%5.83% ) . no significant difference between two groups was concluded . all cause death rate , as well as cardiac death or mi rate ( 0 vs. 3.88% , p = 0.058 ) at 24 months , was lower in aes group compared with ses group ( 0 vs. 4.85% , p = 0.028 ) . other adverse events including tlr , cerebral hemorrhage , noncardiac death , stent thrombosis , and angina pectoris were similar in both groups [ table 4 ] . 85 patients with 101 lesions in aes group ( 80.95% ) and 57 patients with 62 lesions in ses group ( 53.27% ) underwent angiographic follow - up at 9 months after stent implantation . in - segment late loss was 0.20 0.22 mm in aes group and 0.10 0.35 mm ses group . in - stent late loss was 0.29 0.52 mm in aes group and 0.10 0.25 mm in ses group . the difference in in - stent late loss between two groups was 0.18 mm ( 95% confidence interval : 0.0770.313 mm ) , which was larger than the noninferiority margin as 0.15 mm . binary in - stent and in - segment restenosis rate were not different between the two groups ; however , diameter stenosis was statistically greater in aes group than ses group for both in - stent ( 21.05% 18.70% vs. 12.01% 16.26% , p = 0.002 ) and in - segment ( 22.86% 11.34% vs. 17.83% 13.59% , p = 0.012 ) [ table 5 ] . clinical outcomes , n ( % ) tvf : target vessel failure ; mi : myocardial infarction ; tlr : target lesion revascularization ; st : stent thrombosis ; aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . nine - month angiographic follow - up results aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . the main findings of the current study were as below : ( 1 ) the efficacy of aes and ses was similar over the follow - up period , with no significant difference observed in the incidence of tvf . ( 2 ) aes group has a significant lower rate of all - cause death than ses group , but not in lll and in - stent restenosis . although both ses and paclitaxel - eluting stents have greatly reduced in - stent restenosis , the first generation of des has led to concern for increased risk of late and vlst due to delayed arterial healing . in spite of the unclear mechanism , both sirolimus and paclitaxel were thought to play an important role on both delayed healing and endothelial dysfunction , which may cause the increasing risk of lst . despite the lower rate of late adverse events and better endothelial function of the second generation des , the anti - proliferative drugs they used , such as zotarolimus , everolimus , and biolimus a9 , were still sirolimus derivatives . all of them are specific inhibitors of the mammalian target of rapamycin ( mtor ) signaling pathway , which controls protein synthesis and has an important role in modulating cell division in response to mitogenic stimuli . the inhibition of mtor pathway could greatly inhibit the proliferation of vsmc , however it could also inhibit the proliferation and migration of endothelial cell ( ec ) , which causes delayed healing and endothelial dysfunction . hence , the attempt to using a novel drug , which has diverse pathways to inhibiting vsmc proliferation , could possibly reduce the damage to ecs , and leads to better clinical outcomes . arsenic trioxide , a natural substance that was used in the treatment of apl , also proved to induce cellular apoptosis in vsmcs through intracellular reactive oxygen species formation and free ca increase in vitro . previous study suggested that the aes could significantly suppress the in - stent restenosis in animal models as well . besides , the dosage of arsenic trioxide in our eluting stent was only 40 g , which was much less than the safe amount administered systemically ( 0.15 mgkgday ) . in our study , no adverse events correlated with arsenic trioxide toxicity occurred . though further observation for long - term safety is needed , aes was safe to use on the basis of current evidence . the primary end point tvf was similar between two groups in 24-month follow - up ( 6.67% vs. 5.83% , p = 0.802 ) . we note that all tvf ( actually all tlr ) events occurred within the 9-month follow - up in aes group , in contrast to the gradually increasing incidence in ses group , which could be explained as late catch - up phenomenon . although early observation of tlr showed possible greater neointimal hyperplasia with aes , no events after 12 months indicated the potential advantage of aes in long - term efficacy . notably , all - cause death ( 0 vs. 4.85% , p = 0.028 ) and cardiac death or mi events ( 0 vs. 3.88% , p = 0.058 ) , mostly happening after 1 year , were lower in aes group . among the four cardiac death events , three of them were attributed to definite or probable late or vlst , although there were no significant differences between two groups . possible explanation of this finding may be the comparative lower toxicity of arsenic trioxide to ec and rapid elution of the drug from a biodegradable polymer . although des remarkably reduced restenosis and repeated revascularization , the occurrence of late events , such as lst and vlst , was the major limitation of des . the reduction of cardiac death and mi , which seemed to be mainly driven by a reduction in vlst , could greatly shift the risk / benefit balance in favor of des . hence , the less long - term adverse events could be a potential advantage of aes over ses . in - stent lll at 9-month angiographic follow - up was significantly higher in patients treated with aes as compared those with ses ( 0.29 0.52 mm vs. 0.10 0.25 mm , p = 0.008 ) , as well as in - segment lll ( 0.20 0.22 mm vs. 0.10 0.35 mm , p = 0.026 ) . the reasons for higher lll observed with aes in this trial compared with ses are not completely understood . it might be due to differences in biological activity of arsenic trioxide compared with sirolimus , although in vitro cell culture experiments and animal studies would suggest at least equivalent potencies in suppressing smooth muscle cell proliferation and even additionally inducing cell apoptosis . another potential reason for the difference is the too short elution time of arsenic trioxide from the plla . in previous study , arsenic trioxide could reach a plateau after 8 days in contrast to 14 days of paclitaxel in vitro . more rapid elution time leads to insufficient tissue exposure to the therapeutic agents , resulting inadequate suppression of inflammation and intimal hyperplasia induced by injury responses . the endeavor zotarolimus - eluting stent ( e - zes ) , which also has a short release duration , suffered an even higher in - stent lll of around 0.60 mm and higher tlr rate compared to ses . hence , it has reason to believe that the higher lll of the aes group is caused by the quick release of drugs and polymers . third , low rate of angiographic follow - up in ses group ( 53.27% ) may result in an overestimate of the difference between two groups . despite the higher restenosis rate , the lower late events and stable tlr rate let us consider aes as a potential interestingly , in spite of the higher tlr of e - zes compared with des in the 1 year , the rates of cardiac death / mi , tlr , and definite / probable stent thrombosis were significantly lower with e - zes in the 5-year time frame . hence , the long - term efficacy and safety of aes are promising , and the attempt to using novel arsenic trioxide as an alternative to traditional sirolimus derivatives remains bright prospects . first , patients included in this study were selected strictly by angiographic and clinical characteristics , so the study population may not represent the real - world patients with coronary artery diseases , including mi . meanwhile , the history of some risk factors such as diabetes was not included in this study , and it may cause bias of the result . second , the angiographic follow - up rate was unsatisfactory in ses group and may have resulted in an underestimation of the occurrence of angiographic restenosis in a controlled group . in addition , around 20% of patients were lost to clinical follow - up during the 270 days , and it may also affect the final conclusion . third , ivus , oct , and other intracoronary imaging examinations were not used to evaluate the stent - treated lesion . besides that , the trial is a lack of a long - term angiographic endpoint , which can be used to confirm the long - term safety of aes . prospective observational and controlled clinical trials with a larger size may contribute to our understanding of the performance of aes and may provide insight into outcomes of the real - world use of aes . the comparison group used in this study enrolled patients using the first - generation ses , which was in routine clinical use when the study was undertaken while now it is used relatively infrequently in clinical practice . the 2-year follow - up of the randomized controlled trial demonstrates the efficacy and safety of aes compared with ses for the treatment of de novo coronary lesions . despite the higher lll , aes group shows similar tvf rate and significant reduction of all - caused death rate .

background : despite great reduction of in - stent restenosis , first - generation drug - eluting stents ( dess ) have increased the risk of late stent thrombosis due to delayed endothelialization . arsenic trioxide , a natural substance that could inhibit cell proliferation and induce cell apoptosis , seems to be a promising surrogate of sirolimus to improve des performance . this randomized controlled trial was to evaluate the efficacy and safety of a novel arsenic trioxide - eluting stent ( aes ) , compared with traditional sirolimus - eluting stent ( ses).methods : patients with symptoms of angina pectoris were enrolled and randomized to aes or ses group . the primary endpoint was target vessel failure ( tvf ) , and the second endpoint includes rates of all - cause death , cardiac death or myocardial infarction , target lesion revascularization ( tlr ) by telephone visit and late luminal loss ( lll ) at 9-month by angiographic follow-up.results:from july 2007 to 2009 , 212 patients were enrolled and randomized 1:1 to receive either aes or ses . at 2 years of follow - up , tvf rate was similar between aes and ses group ( 6.67% vs. 5.83% , p = 0.980 ) . frequency of all - cause death was significantly lower in aes group ( 0 vs. 4.85% , p = 0.028 ) . there was no significant difference between aes and ses in frequency of tlr and in - stent restenosis , but greater in - stent lll was observed for aes group ( 0.29 0.52 mm vs. 0.10 0.25 mm , p = 0.008).conclusions : after 2 years of follow - up , aes demonstrated comparable efficacy and safety to ses for the treatment of de novo coronary artery lesions .

I M Study design and population Randomization and procedure Follow-up End points and definition Statistical analysis R Baseline characteristics and procedural results Clinical outcomes and angiographic characteristics D

the introduction of drug - eluting stents ( dess ) led the interventional cardiology to a new era , in which the pci became an alternative to coronary artery bypass surgery even in complex lesions . a new des platform combining the approved 316l stainless stent platform with a novel anti - proliferative agent , arsenic trioxide , and a biodegradable poly - l - lactic acid ( plla ) polymer has been developed . arsenic trioxide , a chemotherapy drug that has been widely used in the treatment of acute promyelocytic leukemia ( apl ) , was proved to inhibit cell growth and induce cell apoptosis . our previous study had proved this stent system significantly suppressed in - stent restenosis by reducing proliferation and inducing apoptosis of vascular smooth muscle cells ( vsmcs ) compared to uncoated stents in a rabbit iliac artery injury model . the trial was a multicenter , perspective , randomized controlled trial designed to compare clinical and angiographic outcomes between arsenic trioxide - eluting stents ( aess ) and sirolimus - eluting stents ( ses ) [ figure 1 ] . aes : arsenic trioxide - eluting stent(s ) ; ses : sirolimus - eluting stent(s ) ; tvf : target vessel failure ; lll : late luminal loss . patients 18 and < 80 years of age were eligible for enrollment if they showed symptoms of angina pectoris and/or signs of ischemia or angina and met the main inclusion criteria of coronary angiography : ( 1 ) lesion length 37 mm ; ( 2 ) reference vessel diameter ( rvd ) 2.04.0 mm ; ( 3 ) a diameter stenosis 70% or total occlusion lesion . exclusion criteria included cardiac shock , congenital heart disease , severe valvular disease , left ventricular ejection fraction < 30% , allergy to antiplatelet drugs , heparin , stainless steel , contrast agents , surgery in 6 weeks , acute myocardial infarction ( mi ) within 2 weeks , transient ischemic attack or stroke within 7 days , wbc < 4.0 10/l , pbc < 100 10/l , 1.5 times higher than normal value of alanine aminotransferases , aspartate aminotransferase , or alkaline phosphate , using thrombolytic agents or gpiib / iiia antagonists in 1 week , gastrointestinal and fundus bleeding within 6 months , true bifurcation lesion with side branch diameter > 2.00 mm , life expectancy < 1 year , stented target lesion , inability or unwillingness to comply with all protocol - required procedure , and a positive pregnancy test within 24 h before enrollment . at follow - up , patients were specifically questioned regarding any adverse events or angina symptoms or other interventional therapy . the primary end point was target vessel failure ( tvf ) , a composite of cardiac death , target vessel mi , clinically driven target vessel revascularization ( defined as a repeated revascularization due to either a > 50% restenosis in the treated lesion with symptoms of ischemia or for a > 70% restenosis with or without symptoms within the entire major coronary vessel proximal and distal to a target lesion , including upstream and downstream branches and the target lesion itself ) . the secondary endpoints included late luminal loss ( lll ) , procedure success rate , in - stent restenosis rate , target lesion revascularization ( tlr ) , all - cause death , cardiac death or mi . the lll was defined as the difference between postprocedural and 9-month angiography follow - up minimal lumen diameter . cardiac death was defined as death due to any of the following : acute mi ; cardiac perforation / pericardial tamponade ; arrhythmia or conduction abnormality . the trial was designed to test the efficacy of aes compared with ses with respect to the primary endpoint of tvf . the objective of the study was to assess efficacy of aes compared with the ses with regard to the primary endpoint , tvf . based on the previous studies , we expect a mean in - stent lll of 0.40 mm firebird ses . the trial was a multicenter , perspective , randomized controlled trial designed to compare clinical and angiographic outcomes between arsenic trioxide - eluting stents ( aess ) and sirolimus - eluting stents ( ses ) [ figure 1 ] . aes : arsenic trioxide - eluting stent(s ) ; ses : sirolimus - eluting stent(s ) ; tvf : target vessel failure ; lll : late luminal loss . patients 18 and < 80 years of age were eligible for enrollment if they showed symptoms of angina pectoris and/or signs of ischemia or angina and met the main inclusion criteria of coronary angiography : ( 1 ) lesion length 37 mm ; ( 2 ) reference vessel diameter ( rvd ) 2.04.0 mm ; ( 3 ) a diameter stenosis 70% or total occlusion lesion . exclusion criteria included cardiac shock , congenital heart disease , severe valvular disease , left ventricular ejection fraction < 30% , allergy to antiplatelet drugs , heparin , stainless steel , contrast agents , surgery in 6 weeks , acute myocardial infarction ( mi ) within 2 weeks , transient ischemic attack or stroke within 7 days , wbc < 4.0 10/l , pbc < 100 10/l , 1.5 times higher than normal value of alanine aminotransferases , aspartate aminotransferase , or alkaline phosphate , using thrombolytic agents or gpiib / iiia antagonists in 1 week , gastrointestinal and fundus bleeding within 6 months , true bifurcation lesion with side branch diameter > 2.00 mm , life expectancy < 1 year , stented target lesion , inability or unwillingness to comply with all protocol - required procedure , and a positive pregnancy test within 24 h before enrollment . clinical follow - up was performed at 1 , 3 , 6 , 9 , 18 , 24 months by telephone . at follow - up , patients were specifically questioned regarding any adverse events or angina symptoms or other interventional therapy . the primary end point was target vessel failure ( tvf ) , a composite of cardiac death , target vessel mi , clinically driven target vessel revascularization ( defined as a repeated revascularization due to either a > 50% restenosis in the treated lesion with symptoms of ischemia or for a > 70% restenosis with or without symptoms within the entire major coronary vessel proximal and distal to a target lesion , including upstream and downstream branches and the target lesion itself ) . the secondary endpoints included late luminal loss ( lll ) , procedure success rate , in - stent restenosis rate , target lesion revascularization ( tlr ) , all - cause death , cardiac death or mi . the lll was defined as the difference between postprocedural and 9-month angiography follow - up minimal lumen diameter . cardiac death was defined as death due to any of the following : acute mi ; cardiac perforation / pericardial tamponade ; arrhythmia or conduction abnormality . the trial was designed to test the efficacy of aes compared with ses with respect to the primary endpoint of tvf . the objective of the study was to assess efficacy of aes compared with the ses with regard to the primary endpoint , tvf . based on the previous studies , we expect a mean in - stent lll of 0.40 mm firebird ses . the standard deviation ( sd ) for lll was presumed to be 0.45 mm , so the noninferiority margin was set at 0.15 mm ( equaling 35% of 0.45 mm ) . between july 2009 and january 2010 , 212 patients and 265 lesions were enrolled into this randomized study at eight sites across china . at 24 months , clinical follow - up was available for 105 patients in aes group and 103 patients in ses group . of a total of 265 treated lesions , approximately half of them are type b2/c lesions with mean rvds of 3.01 0.36 mm and lesion lengths of 19.97 8.20 mm ( aes ) and 19.52 8.13 mm ( ses ) . there was no significant difference between the baseline target lesions , but more severe diameter stenosis was observed in aes group ( 87.09 8.86% vs. 83.36 9.00% , p = 0.001 ) [ table 2 ] . baseline patient characteristics ecg : electrocardiogram ; ucg : ultrasound cardiogram ; aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . lesion and procedure characteristics ds : diameter stenosis ; rvd : reference vessel diameter ; aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent ; acc : american college of cardiology ; aha : american heart association . stent implantation data aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . the follow - up rate was more than 80% through the 24-month follow - up . the primary endpoint tvf in aes group was stable ( 6.67% ) , but an increasing trend was observed in ses group ( 1.87%5.83% ) . all cause death rate , as well as cardiac death or mi rate ( 0 vs. 3.88% , p = 0.058 ) at 24 months , was lower in aes group compared with ses group ( 0 vs. 4.85% , other adverse events including tlr , cerebral hemorrhage , noncardiac death , stent thrombosis , and angina pectoris were similar in both groups [ table 4 ] . 85 patients with 101 lesions in aes group ( 80.95% ) and 57 patients with 62 lesions in ses group ( 53.27% ) underwent angiographic follow - up at 9 months after stent implantation . in - segment late loss was 0.20 0.22 mm in aes group and 0.10 0.35 mm ses group . in - stent late loss was 0.29 0.52 mm in aes group and 0.10 0.25 mm in ses group . the difference in in - stent late loss between two groups was 0.18 mm ( 95% confidence interval : 0.0770.313 mm ) , which was larger than the noninferiority margin as 0.15 mm . binary in - stent and in - segment restenosis rate were not different between the two groups ; however , diameter stenosis was statistically greater in aes group than ses group for both in - stent ( 21.05% 18.70% vs. 12.01% 16.26% , p = 0.002 ) and in - segment ( 22.86% 11.34% vs. 17.83% 13.59% , p = 0.012 ) [ table 5 ] . clinical outcomes , n ( % ) tvf : target vessel failure ; mi : myocardial infarction ; tlr : target lesion revascularization ; st : stent thrombosis ; aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . nine - month angiographic follow - up results aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . between july 2009 and january 2010 , 212 patients and 265 lesions were enrolled into this randomized study at eight sites across china . at 24 months , clinical follow - up was available for 105 patients in aes group and 103 patients in ses group . there was no significant difference between the baseline target lesions , but more severe diameter stenosis was observed in aes group ( 87.09 8.86% vs. 83.36 9.00% , p = 0.001 ) [ table 2 ] . baseline patient characteristics ecg : electrocardiogram ; ucg : ultrasound cardiogram ; aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . lesion and procedure characteristics ds : diameter stenosis ; rvd : reference vessel diameter ; aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent ; acc : american college of cardiology ; aha : american heart association . stent implantation data aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . the primary endpoint tvf in aes group was stable ( 6.67% ) , but an increasing trend was observed in ses group ( 1.87%5.83% ) . all cause death rate , as well as cardiac death or mi rate ( 0 vs. 3.88% , p = 0.058 ) at 24 months , was lower in aes group compared with ses group ( 0 vs. 4.85% , p = 0.028 ) . other adverse events including tlr , cerebral hemorrhage , noncardiac death , stent thrombosis , and angina pectoris were similar in both groups [ table 4 ] . 85 patients with 101 lesions in aes group ( 80.95% ) and 57 patients with 62 lesions in ses group ( 53.27% ) underwent angiographic follow - up at 9 months after stent implantation . in - segment late loss was 0.20 0.22 mm in aes group and 0.10 0.35 mm ses group . in - stent late loss was 0.29 0.52 mm in aes group and 0.10 0.25 mm in ses group . binary in - stent and in - segment restenosis rate were not different between the two groups ; however , diameter stenosis was statistically greater in aes group than ses group for both in - stent ( 21.05% 18.70% vs. 12.01% 16.26% , p = 0.002 ) and in - segment ( 22.86% 11.34% vs. 17.83% 13.59% , p = 0.012 ) [ table 5 ] . clinical outcomes , n ( % ) tvf : target vessel failure ; mi : myocardial infarction ; tlr : target lesion revascularization ; st : stent thrombosis ; aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . nine - month angiographic follow - up results aes : arsenic trioxide - eluting stent ; ses : sirolimus - eluting stent . the main findings of the current study were as below : ( 1 ) the efficacy of aes and ses was similar over the follow - up period , with no significant difference observed in the incidence of tvf . ( 2 ) aes group has a significant lower rate of all - cause death than ses group , but not in lll and in - stent restenosis . although both ses and paclitaxel - eluting stents have greatly reduced in - stent restenosis , the first generation of des has led to concern for increased risk of late and vlst due to delayed arterial healing . arsenic trioxide , a natural substance that was used in the treatment of apl , also proved to induce cellular apoptosis in vsmcs through intracellular reactive oxygen species formation and free ca increase in vitro . the primary end point tvf was similar between two groups in 24-month follow - up ( 6.67% vs. 5.83% , p = 0.802 ) . we note that all tvf ( actually all tlr ) events occurred within the 9-month follow - up in aes group , in contrast to the gradually increasing incidence in ses group , which could be explained as late catch - up phenomenon . notably , all - cause death ( 0 vs. 4.85% , p = 0.028 ) and cardiac death or mi events ( 0 vs. 3.88% , p = 0.058 ) , mostly happening after 1 year , were lower in aes group . the reduction of cardiac death and mi , which seemed to be mainly driven by a reduction in vlst , could greatly shift the risk / benefit balance in favor of des . in - stent lll at 9-month angiographic follow - up was significantly higher in patients treated with aes as compared those with ses ( 0.29 0.52 mm vs. 0.10 0.25 mm , p = 0.008 ) , as well as in - segment lll ( 0.20 0.22 mm vs. 0.10 0.35 mm , p = 0.026 ) . it might be due to differences in biological activity of arsenic trioxide compared with sirolimus , although in vitro cell culture experiments and animal studies would suggest at least equivalent potencies in suppressing smooth muscle cell proliferation and even additionally inducing cell apoptosis . the endeavor zotarolimus - eluting stent ( e - zes ) , which also has a short release duration , suffered an even higher in - stent lll of around 0.60 mm and higher tlr rate compared to ses . third , low rate of angiographic follow - up in ses group ( 53.27% ) may result in an overestimate of the difference between two groups . despite the higher restenosis rate , the lower late events and stable tlr rate let us consider aes as a potential interestingly , in spite of the higher tlr of e - zes compared with des in the 1 year , the rates of cardiac death / mi , tlr , and definite / probable stent thrombosis were significantly lower with e - zes in the 5-year time frame . hence , the long - term efficacy and safety of aes are promising , and the attempt to using novel arsenic trioxide as an alternative to traditional sirolimus derivatives remains bright prospects . first , patients included in this study were selected strictly by angiographic and clinical characteristics , so the study population may not represent the real - world patients with coronary artery diseases , including mi . second , the angiographic follow - up rate was unsatisfactory in ses group and may have resulted in an underestimation of the occurrence of angiographic restenosis in a controlled group . in addition , around 20% of patients were lost to clinical follow - up during the 270 days , and it may also affect the final conclusion . third , ivus , oct , and other intracoronary imaging examinations were not used to evaluate the stent - treated lesion . the 2-year follow - up of the randomized controlled trial demonstrates the efficacy and safety of aes compared with ses for the treatment of de novo coronary lesions . despite the higher lll , aes group shows similar tvf rate and significant reduction of all - caused death rate .

diabetic retinopathy and age - related macular degeneration ( amd ) are primary vision threatening ocular diseases which affect retinal pigment epithelium ( rpe ) , macular region of the retina , choriocapillary , and bruch 's membrane . amd is typically observed in two forms , wet and dry . in wet amd , choroidal neovascularization ( cnv ) occurs due to the leakage of blood and other fluids into the subretinal space which leads to scar formation eventually causing irreversible vision loss . many investigators reported active involvement of vascular endothelial growth factor ( vegf ) , a naturally occurring lipoprotein in various pathophysiological processes including amd and diabetic retinopathy ( dr ) . currently , anti - vegf antibodies such as bevacizumab and ranibizumab are indicated for the treatment of wet amd . bevacizumab is a full - length ( 149 kda ) recombinant humanized murine monoclonal antibody specific to all isoforms of vegf . due to shorter intravitreal half - life of bevacizumab , frequent administrations are inconvenient and cause potential complications like retinal hemorrhage , retinal detachment , endophthalmitis , and more importantly patient noncompliance [ 57 ] . various biodegradable polymeric nanoparticulate formulations have been extensively investigated for controlled delivery of protein therapeutics . biodegradable polymers such as polycaprolactone ( pcl ) , polylactic acid ( pla ) , polyglycolic acid ( pga ) , and polyethylene glycol ( peg ) have been comprehensively studied for the preparation of protein - encapsulated nanoparticles ( nps ) . recently , many investigators have applied various block copolymers such as peg - pcl , pcl - peg - pcl , poly lactide - co - glycolide ( plga ) , and peg - pla for the development of sustained release protein formulations . however , previously published reports indicate that protein / peptide molecules suffer from rapid loss of biological activity during formulation preparation , storage , and/or release [ 1215 ] . acylation , with polymer degradation products ( lactic acid and glycolic acid ) [ 16 , 17 ] , accelerates hydrolysis due to lower ph caused by polymer degradation . moreover , presence of hydrophobic interfaces is a potential reason for the loss of activity and/or irreversible aggregation of protein therapeutics inside the pla , plga , pcl - pla - pcl , and pla - peg - pla based delivery systems . it is imperative to note that any change in protein / peptide structure , either physically or chemically , may cause immunogenicity and toxicity . a consecutive antibody response signifies safety concerns and subsequently restricts the efficacy of subsequent applications . as a result , there is an urgency to develop biocompatible and biodegradable polymeric system which provides sustained release of protein therapeutics at near zero - order rate for longer periods without compromising stability and functional activity of proteins . therefore , the objective of this work is to synthesize and evaluate novel tailor - made pb copolymers for controlled and noninvasive delivery of protein macromolecules in the treatment of posterior segment diseases . various ratios and molecular weights of each block ( pga , peg , pla , and pcl ) were selected for the synthesis to optimize sustained release profile of fitc - bsa , igg , and bevacizumab from nps and thermosensitive gel and combinations . our hypothesis is that release of protein therapeutics from nps is affected by their hydrodynamic diameters . therefore , in this study , we have examined the effect of hydrodynamic diameter of protein molecule on entrapment efficiency and in vitro drug release . nps have been characterized by particle size , polydispersity , entrapment efficiency , drug loading , and in vitro release profiles . furthermore , in order to achieve continuous zero - order drug release , we have prepared and characterized a novel composite formulation comprising drug - loaded nps suspended in thermosensitive gelling aqueous solution . stability of released protein was also examined by cd spectroscopy and in vitro biological assays to ensure therapeutic efficacy of the protein . poly(ethylene glycol ) , ( peg : 1 kda and 4 kda ) , methoxy - peg ( 550 da ) , stannous octoate , -caprolactone , poly(vinyl alcohol ) ( pva ) , and lipopolysaccharide were procured from sigma - aldrich ( st . louis , mo , usa ) . l - lactide and hexamethylenediamine ( hmdi ) were purchased from acros organics ( morris plains , nj , usa ) . lactate dehydrogenase estimation kit and celltiter 96 aqueous nonradioactive cell proliferation assay ( mts ) kit were obtained from takara bio inc . and promega corp . , novel pb copolymers , poly(glycolic acid)-poly(caprolactone)-poly(ethylene glycol)-poly(caprolactone)-poly(glycolic acid ) ( pga - pcl - peg - pcl - pga , i.e. , pb - a ) , poly(lactic acid)-poly(caprolactone)-poly(ethylene glycol)-poly(caprolactone)-poly(lactic acid ) ( pla - pcl - peg - pcl - pla , i.e. , pb - b ) , and poly(ethylene glycol)-poly(caprolactone)-poly(lactic acid)- poly(caprolactone)-poly(ethylene glycol ) , ( peg - pcl - pla - pcl - peg , i.e. , pb - c ) , were synthesized by ring - opening bulk polymerization method . pb copolymers for preparation of nps , i.e. , pb - a and pb - b , were synthesized in two steps by sequential ring - opening polymerization . peg ( 1 kda and 4 kda ) was utilized as macroinitiator and stannous octoate was utilized as catalyst . in the first step , triblock ( tb ) copolymer pcl - peg - pcl ( figure 1 , step 1 ) was synthesized by polymerization of -caprolactone on two open hydroxyl ends of peg . in brief , peg ( 1 g , 1 kda for pb - a ) ( 2 g , 4 kda for pb - b ) was dissolved in anhydrous toluene followed by distillation to remove residual moisture . -caprolactone ( 15 g for pb - a and 5.7 g for pb - b ) and stannous octoate ( 0.5% w / w ) were added to anhydrous peg and temperature was raised to 130c . after 24 h , reaction mixture was dissolved in methylene chloride followed by precipitation in cold petroleum ether . the precipitated polymer was filtered and dried for 24 h under vacuum at room temperature . in the second step , pcl - peg - pcl tb copolymer was reacted with glycolide and l - lactide to prepare pb - a ( figure 1 , step 2 ) and pb - b ( figure 2 ) copolymers , respectively . tb copolymer and glycolide ( 0.6 g)/l - lactide ( 3.45 g ) were added in round bottom flask and temperature was raised to 130c under inert atmosphere . to this reaction mixture , stannous octoate ( 0.5% w / w ) was added and reaction was allowed to run for 24 h. pb copolymer was then purified by cold ether precipitation method as described in the first step . the polymer was dried under vacuum and stored at 20c until further use . for the synthesis of thermosensitive gelling polymer , tb copolymer ( mpeg - pcl - pla ) was synthesized by ring - opening bulk copolymerization as described above ( figure 3 ) . -caprolactone ( 8.25 g ) was polymerized at the hydroxyl terminal of mpeg ( 550 da , 5.5 g ) followed by another polymerization with l - lactide ( 2.75 g ) . resulting tb copolymers were coupled utilizing hmdi ( equimolar of mpeg ) as a linker . coupling reaction was carried out at 70c for 8 h. polymers were purified by cold ether precipitation followed by drying under vacuum and stored at 20c . synthesized polymers were characterized by molecular weight and purity by h nmr spectroscopy and gel permeation chromatography ( gpc ) . purity and molecular weight ( mn ) were calculated from the h - nmr spectra . purity , molecular weights , and polydispersity of pb copolymers were further confirmed by gpc analysis . briefly , samples were prepared by dissolving 5 mg of polymeric material in tetrahydrofuran ( thf ) whereas thf was utilized as eluting agent at the flow rate of 1 ml / min . polystyrene samples with narrow molecular weight distribution were considered as standards . cell culture . human retinal pigment epithelial cells ( arpe-19 ) were cultured and maintained according to the previously published protocol from our laboratory . in brief , cells were cultured in dulbecco 's modified eagle 's medium ( dmem)/f-12 medium containing 10% fetal bovine serum ( fbs ) , 15 mm of hepes , 29 mm of sodium bicarbonate , 100 u / l of penicillin , and 100 mg / l of streptomycin . a mouse macrophage cell line , raw-264.7 , was cultured and maintained in dmem supplemented with 10% fbs , 100 u / l of penicillin , and 100 mg / l of streptomycin . the statens seruminstitut rabbit corneal ( sirc ) cell line was selected between passages 410 and 425 . this cell line was grown with cell culture media composed of mem containing 10% fbs , lactalbumin , hepes , sodium bicarbonate , 100 u / l of penicillin , and 100 mg / human conjunctival epithelial cells ( hcec ) were maintained in cell culture flask containing mem earle 's bss medium supplemented by 10% fbs , 100 u / l of penicillin , 100 mg / l of streptomycin , 29 mm of sodium bicarbonate , and 2 mm l - glutamine . choroid - retinal endothelial cells ( rf/6a cells ) were cultured and maintained in cell culture medium composed of rpmi-1640 comprising 10% fbs , 100 u / l of penicillin , and 100 mg / all five cell lines were procured from atcc and maintained according to atcc guideline at 37c , 5% co2 , and 95% humidity . previously published protocol with minor modification was employed to evaluate the cytotoxicity of pb copolymers . briefly , 10 mg / ml of pb copolymers ( pb - a and pb - b ) was dissolved in acn and 100 l of solutions was aliquoted in each of the 96-well plates . plates were exposed overnight under uv light ( laminar flow ) for polymer sterilization as well as evaporation of acn . arpe-19 cells at a density of 1.0 10 were seeded in each well and incubated at 37c and 5% co2 in humidified atmosphere for 48 h. after completion of incubation period , cell supernatants were analyzed for quantification of ldh . more than 10% of ldh release was considered as cytotoxic . to evaluate cytotoxicity of block copolymers on conjunctiva , cornea , and macrophages , ldh release ( % ) was calculated according to ( 1)ldh release(%)=abs . of sampleabs . of negative controlabs . of positive controlabs . of negative control 100 safety and biocompatibility of pb copolymers were further established by performing in vitro cell viability assay ( mts assay ) . as described earlier , pb copolymers solutions at the concentration of 10 mg / ml were prepared , aliquoted , and sterilized . after sterilization , arpe-19 cells were seeded in each well of 96-well plates at a cell density of 1.0 10 and incubated at 37c and 5% co2 in humidified atmosphere for 48 h. at the end of incubation period , cell culture medium was aspirated and cells were incubated for 4 h ( 37c and 5% co2 ) in presence of 100 l of serum - free medium containing 20 l mts solution . similar procedure was repeated with other ocular and macrophage cell lines such as hcec , sirc , and raw-264.7 cells . percent cell viability was calculated according to ( 2)cell viability(%)=abs . of sampleabs . of negative controlabs . of positive controlabs . of negative control 100 . in this study , pb copolymers which exhibited more than 90% of cell viability were considered nontoxic and suitable for ocular applications . pb copolymers were dissolved in acn at the concentration of 10 mg / ml . two hundred l was aliquoted in each well of 48-well cell culture plates which were incubated overnight under uv lights ( laminar flow ) for acn evaporation and sterilization of resulting polymer film . after sterilization , raw-264.7 cells ( 5.0 10 ) were seeded in each well of cell culture plate and incubated for 24 h at 37c and 5% co2 . cell supernatants were analyzed for the presence of cytokines , that is , tnf- , il-6 , and il-1. lipopolysaccharide ( lps ) was utilized as positive control whereas cells without treatment were considered as negative control . standard calibration curves for tnf- , il-6 , and il-1 were prepared in ranges of 10750 pg / ml , 5500 pg / ml , and 10500 pg / ml , respectively . igg - loaded pb nps were prepared by w1/o / w2 double emulsion solvent evaporation method . briefly , predetermined quantity of igg ( 10 mg ) was dissolved in 0.1 m phosphate buffer saline ( pbs , ph 7.4 ) ( 1 ml ) containing 50 l of tween-80 ( w1 phase ) . in order to prepare organic phase , 100 mg of pb copolymer was solubilized in 4 ml of dichloromethane ( dcm ) containing 50 l of span-20 ( organic phase ) . primary emulsion ( w1/o ) was prepared by dropwise addition of w1 phase to the organic phase under constant sonication applied with probe - sonicator for 1 min at 4 w output . to avoid excessive heating and possible degradation of protein , preparation of emulsion resulting w1/o primary emulsion was then added dropwise in 20 ml of 2% polyvinyl alcohol ( pva ) solution ( w2 phase ) under constant sonication for 4 min at 5 w output . double emulsion ( w1/o / w2 ) was stirred at room temperature for 30 min followed by evaporation of dcm under low pressure . once dcm was evaporated , nps were centrifuged for 30 min at 20000 rpm and 4c followed by two washing cycles with distilled deionized water ( ddw ) . finally , igg - loaded nps were freeze - dried in presence of 5% mannitol ( cryoprotectant ) and stored at 20c until further use . nps were prepared with two pb copolymers , that is , pb - a and pb - b . a similar protocol was followed to prepare fitc - bsa and bevacizumab - loaded nps . nps were characterized by particle size , entrapment efficiency ( ee% ) , drug loading ( dl% ) , and in vitro drug release pattern . freeze - dried nps were dispersed in ddw ( 1 mg / ml ) and analyzed for their size and distribution . particle size was determined by zeta sizer ( zetasizer nano zs , malvern instruments ltd . , protein - encapsulated freeze - dried nps were evaluated for the estimation of entrapment efficiency ( ee ) and drug loading ( dl ) . ee was estimated by the amount of protein in the supernatants obtained from np preparation . micro - bca protein estimation kit was employed for estimation of total protein . for analysis of dl , 2 mg equivalent protein - loaded nps was dissolved in 200 l of dimethyl sulfoxide ( dmso ) . standard curve of respective proteins ( igg , fitc - bsa , and bevacizumab ) ranging from 31.25 to 2000 g / ml was prepared in dmso . following equations were utilized for the calculation of ee ( % ) and dl ( % ) : ( 3)ee ( % ) = ( 1amount of drug in supernatanttotal amount of drug)100,dl ( % ) = ( amount of drug in nanoparticlestotal amount of drug and polymer)100 . in vitro release studies . igg - loaded nps were further characterized by their ability to sustain drug release . in order to conduct in vitro drug release studies , 1 mg of igg equivalent freeze - dried nps resulting np suspension was then incubated in water bath equilibrated at 37c . at predefined time intervals , a 200 l supernatant was collected and replaced with the same volume of pbs . nps were then resuspended and release study was continued at 37c . in a second set of in vitro release studies , 1 mg equivalent igg containing nps was suspended in 500 l of aqueous solution of thermosensitive gelling polymer ( pb - c ) ( 20 wt% ) . resulting suspension was incubated in 10 ml vial at 7c for 30 min . once gel was formed , an aliquot ( 5 ml ) of pbs ( preincubated at 37c ) was slowly added . at predetermined time intervals , 1 ml of clear supernatant was collected and replaced with the same volume of fresh pbs ( preincubated at 37c ) . release samples were analyzed by micro - bca for total protein content according to supplier 's instructions . fitc - bsa and bevacizumab - loaded nps were also evaluated for in vitro release kinetics . samples of released fitc - bsa were analyzed by fluorescence spectroscopy with excitation and emission wavelengths of 490 nm and 525 nm , respectively . in vitro release experiments were performed in triplicate and expressed as cumulative drug released ( % ) with time . in order to delineate release mechanism , korsmeyer - peppas equation is as follows : ( 4)mtm= ktn , where k is the kinetic constant and n is the diffusion exponent which describes release mechanism . mt and m represent cumulative protein release at time t and at the equilibrium , respectively . higuchi equation is as follows : ( 5)qt = kt1/2 , where k denotes higuchi rate kinetic constant , qt is the amount of released protein at time t , and t is time in hour . hixson - crowell equation is as follows : ( 6)c01/3ct1/3=kt , where c0 and ct represent initial and remaining amounts of protein in formulation , respectively , k is the constant incorporating surface to volume ratio , and t is time in hour . first - order equation is as follows : ( 7)logc = logc0kt2.303 , where k denotes the first - order rate constant , c0 is the initial protein concentration , and t represents time in hour . zero - order equation is as follows : ( 8)c = k0 t , where k0 is the zero - order rate constant and t is time in hour . released igg was analyzed by cd spectroscopy to examine secondary structure . cd spectra were recorded between wavelengths of 200 and 250 nm at scanning speed of 5 nm / min utilizing 1 cm cell . cd spectrum of pbs was utilized as blank . cell proliferation assay . a cell proliferation assay ( mts ) briefly , cells were seeded at a density of 5 10 cells / well of 96-well cell culture plates . after 24 h of incubation , cells were serum - starved overnight followed by addition of serum - free medium containing 100 ng / ml of vegf and 0.25 mg / ml of released bevacizumab ( test samples ) or 0.25 mg / ml of native bevacizumab ( standard ) . cells without vegf or with native bevacizumab treatment were considered as negative control or cells exposed only to vegf ( 100 ng / ml ) as positive control . next , cells were exposed to 100 l of serum - free medium containing 20 l of mts solution and incubated for 4 h. the absorbance was recorded at 450 nm using a microplate reader . cell migration assay . rf/6a cells were starved overnight ( by exposing it to serum - free medium ) , trypsinized , and suspended in serum - free medium containing 0.25 mg / ml of bevacizumab ( native or released from nps ) . cells ( 5 10 ) were seeded in upper chamber of transwell ( 8.0 m pore size , 10 mm diameter , corning inc . ) and preincubated with cell culture medium . vegf ( 100 ng / ml ) was placed into the lower chamber and cells were incubated for 24 h at 37c and 5% co2 . nonmigrated cells were removed from upper chamber by cotton swab and concentration of migrated cells was estimated by alamarblue assay . it was performed according to supplier 's protocol . moreover , for visual evidence , migrated cells were also stained with methylene blue and images were taken with leica dmi3000b inverted microscope ( germany ) . each experiment was repeated three times . pb copolymers ( pb - a and pb - b ) were successfully synthesized by ring - opening bulk copolymerization of -caprolactone and l - lactide / glycolide . in the first step , tb copolymers ( pcl - peg - pcl ) purified tb copolymers were then utilized for the synthesis of respective pb copolymers , that is , pb - a ( pga - pcl - peg - pcl - pga ) and pb - b ( pla - pcl - peg - pcl - pla ) . purity and molecular weights ( mn ) of pb copolymers were calculated by h - nmr spectroscopy . as described in figures 4 and 5 , typical h - nmr signals of pcl blocks were observed at 1.40 , 1.65 , 2.30 , and 4.06 ppm depicting methylene protons of (ch2)3 , oco pb - a exhibited cluster of singlets between 4.6 and 4.9 ppm representing methylene protons ( ch2 ) of pga units . pb copolymer with pla units as terminals ( pb - b ) demonstrated two additional peaks at 1.50 ( ch3 ) and 5.17 ( ch ) ppm . molar ratios of pb - a and pb - b were calculated from the integration values of peg ( 3.65 ppm ) , pcl ( 2.30 ppm ) , and pla ( 5.17 ppm ) or pga ( 4.64.9 ppm ) . h - nmr spectra of pb - c ( figure 6 ) exhibit typical proton signals of peg , pcl , and pla . an additional peak at 3.38 ppm was denoted to terminal methyl of ( och3 ) of peg and utilized for the molecular weight ( mn ) calculation of pb - c copolymer . as described in table 1 , polydispersity ( pdi ) of all the polymers was below 1.45 suggesting narrow distribution of molecular weights . moreover , block copolymers depicted a single peak in gpc chromatogram ( figure 7 ) indicating monodistribution of molecular weight and absence of any homopolymers such as pla , pga , pcl , and peg . molecular weights ( mn ) of pb - a , pb - b , and pb - c calculated with h - nmr spectroscopy and gpc were very similar to theoretical molecular weights ( table 1 ) . therefore , theoretical molecular weights are mentioned instead of calculated molecular weights in this report . in order to investigate compatibility of pb polymeric materials with biological system ( ocular cell lines ) , arpe-19 , sirc , hcec , and raw-264.7 cells for 48 h were treated with 10 mg / ml of pb - a and pb - b for 48 h. ldh is a cytoplasmic enzyme , secreted in cell culture medium following cell - membrane damage . less than 10% of ldh release was observed after 48 h exposure indicating negligible toxicity with any of the ocular cell lines ( figure 8) . noticeably , results were comparable with negative controls . to further confirm our observation , in addition to ldh assay , mts cell viability studies were also performed utilizing a similar protocol . in mts assay , results in figure 9 demonstrate that more than 90% cell viability ( for all the cell lines ) after 48 h exposure to polymeric materials suggesting excellent safety profile of block copolymers for ocular applications . many investigators have utilized in vitro cell culture model ( raw-264.7 ) for the estimation of biocompatibility of polymeric materials intended for human applications . in this study we have examined various cytokines release such as tnf- , il-6 , and il-1 in culture supernatant following 24 h exposure to pb - a and pb - b copolymers . results depicted in figure 10 indicate release of tnf- ( ~200 pg / ml ) in both groups , that is , pb - a and pb - b . however , these values are comparable to negative control ( cells without treatment ) with no significant difference . similarly , negligible release of il-6 and il-1 was observed suggesting that these copolymers are safe for human use . igg , fitc - bsa , and bevacizumab - encapsulated pb nps were prepared by w1/o / w2 double emulsion solvent evaporation method . nps prepared from pb copolymers were ranging in diameter from 320 to 355 nm ( table 2 ) . no significant effect of polymer composition ( pb - a / pb - b ) or type of protein molecule ( igg / bsa / bevacizumab ) was evident on particle size . these results suggest that hydrodynamic diameter of protein therapeutics or polymer composition has no significant effect on particle size or distribution . entrapment efficiency ( ee ) and drug loading ( dl ) are highly influenced by various parameters including copolymer composition ( hydrophobicity of polymer ) and phase volume ratios ( w1 , o , and w2 ) . in order to understand the effect of hydrophobicity of copolymers on ee and dl , we have prepared and evaluated igg and fitc - bsa - loaded nps utilizing pb - a and pb - b . pb - a copolymer comprising pga ( hydrophilic block ) is relatively hydrophilic compared to pb - b copolymer comprising pla ( hydrophobic block ) . as presented in table 2 , encapsulation of igg or fitc - bsa in pb - a nps was ~40% and ~35% , respectively . however , pb - b nps exhibited significantly higher encapsulation of ~70% and ~69% for igg and fitc - bsa , respectively . this is possibly due to the fact that , during preparation of nps ( solvent evaporation ) , hydrophobicity may allow faster precipitation of pb - b copolymer to form nps preventing diffusion of igg / fitc - bsa from w1 phase to external aqueous ( w2 ) phase . due to higher hydrophilicity , pb - a copolymer may remain hydrated with w1 and w2 phases ( during nps preparation ) allowing escape of igg / fitc - bsa in external phase resulting in poor ee . however , no effect of hydrodynamic diameter of igg or fitc - bsa on ee or dl was observed . it is very plausible that both of the proteins are too large ( 66 kda ) to show any significant effect of hydrodynamic diameter on ee or dl . bevacizumab - loaded pb - b nps exhibited ~67% of ee and ~6% of dl , very similar to igg - loaded pb - b nps ( table 2 ) suggesting that similar molecules behave similarly during np preparation . in order to evaluate the effect of polymer hydrophobicity , release of fitc - bsa and igg from pb - a and pb - b nps as described in figure 11 , both nps ( pb - a and pb - b ) demonstrated biphasic release profile , that is , initial burst release followed by sustained release . pb - a nps exhibited significantly higher burst release ( ~56% ) of fitc - bsa relative to pb - b nps ( ~48% ) . in the second phase , pb - b nps provided sustained bsa release for ~36 days whereas pb - a nps prolonged the release for only ~27 days . similar effect of polymer hydrophobicity was observed with igg - encapsulated nps where pb - b nps displayed longer release ( ~44 days ) than pb - a nps ( ~30 days ) ( figure 12 ) . as described earlier , pga based pb - a copolymer is hydrophilic compared to pla based pb - b copolymer . therefore , it is anticipated that pb - a nps possibly have higher affinity for the protein molecules which may result in higher amount of surface adsorbed drug . moreover , being hydrophilic , pb - a nps may be easily hydrated which may allow rapid diffusion of water molecules through polymeric matrix . therefore , higher amounts of surface adsorbed proteins and higher affinity towards water molecules may have contributed to the higher burst release and shorter duration of release from in pb - a nps . hydrodynamic diameter of protein therapeutics may have significant effect on drug release pattern . in order to examine this possibility we have compared in vitro release profiles of fitc - bsa ( 66 kda ) and igg ( 150 kda ) ( figure 13 ) from pb - b nps . results indicate significantly higher burst release and shorter release duration for fitc - bsa relative to igg from their respective nps . fitc - bsa has smaller hydrodynamic diameter compared to igg and may lead to more rapid diffusion through the polymeric matrix of nps . to further confirm this hypothesis , we have compared in vitro release profile of igg and bevacizumab ( 149 kda ) from their respective pb - b nps . since igg and bevacizumab are full - length antibodies with similar hydrophilicity and molecular weight ( hydrodynamic diameter ) , both protein molecules behave similarly during nps preparation and during release . these results suggest that parallel molecules ( igg or bevacizumab ) may not generate any difference in drug release pattern whereas there is a significant effect of hydrodynamic diameter ( fitc - bsa or igg ) on drug release . any nanoparticulate or microparticulate system can have certain amount of surface adsorbed drug , which is released within the first 24 h leading to burst effect . being very potent by nature , burst release of protein therapeutics may produce side effects . therefore , pharmaceutical scientists are focused on developing a formulation which can eliminate or minimize burst effect and offer zero - order drug release throughout the release period . in order to achieve zero - order drug release profile , protein - encapsulated pb - b nps were suspended in an aqueous solution of thermosensitive gelling polymer ( pb - c ) . thermosensitive gelling solution was composed of 20 wt% pb - c copolymer in ddw . aqueous solution of pb - c copolymer remains in liquid state around room temperature but immediately transforms to hydrogel at body temperature ( sol - gel transition ) . protein - loaded nps were suspended in 20 wt% gelling solution and then brought to 37c which immediately transitioned the solution to solid hydrogel entrapping nps throughout the polymeric matrix . these composite formulations comprised of protein - loaded pb - b nps ( fitc - bsa and igg ) suspended in thermosensitive gel were evaluated for release pattern . as depicted in figure 15 , burst release of fitc - bsa from composite formulation was negligible ( ~10% ) relative to burst release observed from pb - b nps ( ~48% ) . in addition , release of fitc - bsa was prolonged over 40 days . a similar pattern was also observed for a composite formulation comprising igg - loaded pb - b nps suspended in 20 wt% thermosensitive gelling solution ( figure 16 ) . composite formulation of igg exhibited negligible burst release followed by zero - order release up to 60 days . this behavior may be due to the fact that when the nps were suspended into the gel matrix the matrix serves as an additional diffusion barrier for the surface adsorbed drug . the gel matrix might deter quick hydration and dumping of surface adsorbed dose eliminating burst effect . it also leads to zero - order drug release throughout the release period . in order to evaluate drug release mechanism , we have fitted in vitro drug release data in five different release kinetic models , that is , korsmeyer - peppas , higuchi , hixson - crowell , zero - order , and first - order models . results , presented in table 3 , indicate that korsmeyer - peppas is the best fit model for all the formulations with r values ranging between 0.977 and 0.997 . moreover , n values in korsmeyer - peppas model for release of fitc - bsa , igg , and bevacizumab from pb - b nps were below 0.43 indicating diffusion controlled release . interestingly , n values for composite formulation of fitc - bsa ( 0.549 ) and igg ( 0.818 ) ( nps suspended in thermosensitive gel ) were between 0.43 and 0.89 suggesting anomalous diffusion . in other words , release of protein therapeutics from composite formulation is controlled by diffusion as well as degradation of polymer . cd spectroscopy is a sensitive and robust analytical technique exploited for the investigation of secondary and to some extent tertiary conformation of proteins . it is sensitive enough to detect minor conformational changes in -helix and -sheets of the protein . therefore , we have utilized this technique to confirm the conformational stability of released igg by comparing with cd spectrum of native igg ( figure 17 ) . cd spectrum of released igg demonstrated minima of 218 nm similar to the native igg . in addition , cd spectra of native and released igg ranging from 200 nm to 250 nm were identical indicating retention of protein conformation during np preparation and after release . rf/6a cells proliferated rapidly in presence of vegf ( 100 ng / ml , + ve control ) . however , proliferation was inhibited in absence of vegf indicating sensitivity of endothelial cells towards growth factor , particularly , vegf ( figure 18 ) . native bevacizumab ( standard group ) strongly inhibited vegf - induced cell proliferation at a concentration of 0.25 mg / ml . a similar level of inhibition was also observed when released ( from nps ) bevacizumab ( test / sample group ) was exposed for 24 h to vegf treated cells . moreover , inhibitory effects of released and native bevacizumab were not significantly different than ve control . vegf carries chemoattractant property which stimulates migration of rf/6a cells across a porous membrane towards a vegf stimulus . biological activity of released bevacizumab was further evaluated by vegf - induced cell migration assay . as described in figure 19 , a test / sample group ( released bevacizumab ) exhibited significant inhibition of cell migration across transwell membrane compared to vegf treated group ( + ve control ) . results indicated that this inhibitory effect was not significantly different than ve control or a standard group ( cells treated with native bevacizumab ) . for visual evidence , migrated cells were stained with methylene blue and images were prepared . cell proliferation and migration assays clearly suggest that bevacizumab activity is retained during np preparation and release . previous reports suggest that pla and/or pga based copolymers produce large molar mass of lactic acid and/or glycolic acid [ 16 , 17 ] . retention of protein stability ( igg and bevacizumab ) in pb nps can be attributed to lower molar mass of pla or pga blocks which produce very low amounts of lactic acid or glycolic acid , thereby eliminating or reducing the possibilities of protein degradation . this research article discusses synthesis and characterization of novel pb copolymers for the preparation of nps and thermosensitive gel . in order to eliminate burst release phase , a novel composite formulation comprised of protein - loaded pb nps suspended in pb thermosensitive gel fitc - bsa and igg - encapsulated nps suspended in thermosensitive gel demonstrated continuous zero - order release avoiding any possibility of dose - dependent toxicity . bevacizumab - loaded nps without gel demonstrated the same patterns of burst release and sustained release as those of igg - embedded nps . retention of functional activity of bevacizumab by in vitro cell proliferation and migration suggests that this approach can act as a platform for the ocular delivery of therapeutic macromolecules .

the objective of this research article is to report the synthesis and evaluation of novel pentablock copolymers for controlled delivery of macromolecules in the treatment of posterior segment diseases . novel biodegradable pb copolymers were synthesized by sequential ring - opening polymerization . various ratios and molecular weights of each block ( polyglycolic acid , polyethylene glycol , polylactic acid , and polycaprolactone ) were selected for synthesis and to optimize release profile of fitc - bsa , igg , and bevacizumab from nanoparticles ( nps ) and thermosensitive gel . nps were characterized for particle size , polydispersity , entrapment efficiency , and drug loading . in vitro release study of proteins from nps alone and composite formulation ( nps suspended in thermosensitive gel ) was performed . composite formulations demonstrated no or negligible burst release with continuous near zero - order release in contrast to nps alone . hydrodynamic diameter of protein therapeutics and hydrophobicity of pb copolymer exhibited significant effect on entrapment efficiency and in vitro release profile . cd spectroscopy confirmed retention of structural conformation of released protein . biological activity of released bevacizumab was confirmed by in vitro cell proliferation and cell migration assays . it can be concluded that novel pb polymers can serve a platform for sustained delivery of therapeutic proteins .

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

currently , anti - vegf antibodies such as bevacizumab and ranibizumab are indicated for the treatment of wet amd . various biodegradable polymeric nanoparticulate formulations have been extensively investigated for controlled delivery of protein therapeutics . biodegradable polymers such as polycaprolactone ( pcl ) , polylactic acid ( pla ) , polyglycolic acid ( pga ) , and polyethylene glycol ( peg ) have been comprehensively studied for the preparation of protein - encapsulated nanoparticles ( nps ) . moreover , presence of hydrophobic interfaces is a potential reason for the loss of activity and/or irreversible aggregation of protein therapeutics inside the pla , plga , pcl - pla - pcl , and pla - peg - pla based delivery systems . as a result , there is an urgency to develop biocompatible and biodegradable polymeric system which provides sustained release of protein therapeutics at near zero - order rate for longer periods without compromising stability and functional activity of proteins . therefore , the objective of this work is to synthesize and evaluate novel tailor - made pb copolymers for controlled and noninvasive delivery of protein macromolecules in the treatment of posterior segment diseases . various ratios and molecular weights of each block ( pga , peg , pla , and pcl ) were selected for the synthesis to optimize sustained release profile of fitc - bsa , igg , and bevacizumab from nps and thermosensitive gel and combinations . our hypothesis is that release of protein therapeutics from nps is affected by their hydrodynamic diameters . therefore , in this study , we have examined the effect of hydrodynamic diameter of protein molecule on entrapment efficiency and in vitro drug release . nps have been characterized by particle size , polydispersity , entrapment efficiency , drug loading , and in vitro release profiles . furthermore , in order to achieve continuous zero - order drug release , we have prepared and characterized a novel composite formulation comprising drug - loaded nps suspended in thermosensitive gelling aqueous solution . stability of released protein was also examined by cd spectroscopy and in vitro biological assays to ensure therapeutic efficacy of the protein . , novel pb copolymers , poly(glycolic acid)-poly(caprolactone)-poly(ethylene glycol)-poly(caprolactone)-poly(glycolic acid ) ( pga - pcl - peg - pcl - pga , i.e. , pb - c ) , were synthesized by ring - opening bulk polymerization method . pb copolymers for preparation of nps , i.e. , pb - a and pb - b , were synthesized in two steps by sequential ring - opening polymerization . in the first step , triblock ( tb ) copolymer pcl - peg - pcl ( figure 1 , step 1 ) was synthesized by polymerization of -caprolactone on two open hydroxyl ends of peg . -caprolactone ( 15 g for pb - a and 5.7 g for pb - b ) and stannous octoate ( 0.5% w / w ) were added to anhydrous peg and temperature was raised to 130c . in the second step , pcl - peg - pcl tb copolymer was reacted with glycolide and l - lactide to prepare pb - a ( figure 1 , step 2 ) and pb - b ( figure 2 ) copolymers , respectively . to this reaction mixture , stannous octoate ( 0.5% w / w ) was added and reaction was allowed to run for 24 h. pb copolymer was then purified by cold ether precipitation method as described in the first step . for the synthesis of thermosensitive gelling polymer , tb copolymer ( mpeg - pcl - pla ) was synthesized by ring - opening bulk copolymerization as described above ( figure 3 ) . purity and molecular weight ( mn ) were calculated from the h - nmr spectra . purity , molecular weights , and polydispersity of pb copolymers were further confirmed by gpc analysis . choroid - retinal endothelial cells ( rf/6a cells ) were cultured and maintained in cell culture medium composed of rpmi-1640 comprising 10% fbs , 100 u / l of penicillin , and 100 mg / all five cell lines were procured from atcc and maintained according to atcc guideline at 37c , 5% co2 , and 95% humidity . previously published protocol with minor modification was employed to evaluate the cytotoxicity of pb copolymers . briefly , 10 mg / ml of pb copolymers ( pb - a and pb - b ) was dissolved in acn and 100 l of solutions was aliquoted in each of the 96-well plates . to evaluate cytotoxicity of block copolymers on conjunctiva , cornea , and macrophages , ldh release ( % ) was calculated according to ( 1)ldh release(%)=abs . of negative control 100 safety and biocompatibility of pb copolymers were further established by performing in vitro cell viability assay ( mts assay ) . as described earlier , pb copolymers solutions at the concentration of 10 mg / ml were prepared , aliquoted , and sterilized . pb copolymers were dissolved in acn at the concentration of 10 mg / ml . in order to prepare organic phase , 100 mg of pb copolymer was solubilized in 4 ml of dichloromethane ( dcm ) containing 50 l of span-20 ( organic phase ) . finally , igg - loaded nps were freeze - dried in presence of 5% mannitol ( cryoprotectant ) and stored at 20c until further use . a similar protocol was followed to prepare fitc - bsa and bevacizumab - loaded nps . nps were characterized by particle size , entrapment efficiency ( ee% ) , drug loading ( dl% ) , and in vitro drug release pattern . freeze - dried nps were dispersed in ddw ( 1 mg / ml ) and analyzed for their size and distribution . , protein - encapsulated freeze - dried nps were evaluated for the estimation of entrapment efficiency ( ee ) and drug loading ( dl ) . ee was estimated by the amount of protein in the supernatants obtained from np preparation . standard curve of respective proteins ( igg , fitc - bsa , and bevacizumab ) ranging from 31.25 to 2000 g / ml was prepared in dmso . in vitro release studies . nps were then resuspended and release study was continued at 37c . in a second set of in vitro release studies , 1 mg equivalent igg containing nps was suspended in 500 l of aqueous solution of thermosensitive gelling polymer ( pb - c ) ( 20 wt% ) . fitc - bsa and bevacizumab - loaded nps were also evaluated for in vitro release kinetics . samples of released fitc - bsa were analyzed by fluorescence spectroscopy with excitation and emission wavelengths of 490 nm and 525 nm , respectively . in vitro release experiments were performed in triplicate and expressed as cumulative drug released ( % ) with time . higuchi equation is as follows : ( 5)qt = kt1/2 , where k denotes higuchi rate kinetic constant , qt is the amount of released protein at time t , and t is time in hour . hixson - crowell equation is as follows : ( 6)c01/3ct1/3=kt , where c0 and ct represent initial and remaining amounts of protein in formulation , respectively , k is the constant incorporating surface to volume ratio , and t is time in hour . first - order equation is as follows : ( 7)logc = logc0kt2.303 , where k denotes the first - order rate constant , c0 is the initial protein concentration , and t represents time in hour . zero - order equation is as follows : ( 8)c = k0 t , where k0 is the zero - order rate constant and t is time in hour . after 24 h of incubation , cells were serum - starved overnight followed by addition of serum - free medium containing 100 ng / ml of vegf and 0.25 mg / ml of released bevacizumab ( test samples ) or 0.25 mg / ml of native bevacizumab ( standard ) . rf/6a cells were starved overnight ( by exposing it to serum - free medium ) , trypsinized , and suspended in serum - free medium containing 0.25 mg / ml of bevacizumab ( native or released from nps ) . pb copolymers ( pb - a and pb - b ) were successfully synthesized by ring - opening bulk copolymerization of -caprolactone and l - lactide / glycolide . in the first step , tb copolymers ( pcl - peg - pcl ) purified tb copolymers were then utilized for the synthesis of respective pb copolymers , that is , pb - a ( pga - pcl - peg - pcl - pga ) and pb - b ( pla - pcl - peg - pcl - pla ) . purity and molecular weights ( mn ) of pb copolymers were calculated by h - nmr spectroscopy . pb copolymer with pla units as terminals ( pb - b ) demonstrated two additional peaks at 1.50 ( ch3 ) and 5.17 ( ch ) ppm . molar ratios of pb - a and pb - b were calculated from the integration values of peg ( 3.65 ppm ) , pcl ( 2.30 ppm ) , and pla ( 5.17 ppm ) or pga ( 4.64.9 ppm ) . h - nmr spectra of pb - c ( figure 6 ) exhibit typical proton signals of peg , pcl , and pla . as described in table 1 , polydispersity ( pdi ) of all the polymers was below 1.45 suggesting narrow distribution of molecular weights . molecular weights ( mn ) of pb - a , pb - b , and pb - c calculated with h - nmr spectroscopy and gpc were very similar to theoretical molecular weights ( table 1 ) . in order to investigate compatibility of pb polymeric materials with biological system ( ocular cell lines ) , arpe-19 , sirc , hcec , and raw-264.7 cells for 48 h were treated with 10 mg / ml of pb - a and pb - b for 48 h. ldh is a cytoplasmic enzyme , secreted in cell culture medium following cell - membrane damage . in mts assay , results in figure 9 demonstrate that more than 90% cell viability ( for all the cell lines ) after 48 h exposure to polymeric materials suggesting excellent safety profile of block copolymers for ocular applications . igg , fitc - bsa , and bevacizumab - encapsulated pb nps were prepared by w1/o / w2 double emulsion solvent evaporation method . nps prepared from pb copolymers were ranging in diameter from 320 to 355 nm ( table 2 ) . no significant effect of polymer composition ( pb - a / pb - b ) or type of protein molecule ( igg / bsa / bevacizumab ) was evident on particle size . these results suggest that hydrodynamic diameter of protein therapeutics or polymer composition has no significant effect on particle size or distribution . entrapment efficiency ( ee ) and drug loading ( dl ) are highly influenced by various parameters including copolymer composition ( hydrophobicity of polymer ) and phase volume ratios ( w1 , o , and w2 ) . in order to understand the effect of hydrophobicity of copolymers on ee and dl , we have prepared and evaluated igg and fitc - bsa - loaded nps utilizing pb - a and pb - b . as presented in table 2 , encapsulation of igg or fitc - bsa in pb - a nps was ~40% and ~35% , respectively . however , pb - b nps exhibited significantly higher encapsulation of ~70% and ~69% for igg and fitc - bsa , respectively . this is possibly due to the fact that , during preparation of nps ( solvent evaporation ) , hydrophobicity may allow faster precipitation of pb - b copolymer to form nps preventing diffusion of igg / fitc - bsa from w1 phase to external aqueous ( w2 ) phase . due to higher hydrophilicity , pb - a copolymer may remain hydrated with w1 and w2 phases ( during nps preparation ) allowing escape of igg / fitc - bsa in external phase resulting in poor ee . however , no effect of hydrodynamic diameter of igg or fitc - bsa on ee or dl was observed . it is very plausible that both of the proteins are too large ( 66 kda ) to show any significant effect of hydrodynamic diameter on ee or dl . in order to evaluate the effect of polymer hydrophobicity , release of fitc - bsa and igg from pb - a and pb - b nps as described in figure 11 , both nps ( pb - a and pb - b ) demonstrated biphasic release profile , that is , initial burst release followed by sustained release . pb - a nps exhibited significantly higher burst release ( ~56% ) of fitc - bsa relative to pb - b nps ( ~48% ) . hydrodynamic diameter of protein therapeutics may have significant effect on drug release pattern . in order to examine this possibility we have compared in vitro release profiles of fitc - bsa ( 66 kda ) and igg ( 150 kda ) ( figure 13 ) from pb - b nps . results indicate significantly higher burst release and shorter release duration for fitc - bsa relative to igg from their respective nps . fitc - bsa has smaller hydrodynamic diameter compared to igg and may lead to more rapid diffusion through the polymeric matrix of nps . to further confirm this hypothesis , we have compared in vitro release profile of igg and bevacizumab ( 149 kda ) from their respective pb - b nps . since igg and bevacizumab are full - length antibodies with similar hydrophilicity and molecular weight ( hydrodynamic diameter ) , both protein molecules behave similarly during nps preparation and during release . these results suggest that parallel molecules ( igg or bevacizumab ) may not generate any difference in drug release pattern whereas there is a significant effect of hydrodynamic diameter ( fitc - bsa or igg ) on drug release . being very potent by nature , burst release of protein therapeutics may produce side effects . therefore , pharmaceutical scientists are focused on developing a formulation which can eliminate or minimize burst effect and offer zero - order drug release throughout the release period . in order to achieve zero - order drug release profile , protein - encapsulated pb - b nps were suspended in an aqueous solution of thermosensitive gelling polymer ( pb - c ) . protein - loaded nps were suspended in 20 wt% gelling solution and then brought to 37c which immediately transitioned the solution to solid hydrogel entrapping nps throughout the polymeric matrix . these composite formulations comprised of protein - loaded pb - b nps ( fitc - bsa and igg ) suspended in thermosensitive gel were evaluated for release pattern . as depicted in figure 15 , burst release of fitc - bsa from composite formulation was negligible ( ~10% ) relative to burst release observed from pb - b nps ( ~48% ) . in addition , release of fitc - bsa was prolonged over 40 days . a similar pattern was also observed for a composite formulation comprising igg - loaded pb - b nps suspended in 20 wt% thermosensitive gelling solution ( figure 16 ) . composite formulation of igg exhibited negligible burst release followed by zero - order release up to 60 days . in order to evaluate drug release mechanism , we have fitted in vitro drug release data in five different release kinetic models , that is , korsmeyer - peppas , higuchi , hixson - crowell , zero - order , and first - order models . moreover , n values in korsmeyer - peppas model for release of fitc - bsa , igg , and bevacizumab from pb - b nps were below 0.43 indicating diffusion controlled release . interestingly , n values for composite formulation of fitc - bsa ( 0.549 ) and igg ( 0.818 ) ( nps suspended in thermosensitive gel ) were between 0.43 and 0.89 suggesting anomalous diffusion . in other words , release of protein therapeutics from composite formulation is controlled by diffusion as well as degradation of polymer . cd spectroscopy is a sensitive and robust analytical technique exploited for the investigation of secondary and to some extent tertiary conformation of proteins . in addition , cd spectra of native and released igg ranging from 200 nm to 250 nm were identical indicating retention of protein conformation during np preparation and after release . a similar level of inhibition was also observed when released ( from nps ) bevacizumab ( test / sample group ) was exposed for 24 h to vegf treated cells . biological activity of released bevacizumab was further evaluated by vegf - induced cell migration assay . as described in figure 19 , a test / sample group ( released bevacizumab ) exhibited significant inhibition of cell migration across transwell membrane compared to vegf treated group ( + ve control ) . cell proliferation and migration assays clearly suggest that bevacizumab activity is retained during np preparation and release . retention of protein stability ( igg and bevacizumab ) in pb nps can be attributed to lower molar mass of pla or pga blocks which produce very low amounts of lactic acid or glycolic acid , thereby eliminating or reducing the possibilities of protein degradation . this research article discusses synthesis and characterization of novel pb copolymers for the preparation of nps and thermosensitive gel . in order to eliminate burst release phase , a novel composite formulation comprised of protein - loaded pb nps suspended in pb thermosensitive gel fitc - bsa and igg - encapsulated nps suspended in thermosensitive gel demonstrated continuous zero - order release avoiding any possibility of dose - dependent toxicity . retention of functional activity of bevacizumab by in vitro cell proliferation and migration suggests that this approach can act as a platform for the ocular delivery of therapeutic macromolecules .

as the pharmaceutical industry nears the end of its first decade in the 21 century new technologies integrate into old paradigms to develop useful drugs in an increasingly crowded marketplace . at the heart of the pharmaceutical industry is the efficient screening of compound libraries to find molecules with a desired effect while limiting the number of complications . in the past , the majority of these assays were performed using target - based screens to detect molecules of interest by their effects on specific cellular targets . combinatorial chemistry then modified these compounds into drug - like forms with the promise of greater potency and fewer side effects . while this approach has proven somewhat successful , it has done so in an often expensive and inefficient way and is therefore clearly unsustainable . phenotypic drug discovery ( pdd ) , which measures compound effects based upon changes in cellular morphology , has been used increasingly in conjunction with target - based assays to derive additional information about how compound libraries affect the cell . the success of modern phenotypic screens is due to the adaptation of hci to drug discovery , combining computer - driven detection and analysis with immunofluorescent techniques to better characterize cellular phenotypes in response to treatment . the integration of phenotypic and target - based discovery should speed up the discovery process , allowing earlier decisions on molecules of potential interest prior to lengthy development . this synergy decreases the overhead necessary to develop a series of molecules and streamlines the discovery process . for example , although a target - based screen frequently determines the potency of a molecule against one target , and often ignores its activity against others , a phenotypic screen generates additional data about that molecule which would otherwise be missed . for instance compounds with high levels of toxicity may have previously progressed to animal models due to their strong effects against one specific target . with the addition of hci cell - based assays , however , this toxicity could be detected earlier in the development cycle saving valuable time and resources . in addition , compounds with beneficial off - target effects previously missed in target - based screens due to weak activity against a primary target , but with an overall greater phenotypic effect , may be discovered earlier and brought forward as appropriate [ 1 , 2 ] . one of the most powerful , yet frequently overlooked , features of hci is the individualized characterization of each cell , and the subsequent assembly of those individual data points into distinct populations . the data obtained from each cell are therefore not viewed in isolation , but rather each cell becomes part of a newly characterized subpopulation . in addition , hci can easily multiplex divergent immunofluorescent assays to further resolve how a treatment affects multiple aspects of cell biology . the use of these subpopulations , rather than reading the average response of the whole treated population , becomes even more important when dealing with molecules affecting several targets where multiple subpopulations often shift in response in a concentration dependent fashion . two common phenotypic assays are those for cell cycle arrest and apoptosis [ 5 , 6 ] . the obvious phenotypic changes that occur in both of these processes generate distinctive morphologies , and are highly amenable to hci analysis and categorization . when used together these assays distinguish populations of cells that may differ in response to compound treatment due to genotype , cell cycle position , or other niche . here this assay was used to screen a library of commercially available cellular modulators leading to cell cycle arrest in the presence or absence of detectable apoptosis . we demonstrate the differential effects of many of these compounds and display the phenotypic fingerprints for each type of cell cycle arrest . finally we show that the fingerprint data obtained from single cells can be used to classify treatments based upon their phenotypic properties . the combination of these approaches creates an overreaching view of the targeted biology that removes many issues frequently associated with data generated from total population reads and opens up possibilities for the use of targeted agents against multiple simultaneous biologies . hct 116 and hela cells were grown according to atcc guidelines . for the assays described below , adherent cells were plated onto poly - d - lysine coated 96-well dishes ( bd # 356640 ) at a density of 3,000 cells in 100 l of media as determined by a coulter z2 cell and particle counter . all compounds were prepared in ten - point curves using two - fold dilutions and a starting concentration of 5 m . cells were fixed with 3.7% formaldehyde for 20 minutes at 37c and permeabilized with 0.1% triton - x 100 for 10 minutes at 25c . cells were blocked using 1% bovine serum albumin ( bsa ) ( invitrogen # 15260 - 037 ) for 1 hour at 25c . the primary antibodies against phosphorylated - histone h3 ( upstate biolabs # 06 - 570 ) and cyclin b1(bd pharmingen # 624086 ) were diluted in 1% bsa to a final concentration of 5 g / ml and this mixture added to each well for 1 hour at 25c . each well was then washed 3 times with 200 l of pbs , and incubated for 1 hour at 25c with a solution containing 5 g / ml goat -mouse - alexa-555 ( molecular probes # a-21422 ) , 5 g / ml goat -rabbit - alexa-647 ( molecular probes # a-21244 ) to detect phh3 , and 200 ng / ml hoechst 33342 to detect nuclear material ( molecular probes # 21492 ) . tunel analysis was then performed using the roche in situ cell death detection kit with fluorescein ( roche # 11 684 795 910 ) and stored at 4c until analysis . cell images were captured using a cellomics arrayscan vti and analyzed with the target activation bioapplication reading in 4 channels at a magnification of 10x . objects were identified using an algorithm to detect nuclear staining with hoescht dye , and the relative levels and sub - cellular localization of tunel , cyclin b1 and phh3 were determined through the respective intensities and locations of alexa-488 , alexa-555 , and alexa-647 fluorescence . a minimum of 1000 individual cellular images or 20 fields were captured for each condition . for each cell , along with the intensity from each channel , several additional nuclear features were captured including the total nuclear area , the ratio of the perimeter of the nucleus compared to its area , and the length to width ratio of the nucleus . arrayscan data were then log2 transformed and analyzed using unsupervised k - means clustering to group subpopulations with similar profiles [ 8 , 9 ] . all populations were compared to asynchronous untreated control populations across all plates . subpopulation clusters were derived from the entire population of the assay and intensities based on the standard deviation away from the mean of the control population . hct 116 and hela cells were grown according to atcc guidelines . for the assays described below , adherent cells were plated onto poly - d - lysine coated 96-well dishes ( bd # 356640 ) at a density of 3,000 cells in 100 l of media as determined by a coulter z2 cell and particle counter . all compounds were prepared in ten - point curves using two - fold dilutions and a starting concentration of 5 m . cells were fixed with 3.7% formaldehyde for 20 minutes at 37c and permeabilized with 0.1% triton - x 100 for 10 minutes at 25c . cells were blocked using 1% bovine serum albumin ( bsa ) ( invitrogen # 15260 - 037 ) for 1 hour at 25c . the primary antibodies against phosphorylated - histone h3 ( upstate biolabs # 06 - 570 ) and cyclin b1(bd pharmingen # 624086 ) were diluted in 1% bsa to a final concentration of 5 g / ml and this mixture added to each well for 1 hour at 25c . each well was then washed 3 times with 200 l of pbs , and incubated for 1 hour at 25c with a solution containing 5 g / ml goat -mouse - alexa-555 ( molecular probes # a-21422 ) , 5 g / ml goat -rabbit - alexa-647 ( molecular probes # a-21244 ) to detect phh3 , and 200 ng / ml hoechst 33342 to detect nuclear material ( molecular probes # 21492 ) . tunel analysis was then performed using the roche in situ cell death detection kit with fluorescein ( roche # 11 684 795 910 ) and stored at 4c until analysis . cell images were captured using a cellomics arrayscan vti and analyzed with the target activation bioapplication reading in 4 channels at a magnification of 10x . objects were identified using an algorithm to detect nuclear staining with hoescht dye , and the relative levels and sub - cellular localization of tunel , cyclin b1 and phh3 were determined through the respective intensities and locations of alexa-488 , alexa-555 , and alexa-647 fluorescence . a minimum of 1000 individual cellular images or 20 fields were captured for each condition . for each cell , along with the intensity from each channel , several additional nuclear features were captured including the total nuclear area , the ratio of the perimeter of the nucleus compared to its area , and the length to width ratio of the nucleus . arrayscan data were then log2 transformed and analyzed using unsupervised k - means clustering to group subpopulations with similar profiles [ 8 , 9 ] . all populations were compared to asynchronous untreated control populations across all plates . subpopulation clusters were derived from the entire population of the assay and intensities based on the standard deviation away from the mean of the control population . until recently many hci assays determined a response by observing a single parameter , but as the technology advanced additional parameters were added to show a more complete phenotypic change in treated cells . this method was limited , however , as the phenotypic changes were viewed as an aggregate response of the entire treated population . to demonstrate the usefulness of subpopulation analysis in pdd a collection of 44 well - characterized and commercially available cellular modulators were screened . the data obtained from this screen were then analyzed first using traditional well - level averages ( i.e. total population ) , and then by increasingly complex subpopulation analyses . the initial data generated from the average responses of the treated cells displayed the population s primary phenotype . to demonstrate this method cells were treated with an inhibitor of plk1 and four commonly used phenotypic parameters were observed in isolation . plk1 is an important regulator of centrosome maturation and spindle assembly during the mitotic phase of the cell cycle and is critical for exit from mitosis [ 10 , 11 ] . in many , but not all , cases a marker was expected to give its strongest response when exposed to the highest concentration of the agent , and to generate a lesser response as the concentration of that agent decreased . when the entire population is observed at the level of the treated well , concentration response curves are one of the standard methods frequently used to characterize a phenotypic effect . for instance tunel staining , a measure of apoptosis through the use of dna end labeling , increases in response to a wide variety of cytotoxic agents ( fig . 1a ) [ 12 , 13 ] . tunel staining alone , while useful for determining induction of apoptosis , does not fully describe the complete phenotype of a treated cell . the addition of dna intensity shows relative changes in the dna content of treated cells as they replicate their dna and suggests when some of these cells may be arrested in specific cell cycle compartments ( fig . 1b ) . to further define cell cycle position and arrest , the g2/m marker cyclin b1 ( fig . phh3 is a marker for chromosomal condensation present in mitosis and promotes the recruitment of condensin , while cyclin b1 activates cdk1 and allows progression through g2 and into mitosis [ 7 , 14 , 15 ] . the measurement of these four phenotypic parameters illustrate that the highest levels of total dna , tunel , cyclin b1 , and phh3 are found at the highest concentrations of the tested compound and decrease as less compound is added to the cells . these measurements suggest a mitotic and apoptotic response to inhibition of plk1 in this assay , but unfortunately , these data do not differentiate between the multiple phenotypes likely present . while successful in showing a general response , this method lacks the resolution necessary to determine effects at the individual cell level . to characterize individual cellular phenotype response , this method most frequently compares the dna content of a population of cells , but is applicable to a variety of situations found in hci . we have previously shown that a log2 transformation of these data reduces the effects of outliers and increases the ease of analysis . we therefore applied a log2 transformation to generate more meaningful parameter distributions for total dna ( fig . theses distributions compare plk1-inhibitor treated cells with cells treated with dmso alone . in all cases the distribution analyses illustrate that populations have shifted away from the mean control population ( as shown by the vertical blue lines ) . in the case of dna intensity , this shift is towards a much higher dna content containing cells with 4n and 8n dna . these three populations were averaged together as a 4n+ dna population when observed at the well level , demonstrating the improved resolution of this technique when applied to hci . the g2/m marker data shown from the staining of cyclin b1 and phh3 also demonstrate that there are two distinct expression patterns in the data for each of these markers , but that all of the cells have shifted away from the average shown in the control population . when combined with the total dna intensity data , although the tunel staining in these cells presents a uniform distribution , the increase in staining intensity suggests that the g2/m phenotype found upon plk1 inhibition likely leads to apoptosis . the advantage of these subpopulation analyses is the increased resolution of the entire population which distinguishes populations previously undetected through well - level analysis . detecting the differential phenotypes shown using these distribution analyses was impossible when using the total population read . although uniparameter distribution analyses detected the population shifts following treatment , we were unable to link cellular parameters together and show that the same cells that had increases in dna content were also undergoing apoptosis . to more thoroughly examine these subpopulations we used bivariate analyses to combine these parameters in a method similar to that used to display flow cytometry data . these bivariate analyses link parameters from the same cell and demonstrate additional subpopulations that may have been missed in a simpler analysis . the log2 transformed data from the dna total intensity and tunel analysis were compared ( fig . the addition of tunel staining to this analysis demonstrates that apoptosis occurs across the population , but the greatest apoptotic response lies within those cells with 8n dna content . the combination of cyclin b1 intensity data with those from phh3 staining distinguish two populations and demonstrate that the same cells with the greatest expression of cyclin b1 also contain the largest amount of phh3 . the use of these pairwise comparisons to link parameters distinguished additional subpopulations , and further defined the subpopulations detected in the univariate analysis . although in the case of plk1 inhibition the subpopulation with 8n dna content had the highest level of apoptosis , in others it was a different subpopulation with a dissimilar phenotype ( depending upon the treatment , its effect on the cell , and the type of cell studied ) . to further explore how a variety of compounds affected cell cycle arrest and apoptotic response we continued to analyze the set of 44 targeted cancer compounds currently in clinical development ( table 1 ) . compounds were selected for this screen for their kinase target specificity in order to generate phenotypic fingerprints . although many of these compounds generate one specific fingerprint at all concentrations , several of them demonstrate concentration - dependent phenotypic effects resulting in multiple fingerprints , likely due to differing ic50 s against multiple cellular targets . as described above , analysis of these data at the cellular level is crucial to understanding these complex phenotypes in a concentration - dependent setting . although a bivariate analysis began to differentiate phenotypes and subpopulations , even greater resolution was required to discern the many varied phenotypes frequently found in a screening setting . although we were able to link two parameters using a bivariate analysis , generating a new bivariate distribution for every two phenotypic parameters of interest was an unnecessarily complicated procedure . when interested in the overall phenotypic fingerprint of a subpopulation it becomes necessary to use a more refined method of analysis to link multiple parameters into one output . building upon the bivariate analysis , 7 parameters were combined to obtain a phenotypic fingerprint of treatment effects across all of the cellular subpopulations . data were extracted from individual cells and used to create clusters of the cellular subpopulations using unsupervised k - means clustering ( fig . the output from this analysis was a heatmap generated by a user - defined number of clusters based upon similarity of cells to each other across all seven parameters ( like groups with like ) . although a user could request an infinite number of phenotypic clusters , the algorithm limits the output based upon phenotypic similarities present at the population level . if the user defined more clusters than the algorithm could find , the extra clusters contained zero cells . the k - means algorithm determined how the cells cluster based upon the cells in both the control and experimental populations . the colors displayed in the heatmap indicate shifts away from the average of the control population ( numbered at right by standard deviations ) with shifts to red denoting increases and shifts to blue denoting decreases . in the heatmaps , each row contains the data from one cell , and those cells were sorted in each cluster based upon their total dna intensity from high to low . the numbers on the left side of each cluster refer to the fraction of the total number of cells in that cluster . one limitation of this technique as an analytical tool is the number of fluorescent channels that can be measured using fluorescent microscopy in conjunction with the subcellular localization of each fluorescent channel . currently limitations in fluorescent probe technology and the specificity of fluorescent excitation and emission filters impedes our ability to image > 4 unique fluorescent probes at any given time , although we can incorporate subcellular localization of the fluorescent probes to increase the number of potentially useful channels for fingerprint development . to demonstrate the effectiveness of this technique across the cell cycle representative heatmaps were generated for molecules against a number of commonly targeted proteins active in one of the four major cell cycle phases : g1 , s , g2 , and m ( fig . 5 ) . following treatment with an inhibitor of cdk4 , hct-116 cells arrested in the g1 phase of the cell cycle . this arrest was characterized by one predominant cell population having low total , average , and variation of dna intensity and lacking the g2/m markers cyclin b1 and phh3 . these cells also contained relatively small nuclei consistent with g1 arrest and virtually no apoptotic fraction ( as evidenced by a lack of a tunel response ) . g1-arrest phenotype contrasted with that of cells arrested in s phase by an inhibitor of cdk2 . in this case the primary subpopulation of cells , while also lacking high levels of cyclin b1 or phh3 , shifted into a new cluster consisting of slightly higher levels of total dna ( consistent with an s phase arrest ) , but retaining low average and variation of dna intensity . inhibitors of cdk1 have yet another phenotype as cells arrested in the g2 phase of the cell cycle . cells arrested by cdk1 inhibitors exhibited high levels of total dna and a concomitant increase in nuclear area to hold their 4n dna content . as these cells were arrested in g2 and unable to progress through mitosis they expressed high levels of the g2/m markers cyclin b1 and phh3 . at the concentration shown , these cells underwent significant apoptosis as demonstrated by their increased tunel staining . finally , cells arrested through inhibition of plk1 display a mitotic arrest phenotype . along with the increase in total dna intensity found in cells arrested with a compound in g2 , these cells had smaller nuclei due to the condensation of nuclear material . not surprisingly the mitotic markers cyclin b1 and phh3 were at their highest levels in cells arrested in this phase of the cell cycle . the creation of fingerprints specific to individual cell cycle inhibitors is a powerful tool to dissect cellular responses to compound libraries , and also is useful to detect the varying responses to treatment between cell lines . for example , hct-116 and hela cells were both exposed to an inhibitor of cdk4/6 for 48 hours , and the subpopulations from each treatment yielded distinct fingerprints based on the gene expression patterns in each line ( fig . the hct-116 cells were arrested primarily in s phase as shown by their dna content , nuclear area , and low cyclin b1 and phh3 expression , while the hela cells remained unaffected when compared to a control population . this differential effect is due to the prb status of each cell line and how it relates to the given inhibitor . a cdk 4/6 inhibitor arrests the cell cycle through inhibition of rb , a pathway inactivated in hela cells , but intact in hct-116 cells . this form of analysis illustrates the usefulness of heatmaps when used in conjunction with a variety of genetically dissimilar cell lines to determine a compound s mechanism of action . prior analysis of hci data centered around the collection of dna content and protein expression data at the level of the individual cell , but analysis at the level of the entire population . although these readouts with changes in multiple parameters were useful in a screening setting due to their ease of analysis , they became less useful when dissecting a treatment s mechanism of action . the primary issue with well - level analysis is that it frequently masks subtle changes in cellular parameters , whereas subpopulation analysis distinguishes between the subpopulations defined by these subtle but often important changes . data shown as an aggregate response to treatment frequently do not display the effect of the treatment on an individual cell and can mask important phenotypic changes . these aggregate responses often return results similar to those found in elisas which are also unable to differentiate between a large effect in a small subpopulation of cells from a smaller effect in every cell in the population . the differential effects of subpopulations to treatment may be a critical factor when studying diseases where only a subset of the total population is crucial to driving the disease , with the most obvious example found in cancer biology where the majority of the cells respond to chemotherapy while a subset of cancer stem cells remain resistant [ 16 , 17 ] . as a first step towards subpopulation analysis single parameter distributions are a simple tool that can be developed with a minimal amount of statistical and informatics support and can be quickly analyzed by biologists . a bivariate analysis , while adding an additional level of statistical complexity , is well worth the time and resources taken for the analysis . linking changes in parameters from a multiplexed assay is an excellent start to defining subpopulation biology . these uncomplicated analyses illustrate the value of automated statistical tools to ease the analysis of large and complex data sets . although these tools are an excellent first step towards dissecting subpopulations , additional subpopulations affecting the biology of the disease and treatment may remain undetected in the absence of more complex analysis . multiparameter analysis using unsupervised k - means clustering further defines and refines the subpopulations present in any studied population . one advantage to using this method is that a perturbation in any one of the parameters used in the analysis can change the identity and membership of subpopulations and detect additional , previously hidden , subpopulations . the changes in the subpopulations generated by these shifts lead to a degree of sensitivity in this analysis previously unknown to hci . even more important than these subtle population shifts are the slight phenotypic changes which may suggest the mechanism of action of uncharacterized treatments leading to cell cycle arrest , apoptosis , or modulation of cellular signaling . the fingerprints generated using this method of analysis describe important biological phenotypes across a population of treated cells and can be used to advance novel therapeutic treatments simply by following the phenotype of interest . thus , phenotypic effects of traditional lead optimization sar modifications , such as those designed to affect potency , solubility , metabolism , etc , can be tracked relative to the parent molecule through a careful analysis of high content fingerprints . previously we have shown that this analysis can be used effectively both in vitro and in vivo to drive initial drug discovery efforts and is being used in our hands to detect and advance compounds with novel biologies . four phenotypic parameters were measured from hct 116 cells exposed to an inhibitor of plk1 in a concentration response curve for 48 hours . the aggregate phenotypic responses from tunel apoptotic staining ( a ) , total dna intensity ( b ) , cyclin b1 expression ( c ) , and presence of phh3 ( d ) are shown in a concentration range of 5000 nm to 10 nm . distributions were performed of the individual cellular data for the four phenotypic parameters shown in fig . these data were taken from the same hct 116 cells treated with 2500 nm plk1 inhibitor for 48 hours . total dna ( a ) , tunel ( b ) , cyclin b1 ( c ) , and phh3 intensity ( d ) data were log2 transformed and distributed according to intensity . the blue lines shown in the plk1 treatments represent the mean of the control population . ( 2 ) ( green bars at top and side ) were combined to further differentiate subpopulations . total dna intensity data were combined with tunel data ( a ) and cyclin b1 data were combined with phh3 data ( b ) . topographic mapping demonstrates clustering concentrations ( red coloring illustrates closest clustering , and blue furthest clustering ) . the phenotypic fingerprint of each imaged cell is shown from left to right in a single row across the heatmap and is characterized by the 7 parameters : total , average , and variation of dna intensity , nuclear area , tunel apoptotic staining , cyclin b1 expression , and the presence of phh3 . cells were clustered vertically using unsupervised k - means based on those 7 parameters and are grouped within each cluster based upon their total dna intensity from highest to lowest . the blue to red shifts present in the heatmap represent deviations away from the mean of a control population in standard deviations ( shifts to red are increases and shifts to blue decreases ) . inhibition of the cell cycle using kinase inhibitor fell into four main categories demonstrated by the above four compounds . cdk4 inhibition led to a g1 arrest phenotype , cdk2 inhibition led to an s phase arrest phenotype , cdk1 inhibition led to a g2 arrest phenotype , and inhibition of plk1 demonstrates one form of mitotic arrest phenotype . hct 116 and hela cells were both exposed to an inhibitor of cdk4/6 at a concentration of 313 nm for 48 hours . hct 116 cells arrested with a primarily s phase phenotype with hela cells remained unaffected by this treatment .

phenotypic drug discovery , primarily abandoned in the 1980 s in favor of targeted approaches to drug development , is once again demonstrating its value when used in conjunction with new technologies . phenotypic discovery has been brought back to the fore mainly due to recent advances in the field of high content imaging ( hci ) . hci elucidates cellular responses using a combination of immunofluorescent assays and computer analysis which increase both the sensitivity and throughput of phenotypic assays . although hci data characterize cellular responses in individual cells , these data are usually analyzed as an aggregate of the treated population and are unable to discern differentially responsive subpopulations . a collection of 44 kinase inhibitors affecting cell cycle and apoptosis were characterized with a number of univariate , bivariate , and multivariate subpopulation analyses demonstrating that each level of complexity adds additional information about the treated populations and often distinguishes between compounds with seemingly similar mechanisms of action . finally , these subpopulation data were used to characterize compounds as they relate in chemical space .

INTRODUCTION MATERIALS AND METHODS Cell Culture and Compound Treatment Immunofluorescence Fluorescent Imaging and Statistical Analysis RESULTS DISCUSSION Figures and Table

as the pharmaceutical industry nears the end of its first decade in the 21 century new technologies integrate into old paradigms to develop useful drugs in an increasingly crowded marketplace . at the heart of the pharmaceutical industry is the efficient screening of compound libraries to find molecules with a desired effect while limiting the number of complications . in the past , the majority of these assays were performed using target - based screens to detect molecules of interest by their effects on specific cellular targets . phenotypic drug discovery ( pdd ) , which measures compound effects based upon changes in cellular morphology , has been used increasingly in conjunction with target - based assays to derive additional information about how compound libraries affect the cell . the success of modern phenotypic screens is due to the adaptation of hci to drug discovery , combining computer - driven detection and analysis with immunofluorescent techniques to better characterize cellular phenotypes in response to treatment . the integration of phenotypic and target - based discovery should speed up the discovery process , allowing earlier decisions on molecules of potential interest prior to lengthy development . for example , although a target - based screen frequently determines the potency of a molecule against one target , and often ignores its activity against others , a phenotypic screen generates additional data about that molecule which would otherwise be missed . for instance compounds with high levels of toxicity may have previously progressed to animal models due to their strong effects against one specific target . with the addition of hci cell - based assays , however , this toxicity could be detected earlier in the development cycle saving valuable time and resources . in addition , compounds with beneficial off - target effects previously missed in target - based screens due to weak activity against a primary target , but with an overall greater phenotypic effect , may be discovered earlier and brought forward as appropriate [ 1 , 2 ] . one of the most powerful , yet frequently overlooked , features of hci is the individualized characterization of each cell , and the subsequent assembly of those individual data points into distinct populations . in addition , hci can easily multiplex divergent immunofluorescent assays to further resolve how a treatment affects multiple aspects of cell biology . the use of these subpopulations , rather than reading the average response of the whole treated population , becomes even more important when dealing with molecules affecting several targets where multiple subpopulations often shift in response in a concentration dependent fashion . two common phenotypic assays are those for cell cycle arrest and apoptosis [ 5 , 6 ] . the obvious phenotypic changes that occur in both of these processes generate distinctive morphologies , and are highly amenable to hci analysis and categorization . when used together these assays distinguish populations of cells that may differ in response to compound treatment due to genotype , cell cycle position , or other niche . here this assay was used to screen a library of commercially available cellular modulators leading to cell cycle arrest in the presence or absence of detectable apoptosis . we demonstrate the differential effects of many of these compounds and display the phenotypic fingerprints for each type of cell cycle arrest . the combination of these approaches creates an overreaching view of the targeted biology that removes many issues frequently associated with data generated from total population reads and opens up possibilities for the use of targeted agents against multiple simultaneous biologies . each well was then washed 3 times with 200 l of pbs , and incubated for 1 hour at 25c with a solution containing 5 g / ml goat -mouse - alexa-555 ( molecular probes # a-21422 ) , 5 g / ml goat -rabbit - alexa-647 ( molecular probes # a-21244 ) to detect phh3 , and 200 ng / ml hoechst 33342 to detect nuclear material ( molecular probes # 21492 ) . cell images were captured using a cellomics arrayscan vti and analyzed with the target activation bioapplication reading in 4 channels at a magnification of 10x . objects were identified using an algorithm to detect nuclear staining with hoescht dye , and the relative levels and sub - cellular localization of tunel , cyclin b1 and phh3 were determined through the respective intensities and locations of alexa-488 , alexa-555 , and alexa-647 fluorescence . for each cell , along with the intensity from each channel , several additional nuclear features were captured including the total nuclear area , the ratio of the perimeter of the nucleus compared to its area , and the length to width ratio of the nucleus . arrayscan data were then log2 transformed and analyzed using unsupervised k - means clustering to group subpopulations with similar profiles [ 8 , 9 ] . subpopulation clusters were derived from the entire population of the assay and intensities based on the standard deviation away from the mean of the control population . each well was then washed 3 times with 200 l of pbs , and incubated for 1 hour at 25c with a solution containing 5 g / ml goat -mouse - alexa-555 ( molecular probes # a-21422 ) , 5 g / ml goat -rabbit - alexa-647 ( molecular probes # a-21244 ) to detect phh3 , and 200 ng / ml hoechst 33342 to detect nuclear material ( molecular probes # 21492 ) . cell images were captured using a cellomics arrayscan vti and analyzed with the target activation bioapplication reading in 4 channels at a magnification of 10x . objects were identified using an algorithm to detect nuclear staining with hoescht dye , and the relative levels and sub - cellular localization of tunel , cyclin b1 and phh3 were determined through the respective intensities and locations of alexa-488 , alexa-555 , and alexa-647 fluorescence . for each cell , along with the intensity from each channel , several additional nuclear features were captured including the total nuclear area , the ratio of the perimeter of the nucleus compared to its area , and the length to width ratio of the nucleus . arrayscan data were then log2 transformed and analyzed using unsupervised k - means clustering to group subpopulations with similar profiles [ 8 , 9 ] . this method was limited , however , as the phenotypic changes were viewed as an aggregate response of the entire treated population . to demonstrate the usefulness of subpopulation analysis in pdd a collection of 44 well - characterized and commercially available cellular modulators were screened . total population ) , and then by increasingly complex subpopulation analyses . the initial data generated from the average responses of the treated cells displayed the population s primary phenotype . plk1 is an important regulator of centrosome maturation and spindle assembly during the mitotic phase of the cell cycle and is critical for exit from mitosis [ 10 , 11 ] . in many , but not all , cases a marker was expected to give its strongest response when exposed to the highest concentration of the agent , and to generate a lesser response as the concentration of that agent decreased . when the entire population is observed at the level of the treated well , concentration response curves are one of the standard methods frequently used to characterize a phenotypic effect . the addition of dna intensity shows relative changes in the dna content of treated cells as they replicate their dna and suggests when some of these cells may be arrested in specific cell cycle compartments ( fig . to further define cell cycle position and arrest , the g2/m marker cyclin b1 ( fig . the measurement of these four phenotypic parameters illustrate that the highest levels of total dna , tunel , cyclin b1 , and phh3 are found at the highest concentrations of the tested compound and decrease as less compound is added to the cells . these measurements suggest a mitotic and apoptotic response to inhibition of plk1 in this assay , but unfortunately , these data do not differentiate between the multiple phenotypes likely present . to characterize individual cellular phenotype response , this method most frequently compares the dna content of a population of cells , but is applicable to a variety of situations found in hci . we have previously shown that a log2 transformation of these data reduces the effects of outliers and increases the ease of analysis . in the case of dna intensity , this shift is towards a much higher dna content containing cells with 4n and 8n dna . the g2/m marker data shown from the staining of cyclin b1 and phh3 also demonstrate that there are two distinct expression patterns in the data for each of these markers , but that all of the cells have shifted away from the average shown in the control population . the advantage of these subpopulation analyses is the increased resolution of the entire population which distinguishes populations previously undetected through well - level analysis . although uniparameter distribution analyses detected the population shifts following treatment , we were unable to link cellular parameters together and show that the same cells that had increases in dna content were also undergoing apoptosis . to more thoroughly examine these subpopulations we used bivariate analyses to combine these parameters in a method similar to that used to display flow cytometry data . the combination of cyclin b1 intensity data with those from phh3 staining distinguish two populations and demonstrate that the same cells with the greatest expression of cyclin b1 also contain the largest amount of phh3 . the use of these pairwise comparisons to link parameters distinguished additional subpopulations , and further defined the subpopulations detected in the univariate analysis . although in the case of plk1 inhibition the subpopulation with 8n dna content had the highest level of apoptosis , in others it was a different subpopulation with a dissimilar phenotype ( depending upon the treatment , its effect on the cell , and the type of cell studied ) . to further explore how a variety of compounds affected cell cycle arrest and apoptotic response we continued to analyze the set of 44 targeted cancer compounds currently in clinical development ( table 1 ) . although many of these compounds generate one specific fingerprint at all concentrations , several of them demonstrate concentration - dependent phenotypic effects resulting in multiple fingerprints , likely due to differing ic50 s against multiple cellular targets . as described above , analysis of these data at the cellular level is crucial to understanding these complex phenotypes in a concentration - dependent setting . although a bivariate analysis began to differentiate phenotypes and subpopulations , even greater resolution was required to discern the many varied phenotypes frequently found in a screening setting . although we were able to link two parameters using a bivariate analysis , generating a new bivariate distribution for every two phenotypic parameters of interest was an unnecessarily complicated procedure . when interested in the overall phenotypic fingerprint of a subpopulation it becomes necessary to use a more refined method of analysis to link multiple parameters into one output . building upon the bivariate analysis , 7 parameters were combined to obtain a phenotypic fingerprint of treatment effects across all of the cellular subpopulations . data were extracted from individual cells and used to create clusters of the cellular subpopulations using unsupervised k - means clustering ( fig . the output from this analysis was a heatmap generated by a user - defined number of clusters based upon similarity of cells to each other across all seven parameters ( like groups with like ) . although a user could request an infinite number of phenotypic clusters , the algorithm limits the output based upon phenotypic similarities present at the population level . the k - means algorithm determined how the cells cluster based upon the cells in both the control and experimental populations . the colors displayed in the heatmap indicate shifts away from the average of the control population ( numbered at right by standard deviations ) with shifts to red denoting increases and shifts to blue denoting decreases . in the heatmaps , each row contains the data from one cell , and those cells were sorted in each cluster based upon their total dna intensity from high to low . the numbers on the left side of each cluster refer to the fraction of the total number of cells in that cluster . one limitation of this technique as an analytical tool is the number of fluorescent channels that can be measured using fluorescent microscopy in conjunction with the subcellular localization of each fluorescent channel . currently limitations in fluorescent probe technology and the specificity of fluorescent excitation and emission filters impedes our ability to image > 4 unique fluorescent probes at any given time , although we can incorporate subcellular localization of the fluorescent probes to increase the number of potentially useful channels for fingerprint development . to demonstrate the effectiveness of this technique across the cell cycle representative heatmaps were generated for molecules against a number of commonly targeted proteins active in one of the four major cell cycle phases : g1 , s , g2 , and m ( fig . following treatment with an inhibitor of cdk4 , hct-116 cells arrested in the g1 phase of the cell cycle . this arrest was characterized by one predominant cell population having low total , average , and variation of dna intensity and lacking the g2/m markers cyclin b1 and phh3 . in this case the primary subpopulation of cells , while also lacking high levels of cyclin b1 or phh3 , shifted into a new cluster consisting of slightly higher levels of total dna ( consistent with an s phase arrest ) , but retaining low average and variation of dna intensity . inhibitors of cdk1 have yet another phenotype as cells arrested in the g2 phase of the cell cycle . as these cells were arrested in g2 and unable to progress through mitosis they expressed high levels of the g2/m markers cyclin b1 and phh3 . at the concentration shown , these cells underwent significant apoptosis as demonstrated by their increased tunel staining . finally , cells arrested through inhibition of plk1 display a mitotic arrest phenotype . along with the increase in total dna intensity found in cells arrested with a compound in g2 , these cells had smaller nuclei due to the condensation of nuclear material . not surprisingly the mitotic markers cyclin b1 and phh3 were at their highest levels in cells arrested in this phase of the cell cycle . the creation of fingerprints specific to individual cell cycle inhibitors is a powerful tool to dissect cellular responses to compound libraries , and also is useful to detect the varying responses to treatment between cell lines . for example , hct-116 and hela cells were both exposed to an inhibitor of cdk4/6 for 48 hours , and the subpopulations from each treatment yielded distinct fingerprints based on the gene expression patterns in each line ( fig . the hct-116 cells were arrested primarily in s phase as shown by their dna content , nuclear area , and low cyclin b1 and phh3 expression , while the hela cells remained unaffected when compared to a control population . this differential effect is due to the prb status of each cell line and how it relates to the given inhibitor . a cdk 4/6 inhibitor arrests the cell cycle through inhibition of rb , a pathway inactivated in hela cells , but intact in hct-116 cells . this form of analysis illustrates the usefulness of heatmaps when used in conjunction with a variety of genetically dissimilar cell lines to determine a compound s mechanism of action . prior analysis of hci data centered around the collection of dna content and protein expression data at the level of the individual cell , but analysis at the level of the entire population . although these readouts with changes in multiple parameters were useful in a screening setting due to their ease of analysis , they became less useful when dissecting a treatment s mechanism of action . the primary issue with well - level analysis is that it frequently masks subtle changes in cellular parameters , whereas subpopulation analysis distinguishes between the subpopulations defined by these subtle but often important changes . data shown as an aggregate response to treatment frequently do not display the effect of the treatment on an individual cell and can mask important phenotypic changes . these aggregate responses often return results similar to those found in elisas which are also unable to differentiate between a large effect in a small subpopulation of cells from a smaller effect in every cell in the population . the differential effects of subpopulations to treatment may be a critical factor when studying diseases where only a subset of the total population is crucial to driving the disease , with the most obvious example found in cancer biology where the majority of the cells respond to chemotherapy while a subset of cancer stem cells remain resistant [ 16 , 17 ] . as a first step towards subpopulation analysis single parameter distributions are a simple tool that can be developed with a minimal amount of statistical and informatics support and can be quickly analyzed by biologists . a bivariate analysis , while adding an additional level of statistical complexity , is well worth the time and resources taken for the analysis . although these tools are an excellent first step towards dissecting subpopulations , additional subpopulations affecting the biology of the disease and treatment may remain undetected in the absence of more complex analysis . one advantage to using this method is that a perturbation in any one of the parameters used in the analysis can change the identity and membership of subpopulations and detect additional , previously hidden , subpopulations . the changes in the subpopulations generated by these shifts lead to a degree of sensitivity in this analysis previously unknown to hci . even more important than these subtle population shifts are the slight phenotypic changes which may suggest the mechanism of action of uncharacterized treatments leading to cell cycle arrest , apoptosis , or modulation of cellular signaling . the fingerprints generated using this method of analysis describe important biological phenotypes across a population of treated cells and can be used to advance novel therapeutic treatments simply by following the phenotype of interest . thus , phenotypic effects of traditional lead optimization sar modifications , such as those designed to affect potency , solubility , metabolism , etc , can be tracked relative to the parent molecule through a careful analysis of high content fingerprints . previously we have shown that this analysis can be used effectively both in vitro and in vivo to drive initial drug discovery efforts and is being used in our hands to detect and advance compounds with novel biologies . the aggregate phenotypic responses from tunel apoptotic staining ( a ) , total dna intensity ( b ) , cyclin b1 expression ( c ) , and presence of phh3 ( d ) are shown in a concentration range of 5000 nm to 10 nm . distributions were performed of the individual cellular data for the four phenotypic parameters shown in fig . these data were taken from the same hct 116 cells treated with 2500 nm plk1 inhibitor for 48 hours . total dna ( a ) , tunel ( b ) , cyclin b1 ( c ) , and phh3 intensity ( d ) data were log2 transformed and distributed according to intensity . the blue lines shown in the plk1 treatments represent the mean of the control population . total dna intensity data were combined with tunel data ( a ) and cyclin b1 data were combined with phh3 data ( b ) . topographic mapping demonstrates clustering concentrations ( red coloring illustrates closest clustering , and blue furthest clustering ) . the phenotypic fingerprint of each imaged cell is shown from left to right in a single row across the heatmap and is characterized by the 7 parameters : total , average , and variation of dna intensity , nuclear area , tunel apoptotic staining , cyclin b1 expression , and the presence of phh3 . cells were clustered vertically using unsupervised k - means based on those 7 parameters and are grouped within each cluster based upon their total dna intensity from highest to lowest . the blue to red shifts present in the heatmap represent deviations away from the mean of a control population in standard deviations ( shifts to red are increases and shifts to blue decreases ) . inhibition of the cell cycle using kinase inhibitor fell into four main categories demonstrated by the above four compounds . cdk4 inhibition led to a g1 arrest phenotype , cdk2 inhibition led to an s phase arrest phenotype , cdk1 inhibition led to a g2 arrest phenotype , and inhibition of plk1 demonstrates one form of mitotic arrest phenotype . hct 116 cells arrested with a primarily s phase phenotype with hela cells remained unaffected by this treatment .

taurine ( 2-aminoethanesulfonic acid ) , which is essential during the development of mammals , is not incorporated into proteins . it is mainly produced in the liver and kidney and is present in most other tissues including the brain , leukocytes , retina , heart , placenta , and muscle [ 13 ] . taurine is a key element in many diverse processes including development of the brain , retina , and the immune system , osmoregulation , reproduction , membrane stabilization , regulation of cardiac muscle , and inflammation [ 2 , 410 ] . colostrum contains a very high taurine concentration which is required for development of the brain and retina in the newborn human . in response to research findings , taurine is universally added to infant formula as well as to parenteral solutions [ 2 , 12 ] . cysteine is oxidized by cysteine dioxygenase ( cdo ; ec 1.13.11 , mw 24 kd ) to cysteine sulfinic acid which is converted by csad to hypotaurine which is then oxidized to taurine . csad ( ec 4.1.1.29 , mw 51 kd ) is a cytosolic enzyme expressed primarily in liver and kidney [ 1316 ] . the enzyme responsible for the alternative pathway for taurine biosynthesis is ado ( ec 1.13.11.19 , mw 28 kd ) [ 17 , 18 ] . cysteine is incorporated into coenzyme a ( coa ) , followed by the release of cysteamine during coa turnover . hepatic csad and cdo activity is high compared to that in the kidney and brain [ 15 , 1921 ] . the tissue distribution of csad , protein , and mrna , detected by western and northern blot analyses , is consistent with csad activity . ado level is highest in the brain , whereas csad and cdo levels are highest in the liver [ 15 , 17 , 18 , 22 ] . cysteine sulfinic acid decarboxylase ( csad ) is one of rate - limiting enzymes for taurine biosynthesis [ 2 , 13 ] and the level of its activity determines the need for dietary taurine . cats have been used for taurine studies because they have low levels of cdo and csad leading to a dependence on dietary sources of taurine . rodents have high levels of csad [ 1 , 2 , 23 ] and taurine is not essential to their diet . taurine is considered a conditionally essential amino acid in humans and is required in their diet at certain times during development . analysis of murine csad cdna shows that the enzyme has 98% and 90% homology to rat and human csad , respectively [ 14 , 24 ] . since the cat model has limitations including a long gestation period , a heterogeneous genetic background , and a relatively large maintenance expense , we developed a csad knockout mouse ( csad ko ) model to better understand the physiological roles of taurine . this novel murine model was developed to provide insight into the role of taurine in reproduction , innate and adaptive immunity , and brain development . we report here that the absence of a functional csad gene in the csad ko mouse reduces the level of this amino acid by > 80% and has a severe effect on neonatal survival that is reversed by adding taurine ( 0.05% ) to the drinking water . oligonucleotide primers for pcr were obtained from eurofins mwg operon ( huntsville , al ) . primers were designed by primer designer 4 ( scientific and educational software , cary , nc ) . taq polymerase and deoxynucleotides nitrocellulose membranes for western blot and nylon membranes for northern blot were purchased from invitrogen ( carlsbad , ca ) and nen life science products ( boston , ma ) , respectively . membranes for southern blot were obtained from bio - rad ( hercules , ca ) . trizol and rneasy kit for rna extraction and dneasy kit for dna extraction were obtained from invitrogen and qiagen ( valencia , ca ) , respectively . chimeric csad ko mice were produced by injection of cells from a gene trap es cell line ( xp0392 ) into c57bl/6 ( b6 ) blastocysts at the mouse mutant regional resource centers ( mmrrc , uc davis , ca ) which were implanted into a pseudopregnant b6 mouse . nine chimeric mice from mmrrc were mated with b6 ( jackson laboratories , bar harbor , me ) in the animal colony at this institution ( nys institute for basic research in developmental disabilities ) . agouti mice produced by this mating were back - crossed to b6 and offspring were genotyped using pcr to identify germline transmitted csad heterozygotes ( csad ) . mice were fed taurine - free chow ( labdiet , pmi nutrition international , st . all mice were kept under 12 hr day / night with free access to food and water . some experimental animals had exogenous taurine added to their water at 0.05% as indicated in section 3 . for optimum reproductive performance , both females and males used for mating in the taurine - treated groups were supplemented with taurine . all procedures involving live animals were approved by the institutional animal care and use committee of ibr . dna from the spleen was analyzed by southern blotting to confirm the csad ko genotype using a modified method previously described . dna from the spleen was prepared with dneasy kits ( qiagen , valencia , ca ) . dna was digested with ecor v ( promega ) , fractionated by agarose gel electrophoresis , and transferred to a nylon membrane ( bio - rad ) . the membrane was hybridized using csad cdna as a probe to confirm the disruption of the csad gene . template dna was isolated from a tail sample at 3 weeks of age with a dneasy kit ( qiagen ) and analyzed with two sets of primers , -geo and vector insertion site ( vis ) ( table 1 ) , using a pcr kit ( perkin elmer , shelton , ct ) . after an initial of 93c for 3 min , dna was denatured at 93c for 1 min , annealed at 63c for 1 min , and extended at 72c for one and a half min for 30 cycles . the sizes of pcr products using -geo and vis primers were 682 bp and 1038 bp , respectively . dna from ho and csad wild type ( wt ) produced one band from either -geo or vis , respectively , and csad produced bands from both -geo and vis . , total rnas from liquid nitrogen frozen liver were extracted with trizol and rneasy kit . rna was separated by agarose gel electrophoresis and transferred to a nylon membrane ( nen life science product ) . briefly , frozen liver and brain were homogenized with pbs buffer containing igepal ca-630 and protease inhibitors ( roche applied science ) . supernatants from tissue homogenates were separated using sds - page and transferred to a nitrocellulose membrane . the membrane was incubated with rabbit anti - csad antibody and then with alkaline phosphatase labeled goat anti - rabbit secondary antibody after washing the membrane with pbs containing 0.2% tween 20 . polyclonal rabbit anti - csad was produced in our laboratory using c - terminal 15 peptides obtained from synpep corp . the levels of taurine were determined using hplc ( waters , milford , ma ) . after samples were dried using a speedvac ( savant , holbrook , ny ) , they were derivatized using phenylisothiocyanate ( pitc ) and separated using a c18 column with a gradient of acetate buffer containing 2.5% acetonitrile ( ph 6.5 ) and 45% acetonitrile solution containing 15% methanol at 45c . total rnas from the brain and liver in wt and g3 ho at postnatal day 1 ( pd1 ) were extracted using rneasy mini kit ( qiagen ) according to manufacturer 's instructions . biotin - rna was generated using the epicentre target amp - nano labeling kit for illumina expression beadchip ( cat number tan07908 , san diego , ca ) from an input of 300 ng of high quality rna . illumina bead arrays were performed using mouse wg-6 v2 bead chip ( cat number bd-103 - 0204 ) and illumina gene expression kit according to manufacturer 's protocol . raw intensity values were acquired using the hiscan microarray scanner ( illumina ) and imported to genome studio using the gene expression module ( illumina ) . total rna was extracted using rneasy kit ( qiagen ) from the brain and liver at pd 1 and was reverse - transcribed using cdna kit according to the manufacturer 's instruction ( qiagen ) . quantitative real time pcr with 10 ng of cdna were carried out in duplicate in a 7300 real time pcr system ( effendorff , hauppauge , ny ) using the sybr master mix ( qiagen ) and the following cycles : 2 min at 50c , 10 min at 95c , and then 40 cycles each at 95c for 15 s and 60c for 60 s . rt qpcr analysis was also carried out according to manufacturer 's manual using -actin as a control . the ct method was used ; for each gene fold changes were calculated as difference in gene expression of g3 ho and g3 hot compared to that in wt . ct was calculated by subtraction of ct of -actin from ct of the specific gene . ct was calculated by subtraction of ct of wt from ct of g3 ho or h3 hot . significant differences between groups were determined as p < 0.05 using lsd or tukey hsd in post hoc under one way anova . the murine csad gene is located on chromosome 15:102 , 176 , 998 - 102 , 189 , 043 bp ( assembly dec . chimeric mice were derived from an es cell line in which the csad gene had been inactivated by insertional mutagenesis ( xp0392 , mmrrc : 022638-ucd sanger gene trap line ) [ figure 1(a ) ] . es cells were inserted into b6 blastocysts at the mmrrc [ 27 , 28 ] ( work supported by ncrr - nih ) . the genotypes of the resulting offspring were determined by pcr using tail dna with two pairs of primers ( table 1 ) . one primer pair ( -geo ) detects the presence of the gene trap insertion ( vector ) , located between exons 8 and 9 in the csad gene [ figures 1(a ) and 1(c ) ] , and the other primer pair ( vis ) detects the wild type ( wt ) sequence at the insertion site and gives no product in the presence of the insertion . the sizes of the -geo and vis pcr products are 682 and 1038 bp , respectively . southern blot analysis of homozygous csad ko mice ( ho ) showed disruption of the csad gene [ 7.1 kb in figure 1(b ) ] . wt and csad showed one band ( 5.9 kb ) and two bands ( 5.9 and 7.1 kb ) , respectively . the csad protein ( 51.0 kd ) and csad mrna were detected in wt and csad but were not detected in ho [ figures 1(e ) and 1(d ) ] . plasma was collected by heart puncture from all three groups at age of 8 weeks . plasma taurine concentration , determined by hplc , was significantly reduced in csad but was drastically reduced by more than 80% in g1 ho and g2 ho compared to wt ( table 2 ) . taurine concentrations were not significantly different in females compared to males and data from 8-week - old males and females were pooled . taurine is not completely absent in ho presumably because there is an alternative pathway for taurine biosynthesis from cysteamine through cysteamine dioxygenase ( ado ) . csad csad mating produced offspring genotypes that were not different from the expected mendelian distribution . to investigate the role of taurine in reproduction , we observed three generations of homozygous csad . homozygotes are abbreviated as follows : g1 ho were born from csad csad ; g2 ho were born from g1 ho g1 ho ; g3 ho were born from g2 ho g2 ho ( figures 2 and 3 ) . there were no significant differences from wild type ( wt ) in the pregnancy rates in different generations of ho mice . there was also no difference in the number of females and males that were bred and the number of pregnancies per female averaged 3 - 4 in all groups . in addition , the initial number of pups per litter did not vary significantly across groups ( table 3 ) . although the concentrations of taurine in the plasma were low in g1 ho ( table 2 ) , all of the offspring from g1 ho pairs survived and thrived ( table 3 ) . however , offspring from g2 ho and g3 ho had a very low rate of survival , although a normal number of pups were born alive . all of the pups from eleven out of thirteen litters ( 85% ) born from g2 ho and from nine out of ten litters born from g3 ho ( 90% ) were dead within 24 hrs ( table 3 ) . death / survival was measured at postnatal day one ( pd1 ) ( figure 3 ) . survival rates of g3 and g4 ho litters born from g2 and g3 ho were 15% and 10% , respectively . the surviving pups all showed milk spots ( white material in their stomachs visible through the skin ) while there were no milk spots apparent in the pups that died . birth weights of live ho including g3 ho and g4 ho and wt were not significantly different although g3 and g4 ho pups without milk spots were slightly smaller at pd1 ( ~0.1 g ) compared to those with milk spots . surviving pups from g2 ho and g3 ho grew normally although they were very few in number . since the surviving pups had evidence of successful nursing while the others did not , we cross - fostered ho pups with wt dams and wt pups with ho dams to see whether the fault lay in the dams nursing behavior or the pups ' suckling . when wt neonates born were nursed by g2 ho dams ( that had delivered pups the same day ) , they suckled milk and grew normally . neonates without milk spots born from g2 ho did not survive when they were nursed by wt dams . to see the effect of exogenous taurine we also observed three generations of homozygous csad that were treated with 0.05% taurine in their drinking water ( hot ) ( figure 3 ) . both males and females of g2 hot were treated with taurine ( 0.05% ) in their drinking water from 10 days prior to mating until weaning of their offspring . g3 hot were born from g2 hot and treated with taurine from weaning to their offspring 's weaning . twelve of thirteen litters and fifteen of sixteen litters from g2 and g3 hot , respectively , treated with 0.05% taurine in the drinking water thrived . the survival rates of litters from g2 hot and g3 hot were 92% and 94% , respectively . there was no statistically significant difference in reproduction performance between g2 hot and g3 hot ; pregnancy rates and the number of pups per litter were not different from wt . pups from wt , ht , ho , and hot were weaned 3 to 4 weeks after birth . the high survival rates of litters from both taurine - supplemented groups indicated that proper levels of taurine during gestation are necessary for survival . the death of most pups in the nonsupplemented g2 and g3 ho litters within 24 hours of birth suggests that the lack of taurine in utero may have been the important factor . since the liver is a major source for the production of taurine required for brain development , taurine concentrations in both liver and brain taurine concentrations in the brain and liver were significantly reduced by more than 80% in g2 ho , g3 ho , and g4 ho but not in g1 ho in the brain which were born from heterozygote dams with normal levels of taurine . however , taurine levels in both the liver and brain were increased in mice whose drinking water was supplemented with 0.05% taurine ( g3 hot and g4 hot ) . taurine concentrations were not significantly different in the brain of hot mice compared to wt . although taurine concentrations in the liver from g3 hot and g4 hot increased significantly compared to g3 ho and g4 ho , they were not completely restored to wt levels . although some g3 ho pd1 pups did not show a milk spot , taurine concentrations in the liver and brain were not significantly different in these two groups : offspring with a milk spot ( g3 ho w / ms ) versus offspring without a milk spot ( g3 ho w / o ms ) in the liver and brain , 2.1 0.1 versus 1.8 0.2 and 6.0 0.5 versus 5.5 0.5 , respectively . we used a microarray of more than 9,000 genes to examine the effect of the csad knockout and taurine treatment on gene expression in pd1 ho mice ( figure 3 ) . three hundred genes in the liver and 145 genes in the brain were changed by more than 2-fold in the csad at pd1 ( table 4 ) . genes in the liver were mostly upregulated while the genes in the brain were mostly downregulated . genes selected from microarray data were summarized in table 5 . of the taurine - related genes , csad , cdo , ado and taut , antioxidant enzymes including gpx1 and 3 and prdx 2 and 3 were examined because neonatal death may be caused by oxidative stress . uracil phopshorylase 2 and serine dehydratase , two metabolic enzymes , demonstrated significant upregulation and were further studied using rt qpcr . prolactin and lactoferrin were significantly down - regulated . since these factors are intimately associated with production and function of milk and because milk spots were a predictor for survival , downregulation of these genes taurine - related genes including csad , cdo , ado , and taut were examined using rt qpcr ( table 6 ) . the mrna expression levels of taut in the liver were not affected by taurine treatment while cdo was significantly increased in g3 ho compared to wt . expression of two antioxidant enzymes in the liver , peroxireductase 2 and 3 ( prdx 2 and prdx 3 ) , was not significantly different in csad compared to wt ( table 7 ) . however , glutathione peroxidase 1 and 3 ( gpx1 and gpx3 ) were increased significantly in csad liver and restored to wild type levels in taurine - treated csad animals . since newborn g3 csad often lacked milk spots , a predictor of survival in newborn mice , we examined the expression of lactoferrin ( ltf ) and the prolactin receptor ( prlr ) and found that their expression was significantly decreased in csad animals . prlr expression in the liver was brought to levels higher than wt by taurine treatment , but expression of ltf was not restored ( figure 5 ) . since the absence of a milk spot predicts mortality , we compared the gene expression of pups with a milk spot to those without a milk spot ( table 8) . g3 ho pups without a milk spot ( g3 ho w / o ms ) demonstrated significantly higher expression of cdo , upp2 , and sds compared to those with a milk spot ( g3 ho however , ltf was significantly decreased in g3 ho w / o ms compared to g3 ho w / ms . expression of sds in g3 ho w / ms was significantly increased compared to wt ( table 9 ) . expression of cdo , upp2 , and sds in g3 hot was restored to that in wt . we have generated a new mouse model to study the physiological role of taurine by knocking out the csad gene . disruption of the csad gene in this mouse was documented by southern analysis [ figure 1(b ) ] , the absence of csad mrna and protein was shown by northern and western analyses [ figures 1(d ) and 1(e ) ] , and the taurine concentration in the plasma of csad animals was reduced by > 80% ( table 2 ) . the physiological role of taurine has been studied in the cat , where it is an essential amino acid , and in the rat after treatment with guanidinoethanesulfonate ( ges ) , a competitive inhibitor of taurine transport [ 29 , 30 ] . more recently mouse models of taurine deficiency have been engineered to facilitate this investigation : two taurine transporter knockout mouse models ( taut ko ) show reduced levels of taurine in various tissues in heart , brain , muscle , kidney , and liver [ 3133 ] . these taut ko models demonstrate developmental effects in various organs including the retina , liver , brain , muscle and heart . in addition taurine deficient mice were developed by knocking out the gene for cdo which produces cysteine sulfinic acid , a substrate for csad , from cysteine [ 3436 ] . these mice have severe taurine deficiency and increased catabolism of cysteine to hydrogen sulfide , which leads to pulmonary and pancreatic toxicity . the csad knockout mouse that we describe here shows drastically reduced endogenous taurine biosynthesis and complete dependency on exogenous taurine for normal reproductive performance ( table 3 ) . heterozygous csad ko mice had reduced levels of plasma taurine but csad haploinsufficiency had no detectable effect on reproduction . g1 csad derived from heterozygous parents had much reduced plasma levels of taurine ( table 2 ) but had litters of normal size that were indistinguishable from litters of wt or csad ( table 3 ) . offspring from g2 csad and g3 csad , however , had very low survival rates ( table 3 ) . thus , abnormal reproductive performance appeared from the second generation of taurine deficient csad while reproduction of the first generation was not affected . taurine supplementation in the drinking water ( g2 hot and g3 hot ) prior to , as well as during gestation and after birth , completely restored the pup survival rate . reproduction performance was not significantly different between g2 hot , g3 hot , and wt , indicating that fetus and neonates were healthy when they were supplied with necessary taurine through the placenta and milk . these results strongly indicate that the reduced level of taurine may be responsible for the neonatal mortality . however , since reduced taurine is not 100% lethal and taurine concentrations in g3 ho w / ms and g3 ho w / o ms are not significantly different , there are apparently additional environmental or stochastic factors that influence neonatal mortality . this suggests that the reduced taurine levels impact the suckling ability and it seems likely that this defect is associated with prenatal development . studies have shown that the reproductive performance of cats fed taurine deficient diets ( 0 or 0.01% ) was poor and that they frequently had stillborn or low birth weight kittens with severe neurological abnormalities [ 2 , 37 , 38 ] . the offspring from taurine deficient cats showed abnormal births including stillbirths , abnormal brain and eye development , and poor growth . in contrast , g1 csad born from ht and with low taurine levels grew normally and their offspring also appeared normal ( table 3 ) . a high rate of neonatal death occurred in most of the offspring from g2 csad and g3 csad . these data indicate that low plasma taurine levels like g1 ho ( table 2 ) are not sufficient to induce poor reproductive performance . taurine concentrations at pd1 in the liver and brain from g1 ho were similar to wt ( figures 4(a ) and 4(b ) ) . g1 ho with a taurine rich fetal environment and g2 ho with a taurine poor fetal environment implicate a taurine deficient fetal life for a higher death rate [ table 3 and figures 4(a ) and 4(b ) ] . in the cat model the offspring which show abnormalities or death develop in a low taurine environment due to intrinsically low levels of cdo and csad as well as the absence of dietary taurine . to determine whether oxidants contributed to neonatal death , antioxidant enzymes including gpx1 and 3 as well as prdx 2 and 3 were examined [ 3944 ] . since gpx1 and 3 were significantly increased , oxidants may be contributing factors in the neonatal death of ho mice ( table 7 ) . we used microarray analysis to examine how the mrna expression of more than 9,000 genes might have been altered by the deletion of csad . it is important to keep in mind that these changes could be due to transcriptional differences or differences in mrna processing or mrna stability which may or may not lead to more or less of the protein itself . analysis of the brain and liver from wt and g3 ho at pd1 showed that gene expression in the liver was mostly increased while gene expression in the brain was mostly decreased ( table 4 ) . using rt qpcr gene expression of relevant genes to fetal death was examined at pd1 in wt , g3 ho , and g3 hot ( tables 6 , 7 , 8 , and 9 ) . rt qpcr data in the levels of ado , cdo , and csad are consistent in the brain and liver with data previously described [ 15 , 17 ] . taurine in the liver , brain , and blood of the csad mouse is not completely absent , presumably because of the alternative pathway for taurine biosynthesis through ado ( table 2 and figure 4 ) [ 2 , 17 ] . the mrna expression of ado was not significantly different in both the liver and brain in g3 ho ( table 6 ) and taurine concentrations were markedly reduced in g2 , g3 , and g4 ho [ figures 4(a ) and 4(b ) ] , indicating that the alternative , ado , pathway is a relatively minor source for taurine production . taut was not changed in three groups including wt , g3 csad , and g3 hot , suggesting that the increase of taurine in g3 hot is not mediated by the induction of taut mrna . these data also indicate that taut does not show the increased expression at pd1 that is seen later in taurine deprived / deficient animals . because newborn g3 csad often lacked milk spots , which are a predictor of survival in newborn mice , we examined the gene expression of ltf and prlr which were markedly reduced in microarray analysis ( table 5 ) . ltf and prlr in the liver were decreased significantly in g3 ho but only prlr was restored by taurine supplementation ( g3 hot ) ( figure 5 ) . ltf has innate immune function to protect newborn offspring from infection and is elevated in colostrum [ 4547 ] . prolactin is a lactogenic hormone and regulates the output of insulin - like growth factor-1 . genetic ablation of prlr results in mice which show multiple defects in reproduction leading to infertility , altered maternal behavior , and reduced bone development [ 4850 ] . meanwhile , cdo was increased in g3 ho without milk spots but not in g3 ho with milk spots . these data indicate that increase of cdo in g3 ho is attributable to the absence of milk ( tables 6 and 8) . since uridine phosphorylase 2 ( upp2 ) and serine dehydratase ( sds ) in g3 ho were dramatically increased in microarray analysis ( table 5 ) , these genes were examined using rt qpcr . upp2 and sds were significantly increased and lft was significantly decreased in g3 ho without milk spots , compared to g3 ho with milk spots ( table 8) . upp 2 catalyzes the cleavage of uridine to uracil and ribose which are involved with energy supply and nucleotide synthesis [ 5153 ] . the significance of the increase in upp 2 in g3 ho without milk spot is unclear but may be a consequence of the morbidity of these mice . serine is a substrate for sds with production of pyruvate ( precursor of glucose ) and nh3 [ 5456 ] . the lack of a milk spot and subsequent death indicate starvation which is responsible for the large increase in sds . expression of sds in g3 ho with milk spots was remarkably lower than in g3 ho without milk spots . however , expression of sds in g3 ho with milk spots is significantly increased compared to wt , indicating that low taurine in g3 ho may induce gluconeogenesis by an increase in sds . this result suggests that sds expression is synergistically up - regulated by the ho genotype and by starvation / absence of a milk spot . wt expression levels of these genes were restored in g3 hot , suggesting that taurine corrects abnormalities in nucleotide and amino acid metabolism due to an increase of upp2 and sds ( table 9 ) . ltf is widely present in fluids such as milk and colostrum with a high affinity for iron . lft , a component of the innate immune system , is important in bacteriostasis and required for optimal neutrophil function . the significant decrease of lft in g3 ho w / o ms is consistent with the importance of this protein for survival . taurine concentrations in the brain , liver , and plasma from csad were significantly lower compared to wt and csad . these data indicate that csad is an effective target for lowering taurine to symptomatic levels and that wild - type taurine levels can be restored in the brain and liver of csad ko mice by adding taurine to their drinking water . these data indicate that this csad ko mouse will be a powerful additional model for the analysis of the physiological role of taurine , particularly since taurine requirements can be supplied in food or water .

we engineered a csad ko mouse to investigate the physiological roles of taurine . the disruption of the csad gene was verified by southern , northern , and western blotting . hplc indicated an 83% decrease of taurine concentration in the plasma of csad/. although csad/ generation ( g)1 and g2 survived , offspring from g2 csad/ had low brain and liver taurine concentrations and most died within 24 hrs of birth . taurine concentrations in g3 csad/ born from g2 csad/ treated with taurine in the drinking water were restored and survival rates of g3 csad/ increased from 15% to 92% . the mrna expression of cdo , ado , and taut was not different in csad/ compared to wt and csad mrna was not expressed in csad/. expression of gpx 1 and 3 was increased significantly in csad/ and restored to normal levels with taurine supplementation . lactoferrin and the prolactin receptor were significantly decreased in csad/. the prolactin receptor was restored with taurine supplementation . these data indicated that csad ko is a good model for studying the effects of taurine deficiency and its treatment with taurine supplementation .

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

it is mainly produced in the liver and kidney and is present in most other tissues including the brain , leukocytes , retina , heart , placenta , and muscle [ 13 ] . taurine is a key element in many diverse processes including development of the brain , retina , and the immune system , osmoregulation , reproduction , membrane stabilization , regulation of cardiac muscle , and inflammation [ 2 , 410 ] . colostrum contains a very high taurine concentration which is required for development of the brain and retina in the newborn human . hepatic csad and cdo activity is high compared to that in the kidney and brain [ 15 , 1921 ] . the tissue distribution of csad , protein , and mrna , detected by western and northern blot analyses , is consistent with csad activity . ado level is highest in the brain , whereas csad and cdo levels are highest in the liver [ 15 , 17 , 18 , 22 ] . cats have been used for taurine studies because they have low levels of cdo and csad leading to a dependence on dietary sources of taurine . since the cat model has limitations including a long gestation period , a heterogeneous genetic background , and a relatively large maintenance expense , we developed a csad knockout mouse ( csad ko ) model to better understand the physiological roles of taurine . this novel murine model was developed to provide insight into the role of taurine in reproduction , innate and adaptive immunity , and brain development . we report here that the absence of a functional csad gene in the csad ko mouse reduces the level of this amino acid by > 80% and has a severe effect on neonatal survival that is reversed by adding taurine ( 0.05% ) to the drinking water . for optimum reproductive performance , both females and males used for mating in the taurine - treated groups were supplemented with taurine . dna from the spleen was analyzed by southern blotting to confirm the csad ko genotype using a modified method previously described . the membrane was hybridized using csad cdna as a probe to confirm the disruption of the csad gene . dna from ho and csad wild type ( wt ) produced one band from either -geo or vis , respectively , and csad produced bands from both -geo and vis . total rnas from the brain and liver in wt and g3 ho at postnatal day 1 ( pd1 ) were extracted using rneasy mini kit ( qiagen ) according to manufacturer 's instructions . total rna was extracted using rneasy kit ( qiagen ) from the brain and liver at pd 1 and was reverse - transcribed using cdna kit according to the manufacturer 's instruction ( qiagen ) . quantitative real time pcr with 10 ng of cdna were carried out in duplicate in a 7300 real time pcr system ( effendorff , hauppauge , ny ) using the sybr master mix ( qiagen ) and the following cycles : 2 min at 50c , 10 min at 95c , and then 40 cycles each at 95c for 15 s and 60c for 60 s . the ct method was used ; for each gene fold changes were calculated as difference in gene expression of g3 ho and g3 hot compared to that in wt . the murine csad gene is located on chromosome 15:102 , 176 , 998 - 102 , 189 , 043 bp ( assembly dec . chimeric mice were derived from an es cell line in which the csad gene had been inactivated by insertional mutagenesis ( xp0392 , mmrrc : 022638-ucd sanger gene trap line ) [ figure 1(a ) ] . one primer pair ( -geo ) detects the presence of the gene trap insertion ( vector ) , located between exons 8 and 9 in the csad gene [ figures 1(a ) and 1(c ) ] , and the other primer pair ( vis ) detects the wild type ( wt ) sequence at the insertion site and gives no product in the presence of the insertion . southern blot analysis of homozygous csad ko mice ( ho ) showed disruption of the csad gene [ 7.1 kb in figure 1(b ) ] . wt and csad showed one band ( 5.9 kb ) and two bands ( 5.9 and 7.1 kb ) , respectively . the csad protein ( 51.0 kd ) and csad mrna were detected in wt and csad but were not detected in ho [ figures 1(e ) and 1(d ) ] . plasma taurine concentration , determined by hplc , was significantly reduced in csad but was drastically reduced by more than 80% in g1 ho and g2 ho compared to wt ( table 2 ) . taurine concentrations were not significantly different in females compared to males and data from 8-week - old males and females were pooled . csad csad mating produced offspring genotypes that were not different from the expected mendelian distribution . to investigate the role of taurine in reproduction , we observed three generations of homozygous csad . homozygotes are abbreviated as follows : g1 ho were born from csad csad ; g2 ho were born from g1 ho g1 ho ; g3 ho were born from g2 ho g2 ho ( figures 2 and 3 ) . there was also no difference in the number of females and males that were bred and the number of pregnancies per female averaged 3 - 4 in all groups . although the concentrations of taurine in the plasma were low in g1 ho ( table 2 ) , all of the offspring from g1 ho pairs survived and thrived ( table 3 ) . however , offspring from g2 ho and g3 ho had a very low rate of survival , although a normal number of pups were born alive . all of the pups from eleven out of thirteen litters ( 85% ) born from g2 ho and from nine out of ten litters born from g3 ho ( 90% ) were dead within 24 hrs ( table 3 ) . survival rates of g3 and g4 ho litters born from g2 and g3 ho were 15% and 10% , respectively . surviving pups from g2 ho and g3 ho grew normally although they were very few in number . since the surviving pups had evidence of successful nursing while the others did not , we cross - fostered ho pups with wt dams and wt pups with ho dams to see whether the fault lay in the dams nursing behavior or the pups ' suckling . neonates without milk spots born from g2 ho did not survive when they were nursed by wt dams . to see the effect of exogenous taurine we also observed three generations of homozygous csad that were treated with 0.05% taurine in their drinking water ( hot ) ( figure 3 ) . both males and females of g2 hot were treated with taurine ( 0.05% ) in their drinking water from 10 days prior to mating until weaning of their offspring . g3 hot were born from g2 hot and treated with taurine from weaning to their offspring 's weaning . twelve of thirteen litters and fifteen of sixteen litters from g2 and g3 hot , respectively , treated with 0.05% taurine in the drinking water thrived . the survival rates of litters from g2 hot and g3 hot were 92% and 94% , respectively . there was no statistically significant difference in reproduction performance between g2 hot and g3 hot ; pregnancy rates and the number of pups per litter were not different from wt . the high survival rates of litters from both taurine - supplemented groups indicated that proper levels of taurine during gestation are necessary for survival . the death of most pups in the nonsupplemented g2 and g3 ho litters within 24 hours of birth suggests that the lack of taurine in utero may have been the important factor . since the liver is a major source for the production of taurine required for brain development , taurine concentrations in both liver and brain taurine concentrations in the brain and liver were significantly reduced by more than 80% in g2 ho , g3 ho , and g4 ho but not in g1 ho in the brain which were born from heterozygote dams with normal levels of taurine . taurine concentrations were not significantly different in the brain of hot mice compared to wt . although taurine concentrations in the liver from g3 hot and g4 hot increased significantly compared to g3 ho and g4 ho , they were not completely restored to wt levels . although some g3 ho pd1 pups did not show a milk spot , taurine concentrations in the liver and brain were not significantly different in these two groups : offspring with a milk spot ( g3 ho w / ms ) versus offspring without a milk spot ( g3 ho w / o ms ) in the liver and brain , 2.1 0.1 versus 1.8 0.2 and 6.0 0.5 versus 5.5 0.5 , respectively . we used a microarray of more than 9,000 genes to examine the effect of the csad knockout and taurine treatment on gene expression in pd1 ho mice ( figure 3 ) . three hundred genes in the liver and 145 genes in the brain were changed by more than 2-fold in the csad at pd1 ( table 4 ) . genes in the liver were mostly upregulated while the genes in the brain were mostly downregulated . of the taurine - related genes , csad , cdo , ado and taut , antioxidant enzymes including gpx1 and 3 and prdx 2 and 3 were examined because neonatal death may be caused by oxidative stress . since these factors are intimately associated with production and function of milk and because milk spots were a predictor for survival , downregulation of these genes taurine - related genes including csad , cdo , ado , and taut were examined using rt qpcr ( table 6 ) . the mrna expression levels of taut in the liver were not affected by taurine treatment while cdo was significantly increased in g3 ho compared to wt . expression of two antioxidant enzymes in the liver , peroxireductase 2 and 3 ( prdx 2 and prdx 3 ) , was not significantly different in csad compared to wt ( table 7 ) . however , glutathione peroxidase 1 and 3 ( gpx1 and gpx3 ) were increased significantly in csad liver and restored to wild type levels in taurine - treated csad animals . since newborn g3 csad often lacked milk spots , a predictor of survival in newborn mice , we examined the expression of lactoferrin ( ltf ) and the prolactin receptor ( prlr ) and found that their expression was significantly decreased in csad animals . prlr expression in the liver was brought to levels higher than wt by taurine treatment , but expression of ltf was not restored ( figure 5 ) . g3 ho pups without a milk spot ( g3 ho w / o ms ) demonstrated significantly higher expression of cdo , upp2 , and sds compared to those with a milk spot ( g3 ho however , ltf was significantly decreased in g3 ho w / o ms compared to g3 ho w / ms . expression of sds in g3 ho w / ms was significantly increased compared to wt ( table 9 ) . expression of cdo , upp2 , and sds in g3 hot was restored to that in wt . we have generated a new mouse model to study the physiological role of taurine by knocking out the csad gene . disruption of the csad gene in this mouse was documented by southern analysis [ figure 1(b ) ] , the absence of csad mrna and protein was shown by northern and western analyses [ figures 1(d ) and 1(e ) ] , and the taurine concentration in the plasma of csad animals was reduced by > 80% ( table 2 ) . the physiological role of taurine has been studied in the cat , where it is an essential amino acid , and in the rat after treatment with guanidinoethanesulfonate ( ges ) , a competitive inhibitor of taurine transport [ 29 , 30 ] . more recently mouse models of taurine deficiency have been engineered to facilitate this investigation : two taurine transporter knockout mouse models ( taut ko ) show reduced levels of taurine in various tissues in heart , brain , muscle , kidney , and liver [ 3133 ] . these mice have severe taurine deficiency and increased catabolism of cysteine to hydrogen sulfide , which leads to pulmonary and pancreatic toxicity . heterozygous csad ko mice had reduced levels of plasma taurine but csad haploinsufficiency had no detectable effect on reproduction . g1 csad derived from heterozygous parents had much reduced plasma levels of taurine ( table 2 ) but had litters of normal size that were indistinguishable from litters of wt or csad ( table 3 ) . offspring from g2 csad and g3 csad , however , had very low survival rates ( table 3 ) . thus , abnormal reproductive performance appeared from the second generation of taurine deficient csad while reproduction of the first generation was not affected . taurine supplementation in the drinking water ( g2 hot and g3 hot ) prior to , as well as during gestation and after birth , completely restored the pup survival rate . reproduction performance was not significantly different between g2 hot , g3 hot , and wt , indicating that fetus and neonates were healthy when they were supplied with necessary taurine through the placenta and milk . these results strongly indicate that the reduced level of taurine may be responsible for the neonatal mortality . however , since reduced taurine is not 100% lethal and taurine concentrations in g3 ho w / ms and g3 ho w / o ms are not significantly different , there are apparently additional environmental or stochastic factors that influence neonatal mortality . the offspring from taurine deficient cats showed abnormal births including stillbirths , abnormal brain and eye development , and poor growth . a high rate of neonatal death occurred in most of the offspring from g2 csad and g3 csad . these data indicate that low plasma taurine levels like g1 ho ( table 2 ) are not sufficient to induce poor reproductive performance . taurine concentrations at pd1 in the liver and brain from g1 ho were similar to wt ( figures 4(a ) and 4(b ) ) . in the cat model the offspring which show abnormalities or death develop in a low taurine environment due to intrinsically low levels of cdo and csad as well as the absence of dietary taurine . to determine whether oxidants contributed to neonatal death , antioxidant enzymes including gpx1 and 3 as well as prdx 2 and 3 were examined [ 3944 ] . since gpx1 and 3 were significantly increased , oxidants may be contributing factors in the neonatal death of ho mice ( table 7 ) . we used microarray analysis to examine how the mrna expression of more than 9,000 genes might have been altered by the deletion of csad . analysis of the brain and liver from wt and g3 ho at pd1 showed that gene expression in the liver was mostly increased while gene expression in the brain was mostly decreased ( table 4 ) . using rt qpcr gene expression of relevant genes to fetal death was examined at pd1 in wt , g3 ho , and g3 hot ( tables 6 , 7 , 8 , and 9 ) . rt qpcr data in the levels of ado , cdo , and csad are consistent in the brain and liver with data previously described [ 15 , 17 ] . taurine in the liver , brain , and blood of the csad mouse is not completely absent , presumably because of the alternative pathway for taurine biosynthesis through ado ( table 2 and figure 4 ) [ 2 , 17 ] . the mrna expression of ado was not significantly different in both the liver and brain in g3 ho ( table 6 ) and taurine concentrations were markedly reduced in g2 , g3 , and g4 ho [ figures 4(a ) and 4(b ) ] , indicating that the alternative , ado , pathway is a relatively minor source for taurine production . taut was not changed in three groups including wt , g3 csad , and g3 hot , suggesting that the increase of taurine in g3 hot is not mediated by the induction of taut mrna . these data also indicate that taut does not show the increased expression at pd1 that is seen later in taurine deprived / deficient animals . because newborn g3 csad often lacked milk spots , which are a predictor of survival in newborn mice , we examined the gene expression of ltf and prlr which were markedly reduced in microarray analysis ( table 5 ) . ltf and prlr in the liver were decreased significantly in g3 ho but only prlr was restored by taurine supplementation ( g3 hot ) ( figure 5 ) . prolactin is a lactogenic hormone and regulates the output of insulin - like growth factor-1 . meanwhile , cdo was increased in g3 ho without milk spots but not in g3 ho with milk spots . these data indicate that increase of cdo in g3 ho is attributable to the absence of milk ( tables 6 and 8) . since uridine phosphorylase 2 ( upp2 ) and serine dehydratase ( sds ) in g3 ho were dramatically increased in microarray analysis ( table 5 ) , these genes were examined using rt qpcr . upp2 and sds were significantly increased and lft was significantly decreased in g3 ho without milk spots , compared to g3 ho with milk spots ( table 8) . the significance of the increase in upp 2 in g3 ho without milk spot is unclear but may be a consequence of the morbidity of these mice . serine is a substrate for sds with production of pyruvate ( precursor of glucose ) and nh3 [ 5456 ] . expression of sds in g3 ho with milk spots was remarkably lower than in g3 ho without milk spots . however , expression of sds in g3 ho with milk spots is significantly increased compared to wt , indicating that low taurine in g3 ho may induce gluconeogenesis by an increase in sds . wt expression levels of these genes were restored in g3 hot , suggesting that taurine corrects abnormalities in nucleotide and amino acid metabolism due to an increase of upp2 and sds ( table 9 ) . the significant decrease of lft in g3 ho w / o ms is consistent with the importance of this protein for survival . taurine concentrations in the brain , liver , and plasma from csad were significantly lower compared to wt and csad . these data indicate that csad is an effective target for lowering taurine to symptomatic levels and that wild - type taurine levels can be restored in the brain and liver of csad ko mice by adding taurine to their drinking water . these data indicate that this csad ko mouse will be a powerful additional model for the analysis of the physiological role of taurine , particularly since taurine requirements can be supplied in food or water .

epididymis is a site for a variety of non - neoplastic and neoplastic lesions , predominantly the former . clinically most of these lesions produce worrisome palpable nodules , and fine - needle aspiration is just the procedure that seeks to confirm the non - neoplastic nature of most lesions and guide patient management strategies accordingly . we hereby report our experience with fine needle aspiration cytology ( fnac ) in the treatment of 40 patients with epididymal nodules from a general hospital , with a view to highlight the simplicity of the technique and high accuracy of diagnosis . a total of 40 patients with epididymal nodules were aspirated during a period of eight years . aspiration was done with the patient in supine position and without local anesthesia , by the pathologist only . two - to - three passes were made in each case . only in cases where patient complained of pain or the nodules were tender , the urologic surgeon accompanied the patient and gave a cord block or local anesthesia as required . in case of cystic swellings , in all the cases , wet - fixed papanicolaou - stained and air - dried giemsa - stained smears were studied . the ages of patients were in the wide range of 10 to 45 years ; however , 28 ( 70% ) patients were young adults aged 21 - 30 years . four patients each ( 10% ) were aged < 20 years , 31 - 40years and > 40 years , respectively . the inflammatory lesions occurred in the 21 - 30 years age group , whereas cystic nodules were common in the 31 - 40 years age group . most of the patients had painless nodules , except in 3 cases of acute non - specific epididymitis , where the lesion was tender and painful . out of the 40 cases , 38 cases were of non - neoplastic lesions , and there were 2 ( 5% ) cases of adenomatoid tumors . the cytological finding in 1 of these 2 cases of adenomatoid tumors of the epididymis reported in this study has been published earlier . non - neoplastic lesions constituted 14 ( 35% ) cases of tuberculous granulomatous inflammation ; 10 , of cystic nodules ( 9 spermatoceles and 1 encysted hydrocele ) ; 5 lesions , of non - specific inflammation ; 3 ( 7.5% ) , of filarial infection ; 3 , of sperm granulomas ; and 3 , of probable adenomatous hyperplasia of rete testes . except for the 2 adenomatoid tumors , 1 adenomatous hyperplasia and 1 tuberculous epididymitis , histopathological confirmation was not obtained ; however , accuracy of diagnosis could be judged from the clinical response to treatment , and such clinico - cytologic or histologic correlation was obtained in 78% of the non - neoplastic cases . smears revealed epithelioid granulomas with or without langhans giant cells in 6 patients , whereas caseous necrotic materials with polymorphs and degenerating granulomas were seen in 8 patients [ figure 1a ] . the ziehl - neelsen staining on air - dried smears revealed acid - fast bacilli in 6 of the 12 patients [ figure 1b ] . in 5 patients , all the patients received anti - tuberculous treatment , and in 5 patients the nodules totally disappeared following completion of treatment ; while in the remaining , partial response was observed . only 1 patient underwent curative surgical excision for non - responsive disease [ figures 1c and d ] . ( a ) tuberculous epididymitis : smear showing epithelioid cell granuloma [ arrow ] against caseous necrotic background ( pap stain , 400 ) . ( b ) smear showing only caseous necrosis and inset showing acid - fast tubercle bacilli [ arrowhead ] ( ziehl neelsen stain , 1000 ) . ( d ) histopathology of the nodule showing epithelioid granulomas ( arrow ) ( h and e , 400 ) cytology in these patients revealed acute inflammatory cells in 3 cases [ figure 2a ] and chronic inflammation in the other 2 cases . in addition , native tall columnar glandular epithelium with intra - cytoplasmic polymorphs was seen in acute epididymitis . all showed good response after one week , with shrinkage in size following antibiotic treatment . ( a ) non - specific epididymitis : cytology showing acute inflammatory cells ( pap stain 100 ) . ( b ) microfilarial parasite in epididymis showing adult worm identified by the hyaline sheath [ arrow ] . the hyaline sheath shows multiple larvae within it , indicated by arrowhead ( giemsa stain , 400 ) . ( c ) spermatic granuloma : smear showing plenty of degenerating sperms amongst inflammatory cells , giant cells and histiocytes , few of which had also engulfed the sperms . ( d ) histopathology of spermatic granuloma showing granulomas around the sperms [ arrow ] ( h and e , 100 ) all these patients presented with nodular swelling of the epididymis 2 - 3 cm in size . an entire adult worm was aspirated along with the hyaline sheath of the parasite in 1 patient [ figure 2b ] . while the background eosinophils were variable in all aspirates , only 1 patient had presence of peripheral blood eosinophilia . sized nodules which on aspiration showed plenty of degenerating sperms amongst inflammatory cells ; foreign body type of giant cells ; and histiocytes , most of these patients had cystic swellings that yielded clear straw - colored fluid , with decrease in size following aspiration . lack of inflammation and foreign - body giant cells helped to distinguish the condition from a sperm granuloma . three of the patients , on enquiry , gave past history of surgery for inguinal hernia [ figures 3a and b ] . ( a ) spermatocele : cytologic smears showing numerous sperms against a clear background ( giemsa stain , 100 ) . ( b ) high magnification of the same demonstrating the sperms ( giemsa stain , x400 ) . ( c ) encysted hydrocele : lymphocytes in the background and mesothelial cells [ arrow ] seen ( giemsa stain , 100 ) . ( d ) adenomatous hyperplasia : smears showing clusters of polygonal cells with vesicular nuclei and a moderate amount of cytoplasm along with sperm heads [ arrow ] ( giemsa stain , 400 ) this solitary patient came with cystic epididymal swelling 4 cm in size , and aspiration drew a clear fluid with plenty of lymphocytes and few mesothelial cells [ figure 3c ] . though the lesions were thought to be in epididymis , subsequent histology in 1patient confirmed the diagnosis of adenomatous hyperplasia of the rete testes and assisted us in extrapolating the results in the other 2 cases . smears from these nodules revealed cellular aspirates with clusters of polygonal - to - cuboidal epithelial cells with vesicular nucleus and a moderate amount of cytoplasm along with sperm heads sticking to the epithelial cells [ figure 3d ] . as opposed to an aspirate from adenomatoid tumor , the nuclei of cells were smaller and the cytoplasm was less abundant . histology in 1 case revealed tubulopapillary epithelial proliferation of rete testis lined by cuboidal bland cells . sized painless nodules which on cytology showed clusters of oval - to - polygonal cells with moderate - to - abundant optically clear or vacuolated cytoplasm with molding nuclear membrane [ figures 4a c ] . the nuclear chromatin was finely dispersed ; and in the giemsa stain , the para - nuclear cytoplasm appeared pink . histopathology confirmed the cytological diagnosis of adenomatoid tumor in both cases [ figure 4d ] . adenomatoid tumor of the epididymis : ( a ) multi - layering of tumor cells ( giemsa stain , 400 ) ( b ) monolayered tumor cells , in cords ( pap stain , 100 ) ( c ) cells with optically clear cytoplasm [ arrow ] ( giemsa stain , 400 ) . ( d ) histopathology of adenomatoid tumor of the same case ( h and e , 400 ) out of the 40 cases , 38 cases were of non - neoplastic lesions , and there were 2 ( 5% ) cases of adenomatoid tumors . the cytological finding in 1 of these 2 cases of adenomatoid tumors of the epididymis reported in this study has been published earlier . non - neoplastic lesions constituted 14 ( 35% ) cases of tuberculous granulomatous inflammation ; 10 , of cystic nodules ( 9 spermatoceles and 1 encysted hydrocele ) ; 5 lesions , of non - specific inflammation ; 3 ( 7.5% ) , of filarial infection ; 3 , of sperm granulomas ; and 3 , of probable adenomatous hyperplasia of rete testes . except for the 2 adenomatoid tumors , 1 adenomatous hyperplasia and 1 tuberculous epididymitis , histopathological confirmation was not obtained ; however , accuracy of diagnosis could be judged from the clinical response to treatment , and such clinico - cytologic or histologic correlation was obtained in 78% of the non - neoplastic cases . smears revealed epithelioid granulomas with or without langhans giant cells in 6 patients , whereas caseous necrotic materials with polymorphs and degenerating granulomas were seen in 8 patients [ figure 1a ] . the ziehl - neelsen staining on air - dried smears revealed acid - fast bacilli in 6 of the 12 patients [ figure 1b ] . in 5 patients , all the patients received anti - tuberculous treatment , and in 5 patients the nodules totally disappeared following completion of treatment ; while in the remaining , partial response was observed . only 1 patient underwent curative surgical excision for non - responsive disease [ figures 1c and d ] . ( a ) tuberculous epididymitis : smear showing epithelioid cell granuloma [ arrow ] against caseous necrotic background ( pap stain , 400 ) . ( b ) smear showing only caseous necrosis and inset showing acid - fast tubercle bacilli [ arrowhead ] ( ziehl neelsen stain , 1000 ) . ( d ) histopathology of the nodule showing epithelioid granulomas ( arrow ) ( h and e , 400 ) cytology in these patients revealed acute inflammatory cells in 3 cases [ figure 2a ] and chronic inflammation in the other 2 cases . in addition , native tall columnar glandular epithelium with intra - cytoplasmic polymorphs was seen in acute epididymitis . all showed good response after one week , with shrinkage in size following antibiotic treatment . ( a ) non - specific epididymitis : cytology showing acute inflammatory cells ( pap stain 100 ) . ( b ) microfilarial parasite in epididymis showing adult worm identified by the hyaline sheath [ arrow ] . the hyaline sheath shows multiple larvae within it , indicated by arrowhead ( giemsa stain , 400 ) . ( c ) spermatic granuloma : smear showing plenty of degenerating sperms amongst inflammatory cells , giant cells and histiocytes , few of which had also engulfed the sperms . ( d ) histopathology of spermatic granuloma showing granulomas around the sperms [ arrow ] ( h and e , 100 ) all these patients presented with nodular swelling of the epididymis 2 - 3 cm in size . an entire adult worm was aspirated along with the hyaline sheath of the parasite in 1 patient [ figure 2b ] . while the background eosinophils were variable in all aspirates , sized nodules which on aspiration showed plenty of degenerating sperms amongst inflammatory cells ; foreign body type of giant cells ; and histiocytes , most of these patients had cystic swellings that yielded clear straw - colored fluid , with decrease in size following aspiration . lack of inflammation and foreign - body giant cells helped to distinguish the condition from a sperm granuloma . three of the patients , on enquiry , gave past history of surgery for inguinal hernia [ figures 3a and b ] . ( a ) spermatocele : cytologic smears showing numerous sperms against a clear background ( giemsa stain , 100 ) . ( b ) high magnification of the same demonstrating the sperms ( giemsa stain , x400 ) . ( c ) encysted hydrocele : lymphocytes in the background and mesothelial cells [ arrow ] seen ( giemsa stain , 100 ) . ( d ) adenomatous hyperplasia : smears showing clusters of polygonal cells with vesicular nuclei and a moderate amount of cytoplasm along with sperm heads [ arrow ] ( giemsa stain , 400 ) this solitary patient came with cystic epididymal swelling 4 cm in size , and aspiration drew a clear fluid with plenty of lymphocytes and few mesothelial cells [ figure 3c ] . though the lesions were thought to be in epididymis , subsequent histology in 1patient confirmed the diagnosis of adenomatous hyperplasia of the rete testes and assisted us in extrapolating the results in the other 2 cases . smears from these nodules revealed cellular aspirates with clusters of polygonal - to - cuboidal epithelial cells with vesicular nucleus and a moderate amount of cytoplasm along with sperm heads sticking to the epithelial cells [ figure 3d ] . as opposed to an aspirate from adenomatoid tumor , the nuclei of cells were smaller and the cytoplasm was less abundant . histology in 1 case revealed tubulopapillary epithelial proliferation of rete testis lined by cuboidal bland cells . sized painless nodules which on cytology showed clusters of oval - to - polygonal cells with moderate - to - abundant optically clear or vacuolated cytoplasm with molding nuclear membrane [ figures 4a c ] . the nuclear chromatin was finely dispersed ; and in the giemsa stain , the para - nuclear cytoplasm appeared pink . both the cases were diagnosed on cytology , and patients underwent limited excision . histopathology confirmed the cytological diagnosis of adenomatoid tumor in both cases [ figure 4d ] . adenomatoid tumor of the epididymis : ( a ) multi - layering of tumor cells ( giemsa stain , 400 ) ( b ) monolayered tumor cells , in cords ( pap stain , 100 ) ( c ) cells with optically clear cytoplasm [ arrow ] ( giemsa stain , 400 ) . ( d ) histopathology of adenomatoid tumor of the same case ( h and e , 400 ) smears revealed epithelioid granulomas with or without langhans giant cells in 6 patients , whereas caseous necrotic materials with polymorphs and degenerating granulomas were seen in 8 patients [ figure 1a ] . the ziehl - neelsen staining on air - dried smears revealed acid - fast bacilli in 6 of the 12 patients [ figure 1b ] . in 5 patients , all the patients received anti - tuberculous treatment , and in 5 patients the nodules totally disappeared following completion of treatment ; while in the remaining , partial response was observed . only 1 patient underwent curative surgical excision for non - responsive disease [ figures 1c and d ] . ( a ) tuberculous epididymitis : smear showing epithelioid cell granuloma [ arrow ] against caseous necrotic background ( pap stain , 400 ) . ( b ) smear showing only caseous necrosis and inset showing acid - fast tubercle bacilli [ arrowhead ] ( ziehl neelsen stain , 1000 ) . ( d ) histopathology of the nodule showing epithelioid granulomas ( arrow ) ( h and e , 400 ) cytology in these patients revealed acute inflammatory cells in 3 cases [ figure 2a ] and chronic inflammation in the other 2 cases . in addition , native tall columnar glandular epithelium with intra - cytoplasmic polymorphs was seen in acute epididymitis . all showed good response after one week , with shrinkage in size following antibiotic treatment . ( a ) non - specific epididymitis : cytology showing acute inflammatory cells ( pap stain 100 ) . ( b ) microfilarial parasite in epididymis showing adult worm identified by the hyaline sheath [ arrow ] . the hyaline sheath shows multiple larvae within it , indicated by arrowhead ( giemsa stain , 400 ) . ( c ) spermatic granuloma : smear showing plenty of degenerating sperms amongst inflammatory cells , giant cells and histiocytes , few of which had also engulfed the sperms . ( d ) histopathology of spermatic granuloma showing granulomas around the sperms [ arrow ] ( h and e , 100 ) all these patients presented with nodular swelling of the epididymis 2 - 3 cm in size . an entire adult worm was aspirated along with the hyaline sheath of the parasite in 1 patient [ figure 2b ] . while the background eosinophils were variable in all aspirates , sized nodules which on aspiration showed plenty of degenerating sperms amongst inflammatory cells ; foreign body type of giant cells ; and histiocytes , most of these patients had cystic swellings that yielded clear straw - colored fluid , with decrease in size following aspiration . lack of inflammation and foreign - body giant cells helped to distinguish the condition from a sperm granuloma . three of the patients , on enquiry , gave past history of surgery for inguinal hernia [ figures 3a and b ] . ( a ) spermatocele : cytologic smears showing numerous sperms against a clear background ( giemsa stain , 100 ) . ( b ) high magnification of the same demonstrating the sperms ( giemsa stain , x400 ) . ( c ) encysted hydrocele : lymphocytes in the background and mesothelial cells [ arrow ] seen ( giemsa stain , 100 ) . ( d ) adenomatous hyperplasia : smears showing clusters of polygonal cells with vesicular nuclei and a moderate amount of cytoplasm along with sperm heads [ arrow ] ( giemsa stain , 400 ) this solitary patient came with cystic epididymal swelling 4 cm in size , and aspiration drew a clear fluid with plenty of lymphocytes and few mesothelial cells [ figure 3c ] . though the lesions were thought to be in epididymis , subsequent histology in 1patient confirmed the diagnosis of adenomatous hyperplasia of the rete testes and assisted us in extrapolating the results in the other 2 cases . smears from these nodules revealed cellular aspirates with clusters of polygonal - to - cuboidal epithelial cells with vesicular nucleus and a moderate amount of cytoplasm along with sperm heads sticking to the epithelial cells [ figure 3d ] . as opposed to an aspirate from adenomatoid tumor , the nuclei of cells were smaller and the cytoplasm was less abundant . histology in 1 case revealed tubulopapillary epithelial proliferation of rete testis lined by cuboidal bland cells . both the patients had 3 - 4 cm sized painless nodules which on cytology showed clusters of oval - to - polygonal cells with moderate - to - abundant optically clear or vacuolated cytoplasm with molding nuclear membrane [ figures 4a c ] . the nuclear chromatin was finely dispersed ; and in the giemsa stain , the para - nuclear cytoplasm appeared pink . histopathology confirmed the cytological diagnosis of adenomatoid tumor in both cases [ figure 4d ] . adenomatoid tumor of the epididymis : ( a ) multi - layering of tumor cells ( giemsa stain , 400 ) ( b ) monolayered tumor cells , in cords ( pap stain , 100 ) ( c ) cells with optically clear cytoplasm [ arrow ] ( giemsa stain , 400 ) . ( d ) histopathology of adenomatoid tumor of the same case ( h and e , 400 ) although a simple and easy procedure , there are only few literature reports officially documenting the role of cytology in evaluation of epididymal nodules . this could be because epididymal nodules are overall uncommon in clinical practice . as seen in this study , fine - needle aspiration cytology ( fnac ) proved to be useful in the management of patients with epididymal nodules . besides diagnostic purposes , fnac can be used to obtain material for culture studies in patients with infections . fna material could be used to detect organism - specific antigens , e.g. , chlamydial antigens can be detected using specific techniques like immunohistochemistry and in situ hybridization . most of the reports on use of fnac in epididymal nodules are from india , which may be due to the dominance of higher genital infections in poor patients and high prevalence of tuberculous lesions of genitourinary tract . the spectrum of lesions encountered in our study is similar to that reported earlier.[35 ] fnac has an overall sensitivity and specificity of 90% , respectively , for diagnosing chronic epididymal lesions , with a positive predictive value of 87.5% ; similar high accuracy was observed in our study . most ( 95% ) of the cases in our study were of non - neoplastic lesions , which were treated mainly non - surgically . also the cytologic diagnosis of tuberculous epididymitis prompts a search for genitourinary tuberculosis , and thus fnac helps in guiding the investigations and treatment of these patients . most commonly encountered lesion in india is tuberculous epididymitis . as seen , the typical cytology features aid recognition in most cases . in cases where necrosis predominates , it is difficult to distinguish between tuberculosis and acute suppurative inflammation . in such cases , attention to the granular caseous debris and demonstration of afb either in directly prepared smears or in those obtained from urine culture ; or response to anti - tuberculous therapy can aid in accurate recognition . in some cases , the fibrosis associated with the tuberculous inflammation leads to a misdiagnosis clinically of malignancy . fnac thus serves as a minimally invasive technique in the diagnosis of tuberculous epididymitis and epididymo - orchitis and provides adequate material for cytologic and microbiologic examination , avoiding unnecessary surgeries . the second common infection encountered in epididymis in our population appears to be microfilarial infection . the preferential site of location of the adult worms is the intra - scrotal juxtatesticular lymphatic vessels in nests along the lymphatic vessels of the epididymis , spermatic cord and para - testicular region ; and ultrasound with an fnac can serve as a cost - effective solution to rule out a filarial infection in males . a study by mitra et al . described aspirates from 4 scrotal swellings which showed numerous coiled and uncoiled sheathed microfilariae along with neutrophils , eosinophils and few lymphocytes . thus aspirates from epididymal nodules of microfilarial infection can yield microfilaria with variable amount of inflammation , and the cytopathologist should screen all aspirates from epididymal nodules with eosinophils for the presence of this parasite . after the specific infections are ruled out , sperm granulomas dominate the non - specific inflammations . the granulomatous inflammation and spermatozoids ( intra - histiocytic or as extracellular spermatic debris ) are typical for this lesion . presence of epithelioid type of histiocytes is not uncommon ; however , recognition of abundant sperms within the lesion is a good pointer to the diagnosis . additional features included lymphoid cells and lymphocytic debris ( nuclear tangles ) , rare plasma cells and eosinophils . while the prominent tumor in the epididymis is an adenomatoid tumor , some epithelial proliferations either associated with inflammation or with an adenomatous hyperplasia of the rete testis can mimic an adenomatoid tumor . as opposed to an adenomatoid tumor , the epithelial cells in the latter show lesser amounts of cytoplasm which is not vacuolated and arranged in flat sheets ; in contrast to that in adenomatoid tumor , where epithelium is abundant and arranged in branching cords and glandular patterns . the cells are round to oval , having eccentric , vesicular nuclei with a fine granular chromatin along with a pale / vacuolated cytoplasm . likewise an adenomatoid tumor will mimic a host of testicular and para - testicular neoplasms ; however , awareness of typical cytologic findings helps in accurate diagnosis . we have not come across any reference describing adenomatous hyperplasia of rete testis , but absence of typical branching glands of an adenomatoid tumor was a useful clue to rule out the latter . thus fnac serves as an important tool to screen and diagnose all palpable lesions of the epididymis and provides sufficient information for the initiation of treatment without need for an open biopsy . awareness of cytologic findings goes a long way in sub - classifying most of these lesions .

background : the incidence of non neoplastic lesions are much more common in epididymis . clinically , epididymal nodules are easily accessible to fine needle aspiration cytology ( fnac ) procedure . there are very few literature reports documenting the role of cytology in evaluation of epididymal nodules . thus , we studied patients presenting with palpable epididymis nodules in the out patient department ( opd ) from a tertiary care general hospital.aim:this study is aimed to put forth the diagnostic utility of fnac in palpable lesions of epididymis.materials and methods : a total of 40 palpable epididymal nodules were aspirated as a routine opd procedure as part of this study . smears were fixed in isopropyl alcohol and air dried . in all the cases , wet fixed papanicoloau stained and air dried giemsa stained smears were studied . zeihl neelsen stain was performed in cases which yielded caseous aspirate.results:except for two cases of adenomatoid tumor of epididymis all other lesions were nonneoplastic and included 14 cases ( 35% ) of tuberculous granulomatous inflammation , 10 ( 25% ) cystic nodules ( 9 spermatoceles and 1 encysted hydrocele ) , 5 ( 12.5% ) of nonspecific inflammations , 3 ( 7.5% ) filarial infection , 3 ( 7.5% ) sperm granulomas and 3 ( 7.5% ) adenomatous hyperplasia of rete testes . except for the two tumors , one adenomatous hyperplasia and one tuberculous lesion , no other lesion was excised . follow up and response to therapy was available in 78% patients and resolution indicated appropriateness of the diagnosisconclusions : thus , as most of the lesions in epididymis are non neoplastic responding to medical line of treatment and fnac served to aid diagnosis of non specific inflammation and avoid surgical excision in most cases .

Introduction Materials and Methods Results Cytopathological findings Tuberculous epididymitis (n = 14 cases) Non-specific epididymitis (n = 5 cases) Microfilarial infection (n = 3 cases) Sperm granulomas (n = 3 cases) Spermatoceles (n = 9 cases) Encysted hydrocele (n = 1 case) Adenomatous hyperplasia of rete testes (n = 3 cases) Adenomatoid tumor of epididymis (n = 2 cases) Discussion Conclusion

epididymis is a site for a variety of non - neoplastic and neoplastic lesions , predominantly the former . clinically most of these lesions produce worrisome palpable nodules , and fine - needle aspiration is just the procedure that seeks to confirm the non - neoplastic nature of most lesions and guide patient management strategies accordingly . we hereby report our experience with fine needle aspiration cytology ( fnac ) in the treatment of 40 patients with epididymal nodules from a general hospital , with a view to highlight the simplicity of the technique and high accuracy of diagnosis . a total of 40 patients with epididymal nodules were aspirated during a period of eight years . only in cases where patient complained of pain or the nodules were tender , the urologic surgeon accompanied the patient and gave a cord block or local anesthesia as required . in case of cystic swellings , in all the cases , wet - fixed papanicolaou - stained and air - dried giemsa - stained smears were studied . the ages of patients were in the wide range of 10 to 45 years ; however , 28 ( 70% ) patients were young adults aged 21 - 30 years . the inflammatory lesions occurred in the 21 - 30 years age group , whereas cystic nodules were common in the 31 - 40 years age group . most of the patients had painless nodules , except in 3 cases of acute non - specific epididymitis , where the lesion was tender and painful . out of the 40 cases , 38 cases were of non - neoplastic lesions , and there were 2 ( 5% ) cases of adenomatoid tumors . the cytological finding in 1 of these 2 cases of adenomatoid tumors of the epididymis reported in this study has been published earlier . non - neoplastic lesions constituted 14 ( 35% ) cases of tuberculous granulomatous inflammation ; 10 , of cystic nodules ( 9 spermatoceles and 1 encysted hydrocele ) ; 5 lesions , of non - specific inflammation ; 3 ( 7.5% ) , of filarial infection ; 3 , of sperm granulomas ; and 3 , of probable adenomatous hyperplasia of rete testes . except for the 2 adenomatoid tumors , 1 adenomatous hyperplasia and 1 tuberculous epididymitis , histopathological confirmation was not obtained ; however , accuracy of diagnosis could be judged from the clinical response to treatment , and such clinico - cytologic or histologic correlation was obtained in 78% of the non - neoplastic cases . the ziehl - neelsen staining on air - dried smears revealed acid - fast bacilli in 6 of the 12 patients [ figure 1b ] . in 5 patients , all the patients received anti - tuberculous treatment , and in 5 patients the nodules totally disappeared following completion of treatment ; while in the remaining , partial response was observed . only 1 patient underwent curative surgical excision for non - responsive disease [ figures 1c and d ] . ( b ) smear showing only caseous necrosis and inset showing acid - fast tubercle bacilli [ arrowhead ] ( ziehl neelsen stain , 1000 ) . ( d ) histopathology of the nodule showing epithelioid granulomas ( arrow ) ( h and e , 400 ) cytology in these patients revealed acute inflammatory cells in 3 cases [ figure 2a ] and chronic inflammation in the other 2 cases . ( b ) microfilarial parasite in epididymis showing adult worm identified by the hyaline sheath [ arrow ] . ( d ) histopathology of spermatic granuloma showing granulomas around the sperms [ arrow ] ( h and e , 100 ) all these patients presented with nodular swelling of the epididymis 2 - 3 cm in size . an entire adult worm was aspirated along with the hyaline sheath of the parasite in 1 patient [ figure 2b ] . sized nodules which on aspiration showed plenty of degenerating sperms amongst inflammatory cells ; foreign body type of giant cells ; and histiocytes , most of these patients had cystic swellings that yielded clear straw - colored fluid , with decrease in size following aspiration . lack of inflammation and foreign - body giant cells helped to distinguish the condition from a sperm granuloma . three of the patients , on enquiry , gave past history of surgery for inguinal hernia [ figures 3a and b ] . ( b ) high magnification of the same demonstrating the sperms ( giemsa stain , x400 ) . ( c ) encysted hydrocele : lymphocytes in the background and mesothelial cells [ arrow ] seen ( giemsa stain , 100 ) . ( d ) adenomatous hyperplasia : smears showing clusters of polygonal cells with vesicular nuclei and a moderate amount of cytoplasm along with sperm heads [ arrow ] ( giemsa stain , 400 ) this solitary patient came with cystic epididymal swelling 4 cm in size , and aspiration drew a clear fluid with plenty of lymphocytes and few mesothelial cells [ figure 3c ] . though the lesions were thought to be in epididymis , subsequent histology in 1patient confirmed the diagnosis of adenomatous hyperplasia of the rete testes and assisted us in extrapolating the results in the other 2 cases . as opposed to an aspirate from adenomatoid tumor , the nuclei of cells were smaller and the cytoplasm was less abundant . histology in 1 case revealed tubulopapillary epithelial proliferation of rete testis lined by cuboidal bland cells . the nuclear chromatin was finely dispersed ; and in the giemsa stain , the para - nuclear cytoplasm appeared pink . histopathology confirmed the cytological diagnosis of adenomatoid tumor in both cases [ figure 4d ] . adenomatoid tumor of the epididymis : ( a ) multi - layering of tumor cells ( giemsa stain , 400 ) ( b ) monolayered tumor cells , in cords ( pap stain , 100 ) ( c ) cells with optically clear cytoplasm [ arrow ] ( giemsa stain , 400 ) . ( d ) histopathology of adenomatoid tumor of the same case ( h and e , 400 ) out of the 40 cases , 38 cases were of non - neoplastic lesions , and there were 2 ( 5% ) cases of adenomatoid tumors . the cytological finding in 1 of these 2 cases of adenomatoid tumors of the epididymis reported in this study has been published earlier . non - neoplastic lesions constituted 14 ( 35% ) cases of tuberculous granulomatous inflammation ; 10 , of cystic nodules ( 9 spermatoceles and 1 encysted hydrocele ) ; 5 lesions , of non - specific inflammation ; 3 ( 7.5% ) , of filarial infection ; 3 , of sperm granulomas ; and 3 , of probable adenomatous hyperplasia of rete testes . except for the 2 adenomatoid tumors , 1 adenomatous hyperplasia and 1 tuberculous epididymitis , histopathological confirmation was not obtained ; however , accuracy of diagnosis could be judged from the clinical response to treatment , and such clinico - cytologic or histologic correlation was obtained in 78% of the non - neoplastic cases . the ziehl - neelsen staining on air - dried smears revealed acid - fast bacilli in 6 of the 12 patients [ figure 1b ] . in 5 patients , all the patients received anti - tuberculous treatment , and in 5 patients the nodules totally disappeared following completion of treatment ; while in the remaining , partial response was observed . only 1 patient underwent curative surgical excision for non - responsive disease [ figures 1c and d ] . ( b ) smear showing only caseous necrosis and inset showing acid - fast tubercle bacilli [ arrowhead ] ( ziehl neelsen stain , 1000 ) . ( d ) histopathology of the nodule showing epithelioid granulomas ( arrow ) ( h and e , 400 ) cytology in these patients revealed acute inflammatory cells in 3 cases [ figure 2a ] and chronic inflammation in the other 2 cases . ( b ) microfilarial parasite in epididymis showing adult worm identified by the hyaline sheath [ arrow ] . ( d ) histopathology of spermatic granuloma showing granulomas around the sperms [ arrow ] ( h and e , 100 ) all these patients presented with nodular swelling of the epididymis 2 - 3 cm in size . an entire adult worm was aspirated along with the hyaline sheath of the parasite in 1 patient [ figure 2b ] . while the background eosinophils were variable in all aspirates , sized nodules which on aspiration showed plenty of degenerating sperms amongst inflammatory cells ; foreign body type of giant cells ; and histiocytes , most of these patients had cystic swellings that yielded clear straw - colored fluid , with decrease in size following aspiration . lack of inflammation and foreign - body giant cells helped to distinguish the condition from a sperm granuloma . ( b ) high magnification of the same demonstrating the sperms ( giemsa stain , x400 ) . ( c ) encysted hydrocele : lymphocytes in the background and mesothelial cells [ arrow ] seen ( giemsa stain , 100 ) . ( d ) adenomatous hyperplasia : smears showing clusters of polygonal cells with vesicular nuclei and a moderate amount of cytoplasm along with sperm heads [ arrow ] ( giemsa stain , 400 ) this solitary patient came with cystic epididymal swelling 4 cm in size , and aspiration drew a clear fluid with plenty of lymphocytes and few mesothelial cells [ figure 3c ] . though the lesions were thought to be in epididymis , subsequent histology in 1patient confirmed the diagnosis of adenomatous hyperplasia of the rete testes and assisted us in extrapolating the results in the other 2 cases . as opposed to an aspirate from adenomatoid tumor , the nuclei of cells were smaller and the cytoplasm was less abundant . histology in 1 case revealed tubulopapillary epithelial proliferation of rete testis lined by cuboidal bland cells . the nuclear chromatin was finely dispersed ; and in the giemsa stain , the para - nuclear cytoplasm appeared pink . both the cases were diagnosed on cytology , and patients underwent limited excision . histopathology confirmed the cytological diagnosis of adenomatoid tumor in both cases [ figure 4d ] . adenomatoid tumor of the epididymis : ( a ) multi - layering of tumor cells ( giemsa stain , 400 ) ( b ) monolayered tumor cells , in cords ( pap stain , 100 ) ( c ) cells with optically clear cytoplasm [ arrow ] ( giemsa stain , 400 ) . ( d ) histopathology of adenomatoid tumor of the same case ( h and e , 400 ) smears revealed epithelioid granulomas with or without langhans giant cells in 6 patients , whereas caseous necrotic materials with polymorphs and degenerating granulomas were seen in 8 patients [ figure 1a ] . the ziehl - neelsen staining on air - dried smears revealed acid - fast bacilli in 6 of the 12 patients [ figure 1b ] . in 5 patients , all the patients received anti - tuberculous treatment , and in 5 patients the nodules totally disappeared following completion of treatment ; while in the remaining , partial response was observed . only 1 patient underwent curative surgical excision for non - responsive disease [ figures 1c and d ] . ( b ) smear showing only caseous necrosis and inset showing acid - fast tubercle bacilli [ arrowhead ] ( ziehl neelsen stain , 1000 ) . ( d ) histopathology of the nodule showing epithelioid granulomas ( arrow ) ( h and e , 400 ) cytology in these patients revealed acute inflammatory cells in 3 cases [ figure 2a ] and chronic inflammation in the other 2 cases . ( b ) microfilarial parasite in epididymis showing adult worm identified by the hyaline sheath [ arrow ] . ( d ) histopathology of spermatic granuloma showing granulomas around the sperms [ arrow ] ( h and e , 100 ) all these patients presented with nodular swelling of the epididymis 2 - 3 cm in size . an entire adult worm was aspirated along with the hyaline sheath of the parasite in 1 patient [ figure 2b ] . while the background eosinophils were variable in all aspirates , sized nodules which on aspiration showed plenty of degenerating sperms amongst inflammatory cells ; foreign body type of giant cells ; and histiocytes , most of these patients had cystic swellings that yielded clear straw - colored fluid , with decrease in size following aspiration . lack of inflammation and foreign - body giant cells helped to distinguish the condition from a sperm granuloma . three of the patients , on enquiry , gave past history of surgery for inguinal hernia [ figures 3a and b ] . ( c ) encysted hydrocele : lymphocytes in the background and mesothelial cells [ arrow ] seen ( giemsa stain , 100 ) . ( d ) adenomatous hyperplasia : smears showing clusters of polygonal cells with vesicular nuclei and a moderate amount of cytoplasm along with sperm heads [ arrow ] ( giemsa stain , 400 ) this solitary patient came with cystic epididymal swelling 4 cm in size , and aspiration drew a clear fluid with plenty of lymphocytes and few mesothelial cells [ figure 3c ] . though the lesions were thought to be in epididymis , subsequent histology in 1patient confirmed the diagnosis of adenomatous hyperplasia of the rete testes and assisted us in extrapolating the results in the other 2 cases . as opposed to an aspirate from adenomatoid tumor , the nuclei of cells were smaller and the cytoplasm was less abundant . histology in 1 case revealed tubulopapillary epithelial proliferation of rete testis lined by cuboidal bland cells . histopathology confirmed the cytological diagnosis of adenomatoid tumor in both cases [ figure 4d ] . adenomatoid tumor of the epididymis : ( a ) multi - layering of tumor cells ( giemsa stain , 400 ) ( b ) monolayered tumor cells , in cords ( pap stain , 100 ) ( c ) cells with optically clear cytoplasm [ arrow ] ( giemsa stain , 400 ) . ( d ) histopathology of adenomatoid tumor of the same case ( h and e , 400 ) although a simple and easy procedure , there are only few literature reports officially documenting the role of cytology in evaluation of epididymal nodules . this could be because epididymal nodules are overall uncommon in clinical practice . as seen in this study , fine - needle aspiration cytology ( fnac ) proved to be useful in the management of patients with epididymal nodules . most of the reports on use of fnac in epididymal nodules are from india , which may be due to the dominance of higher genital infections in poor patients and high prevalence of tuberculous lesions of genitourinary tract . the spectrum of lesions encountered in our study is similar to that reported earlier. most ( 95% ) of the cases in our study were of non - neoplastic lesions , which were treated mainly non - surgically . also the cytologic diagnosis of tuberculous epididymitis prompts a search for genitourinary tuberculosis , and thus fnac helps in guiding the investigations and treatment of these patients . as seen , the typical cytology features aid recognition in most cases . in cases where necrosis predominates , it is difficult to distinguish between tuberculosis and acute suppurative inflammation . in such cases , attention to the granular caseous debris and demonstration of afb either in directly prepared smears or in those obtained from urine culture ; or response to anti - tuberculous therapy can aid in accurate recognition . in some cases , the fibrosis associated with the tuberculous inflammation leads to a misdiagnosis clinically of malignancy . fnac thus serves as a minimally invasive technique in the diagnosis of tuberculous epididymitis and epididymo - orchitis and provides adequate material for cytologic and microbiologic examination , avoiding unnecessary surgeries . the second common infection encountered in epididymis in our population appears to be microfilarial infection . the preferential site of location of the adult worms is the intra - scrotal juxtatesticular lymphatic vessels in nests along the lymphatic vessels of the epididymis , spermatic cord and para - testicular region ; and ultrasound with an fnac can serve as a cost - effective solution to rule out a filarial infection in males . thus aspirates from epididymal nodules of microfilarial infection can yield microfilaria with variable amount of inflammation , and the cytopathologist should screen all aspirates from epididymal nodules with eosinophils for the presence of this parasite . after the specific infections are ruled out , sperm granulomas dominate the non - specific inflammations . the granulomatous inflammation and spermatozoids ( intra - histiocytic or as extracellular spermatic debris ) are typical for this lesion . additional features included lymphoid cells and lymphocytic debris ( nuclear tangles ) , rare plasma cells and eosinophils . while the prominent tumor in the epididymis is an adenomatoid tumor , some epithelial proliferations either associated with inflammation or with an adenomatous hyperplasia of the rete testis can mimic an adenomatoid tumor . as opposed to an adenomatoid tumor , the epithelial cells in the latter show lesser amounts of cytoplasm which is not vacuolated and arranged in flat sheets ; in contrast to that in adenomatoid tumor , where epithelium is abundant and arranged in branching cords and glandular patterns . likewise an adenomatoid tumor will mimic a host of testicular and para - testicular neoplasms ; however , awareness of typical cytologic findings helps in accurate diagnosis . we have not come across any reference describing adenomatous hyperplasia of rete testis , but absence of typical branching glands of an adenomatoid tumor was a useful clue to rule out the latter . thus fnac serves as an important tool to screen and diagnose all palpable lesions of the epididymis and provides sufficient information for the initiation of treatment without need for an open biopsy . awareness of cytologic findings goes a long way in sub - classifying most of these lesions .

african american subjects were ascertained and tested using the protocols and procedures of the gennid project , as described previously ( 10 ) , from 10 sites : birmingham , al ; little rock , ar ; chicago ; denver , co ; los angeles ( two sites ) ; baltimore , md ; raleigh - durham , nc ; charleston , sc ; and st . pedigrees were ascertained between 1993 and 2003 in three phases , all requiring two affected siblings . phase 2 included nuclear families with affected siblings , parents if available , or unaffected siblings where both parents were not available . age of diagnosis was obtained by history using a standardized questionnaire ( 10 ) . physical examination data and after removal of apparent sample discrepancies , the sample was reduced to 1,450 individuals , of whom 73 were the only members of the families and 9 were unrelated to any other pedigree member . additional samples were removed subsequently for missing diabetes status ( n = 2 ) , missing current age ( n = 1 ) , age of diagnosis reported < 15 years ( n = 36 ) , or bmi < 14 or > 55 kg / m ( n = 65 ) . relationships included 1,021 full sibpairs and are shown in supplementary table s1 , available in an online appendix at http://dx.doi.org/10.2337/db08-0931 . for the analyses reported , we included 1,082 affected individuals for sibpair analyses and variance component analysis with age of type 2 diabetes diagnosis and 1,344 individuals for analysis of bmi and type 2 diabetes . dna was prepared from lymphoblastoid cell lines by coriell cell repository ( camden , nj ) by salting out ; dna quality was checked by pico - green , and concentration was adjusted for submission to the center for inherited disease research ( cidr ) . genotyping was completed using the illumina golden gate assay to type illumina linkage panel ivb , comprising 6,008 snps , of which 5,958 were released . pedigree structures were examined for 1,413 snps selected for spacing at 2 cm using the eclipse2 program ( 11 ) . likelihoods of eight relationships ( monozygotic twins , full siblings , half siblings , parent - offspring , avuncular , grandparent - grandchild , and unrelated ) were calculated . two pairs of monozygotic twins were identified , 11 individuals were removed due to apparent sample mix - ups , and 89 families with nonpaternity or nonmaternity were identified and corrected . an additional eight pedigrees were merged based on likelihoods supporting first- or second - degree relationships between pairs of pedigrees from the same site . resulting corrections reduced the sample size to 1,450 members of 534 pedigrees , for which 4,004 genotypes ( 0.047% ) were missing . genotyping errors were identified using pedcheck ( 12 ) to remove family members with mendelian inconsistencies and merlin ( 13 ) to identify markers with excess recombination . these steps removed 6,435 genotypes ( 0.075% of the total ) . for seven markers , an additional 37 snps showed deviations from hardy - weinberg equilibrium at p < 0.005 , and 31 showed deviations with 0.005 < p < 0.01 . removal of these possibly poor - quality snps had minimal or no effect on logarithm of odds ( lod ) scores ( data not shown ) . we conducted linkage analysis using variance component likelihood analysis for the traits type 2 diabetes , type 2 diabetes age of diagnosis / onset , and bmi and nonparametric linkage ( npl ) analysis implemented in merlin ( 13 ) for type 2 diabetes . we report variance component linkage analysis using 5,914 markers and npl analyses using 5,870 markers . for variance component analysis sample size was 1,344 for type 2 diabetes and bmi and 1,082 for age of type 2 diabetes diagnosis . briefly , we modeled type 2 diabetes risk to account for age of type 2 diabetes diagnosis in affected pedigree members using a modification of the age at diagnosis regressive model described elsewhere ( 15 ) . we modeled the logit probability of type 2 diabetes on age , bmi , and sex . both age of type 2 diabetes diagnosis and bmi were modeled as a normal density with mean sd . linkage was tested using multipoint identity by descent probabilities computed in merlin ( 13 ) and by comparing the maximized likelihood with a quantitative trait locus ( qtl ) to that without the qtl . we present best findings from all three methods ; the npl score gave more conservative results . linkage disequilibrium between snps could inflate lod scores for linkage studies . to address this issue , snps were clustered using r thresholds of 0.25 , 0.50 , and 0.70 , as implemented in merlin , assuming no recombination within clusters and no linkage disequilibrium between clusters . using the three thresholds , we identified 338 snps in 147 clusters ( r > 0.7 ) , 462 snps in 202 clusters ( r > 0.5 ) , and 770 snps in 332 clusters ( r > 0.25 ) . the different thresholds had little effect on lod scores ( data not shown ) ; we present results when correcting for clusters of snps with r > 0.7 . ordered subset analysis was conducted using family - specific lod scores from the merlin output for both npl and s - pairs statistics . families were ranked from low to high and from high to low based on average - within - family values for age of type 2 diabetes diagnosis , bmi , waist circumference , waist - to - hip ratio ( whr ) , diabetes duration , and admixture . significant differences from the unranked lod score were calculated based on a minimum of 500 permutations with ranks randomly assigned . association of each marker was calculated using the regression models described above and comparing the model with the snp to one without the snp . genotypes were scored as 0/1/2 for homozygotes , heterozygotes , and rare homozygotes , thus assuming an additive model . an approximate statistic was calculated as twice the natural logarithm of the likelihood ratio with the snp to the likelihood without the snp , and a 1 degree of freedom ( d.f . ) admixture mapping was conducted on 1,450 individuals using the program admixmap ( 17 ) to estimate the proportion of european admixture for each subject . to avoid effects of linkage disequilibrium , markers were selected to optimize the frequency difference between caucasian and african ( yoruban ) hapmap samples . dna was prepared from lymphoblastoid cell lines by coriell cell repository ( camden , nj ) by salting out ; dna quality was checked by pico - green , and concentration was adjusted for submission to the center for inherited disease research ( cidr ) . genotyping was completed using the illumina golden gate assay to type illumina linkage panel ivb , comprising 6,008 snps , of which 5,958 were released . pedigree structures were examined for 1,413 snps selected for spacing at 2 cm using the eclipse2 program ( 11 ) . likelihoods of eight relationships ( monozygotic twins , full siblings , half siblings , parent - offspring , avuncular , grandparent - grandchild , and unrelated ) were calculated . two pairs of monozygotic twins were identified , 11 individuals were removed due to apparent sample mix - ups , and 89 families with nonpaternity or nonmaternity were identified and corrected . an additional eight pedigrees were merged based on likelihoods supporting first- or second - degree relationships between pairs of pedigrees from the same site . resulting corrections reduced the sample size to 1,450 members of 534 pedigrees , for which 4,004 genotypes ( 0.047% ) were missing . genotyping errors were identified using pedcheck ( 12 ) to remove family members with mendelian inconsistencies and merlin ( 13 ) to identify markers with excess recombination . these steps removed 6,435 genotypes ( 0.075% of the total ) . for seven markers , an additional 37 snps showed deviations from hardy - weinberg equilibrium at p < 0.005 , and 31 showed deviations with 0.005 < p < 0.01 . removal of these possibly poor - quality snps had minimal or no effect on logarithm of odds ( lod ) scores ( data not shown ) . we conducted linkage analysis using variance component likelihood analysis for the traits type 2 diabetes , type 2 diabetes age of diagnosis / onset , and bmi and nonparametric linkage ( npl ) analysis implemented in merlin ( 13 ) for type 2 diabetes . we report variance component linkage analysis using 5,914 markers and npl analyses using 5,870 markers . for variance component analysis sample size was 1,344 for type 2 diabetes and bmi and 1,082 for age of type 2 diabetes diagnosis . briefly , we modeled type 2 diabetes risk to account for age of type 2 diabetes diagnosis in affected pedigree members using a modification of the age at diagnosis regressive model described elsewhere ( 15 ) . we modeled the logit probability of type 2 diabetes on age , bmi , and sex . both age of type 2 diabetes diagnosis and bmi were modeled as a normal density with mean sd . linkage was tested using multipoint identity by descent probabilities computed in merlin ( 13 ) and by comparing the maximized likelihood with a quantitative trait locus ( qtl ) to that without the qtl . we present best findings from all three methods ; the npl score gave more conservative results . linkage disequilibrium between snps could inflate lod scores for linkage studies . to address this issue , snps were clustered using r thresholds of 0.25 , 0.50 , and 0.70 , as implemented in merlin , assuming no recombination within clusters and no linkage disequilibrium between clusters . using the three thresholds , we identified 338 snps in 147 clusters ( r > 0.7 ) , 462 snps in 202 clusters ( r > 0.5 ) , and 770 snps in 332 clusters ( r > 0.25 ) . the different thresholds had little effect on lod scores ( data not shown ) ; we present results when correcting for clusters of snps with r > 0.7 . ordered subset analysis was conducted using family - specific lod scores from the merlin output for both npl and s - pairs statistics . families were ranked from low to high and from high to low based on average - within - family values for age of type 2 diabetes diagnosis , bmi , waist circumference , waist - to - hip ratio ( whr ) , diabetes duration , and admixture . significant differences from the unranked lod score were calculated based on a minimum of 500 permutations with ranks randomly assigned . association of each marker was calculated using the regression models described above and comparing the model with the snp to one without the snp . genotypes were scored as 0/1/2 for homozygotes , heterozygotes , and rare homozygotes , thus assuming an additive model . an approximate statistic was calculated as twice the natural logarithm of the likelihood ratio with the snp to the likelihood without the snp , and a 1 degree of freedom ( d.f . ) admixture mapping was conducted on 1,450 individuals using the program admixmap ( 17 ) to estimate the proportion of european admixture for each subject . to avoid effects of linkage disequilibrium , markers were selected to optimize the frequency difference between caucasian and african ( yoruban ) hapmap samples . we first examined three traits , type 2 diabetes , age of type 2 diabetes diagnosis , and bmi , using variance components linkage in 5,914 markers for 1,344 members of 530 families . given the close association of bmi with type 2 diabetes , the likely role in modifying age of type 2 diabetes diagnosis , and as a surrogate for insulin resistance , our primary goal was to determine whether bmi mapped to type 2 diabetes or age of type 2 diabetes diagnosis ( diabesity loci ) . characteristics of the study population are shown in table 2 , and linkage results are shown in fig . 1 . the lod scores > 1.5 for all three traits are summarized in table 3 . type 2 diabetes was most strongly linked to chromosome 2 ( 124132 mb ) , with maxima of 4.53 at 127 cm and 2.98 at 108 cm , and this region showed suggestive linkage to age of type 2 diabetes diagnosis ( lod 1.82 at 84 cm and 1.66 at 115 cm ) . both type 2 diabetes and age of type 2 diabetes diagnosis showed suggestive linkage also to chromosome 13p ( 322 cm ; lod 2.42 for type 2 diabetes and 2.46 for age of type 2 diabetes diagnosis ) . the strongest linkage for age of type 2 diabetes diagnosis was on chromosome 18p ( lod 2.96 ; 5496 cm ) . type 2 diabetes also showed suggestive linkage to chromosome 7p ( 79 cm , 2.93 ) , a region implicated previously ( 18 ) . other suggestive type 2 diabetes peaks were on chromosome 4 ( 135 cm ; lod 2.26 ) , 11 ( 123 cm , 2.36 ) , and 16 ( 56 cm , 2.43 ) . the chromosome 18 linkage for age of type 2 diabetes diagnosis corresponded with previous reports in caucasians ( 19,20 ) . best linkages for bmi were on chromosomes 1 ( lod 2.30 , 69 cm ) , 18 ( 116 cm , at 2.45 ; telomeric to type 2 diabetes and age of type 2 diabetes diagnosis ) , and 21 ( 14 cm ) but did not overlap with type 2 diabetes or age of type 2 diabetes diagnosis linkages except on chromosome 1 ( 7090 cm and 123130 cm ; table 3 ; fig . as expected , npl analysis of type 2 diabetes gave overall smaller peaks and incomplete overlap with variance component scores . on chromosome 7 ( 27 cm ; 1.73 ) , type 2 diabetes linkage corresponded to a region identified in a preliminary report in african american gullah - speaking families ( 18 ) . other regions on chromosome 13 ( 20 cm ; lod 1.63 ) , 16 ( 51 cm ; 1.84 ) , and 22 ( rs815550 ; 1.19 ) were near variance component peaks ( fig . 1 ; supplementary table s2 , available in the online appendix ) . we performed ordered subset analysis on type 2 diabetes npl scores by ranking families ( both from low to high and high to low ) by mean age of type 2 diabetes diagnosis , bmi , waist , whr , diabetes duration , and admixture . locations with scores that increased significantly are shown in table 4 , with the most prominent regions in a subset of at least 100 families on chromosomes 1 ( 30 cm ; admixture ) , 3 ( 11 cm ; age of type 2 diabetes diagnosis ) , 7 ( 131 cm ; whr ) , and 14 ( 57 cm ; duration of diabetes ) . only the region on chromosome 1 overlapped with a variance component linkage signal ( table 4 ) . admixture mapping provides an alternative to family - based linkage to identify genes in recently admixed populations ( 21,22 ) . although our snp - based linkage map was limited for admixture mapping by the relatively small proportion of ancestrally informative markers , the large number of markers nonetheless made mapping with the admixmap program feasible . we tested autosomal markers at 0.05-cm intervals to avoid confounding by linkage disequilibrium . among the resulting 4,486 markers , the strongest association was for snp rs1565728 ( chromosome 12 , 90 cm ; p = 0.0003 ) . no other marker approached bonferroni - corrected experiment - wide significance ( supplemental data and supplementary table s3 , available in the online appendix ) , and no marker that achieved nominal uncorrected significance ( p < 0.05 ) mapped to a linkage peak . individual admixture estimates varied from 0.010 to 0.614 but with mean estimates by study site ranging from 0.069 ( south carolina gullah population ) to 0.201 ( colorado ) ( supplemental data and supplementary table s4 , available in the online appendix ) . heterogeneity of admixture proportions across sites was highly significant ( = 154.76 , p < 5 10 ; 10 df ) . admixture proportion significantly affected both type 2 diabetes risk ( p = 0.01 ) and age of type 2 diabetes diagnosis ( p = 0.003 ) but had no effect on bmi ( p = 0.95 ) . considerable progress has been made in the identification of type 2 diabetes susceptibility genes for individuals of european ancestry . nonetheless , only a small portion of the genetic risk has been explained , and of the 18 reported susceptibility genes ( 4 ) , only tcf7l2 shows any evidence for a role in african - derived populations ( 5 ) . thus , most of the susceptibility genes in high - risk ethnic groups remain to be identified . genome - wide association studies in populations of african descent may identify a subset of these genes , but a much larger number of snps will be required to overcome lower levels of linkage disequilibrium , with accompanying difficulties in separating real signals from noise in populations that are readily available . family - based linkage studies , particularly snp - based methods with higher information content , thus remain an important alternative . based on european data , linkage peaks are unlikely to represent single common variants of large effect sizes . instead , these peaks likely represent multiple rare variants of large effect , copy number polymorphisms , or multiple common variants that together provide a linkage signal . only the latter model is amenable to genome - wide association studies , but even for such variants , the additional information of linkage studies may be essential to select significant associations from the large number of nominally associated snps . previous studies of populations of african descent have been limited . as of this writing , no genome - wide association studies of african - based populations have been reported , and most european susceptibility loci do not appear to be important in african american populations ( 5 ) . among previous linkage studies , ( 8) used a microsatellite map to analyze 675 individuals in 247 families ascertained primarily for diabetic renal disease . the strongest signal from that study ( chromosome 6q , 163 cm ) is considerably telomeric to our strongest chromosome 6 peak ( lod 1.82 at 118 cm ) , whereas the second best signal from that study ( chromosome 22 , 32 cm , lod 1.33 ) is in the same location as a minor peak in our current study ( 33 cm , lod 1.68 ) . , however . a microsatellite scan in west african families ( 9 ) reported signals on chromosome 20 ( lod 1.8 ; 6078 cm ) , which overlapped with a minor peak of similar size in our study at rs1406966 ( lod 1.77 ; 62 cm ) . a second minor peak on chromosome 4 from the west africa study ( lod 1.37 , 125 cm ) also overlapped with a peak on chromosome 4 ( 135 cm ; lod 2.26 ) in our study . ( 7 ) are entirely contained in the present multicenter study , and thus , any overlap in linkage signals would not be independent . more recently , sale et al . ( 18 ) analyzed 471 individuals from the gullah - speaking african american population of south carolina for the same snp markers as the present study . chromosome 7 linkage to type 2 diabetes from that study overlaps with both npl and variance component peaks near 27 , 40 , and 78 cm reported here ( 18 ) ( m. sale , personal communication ) . because some of the gennid samples included gullah - speaking families from south carolina , we repeated our chromosome 7 analysis excluding all samples from south carolina . the maximum lod score dropped from 2.93 to 2.03 , consistent with a contribution to the total score from the south carolina samples . whereas sale et al . ( 18 ) found chromosome 7 to be most strongly implicated in early - onset type 2 diabetes , ordered subset analyses in our sample with families ranked by age of type 2 diabetes diagnosis implicated no peak in this region . in a recently published meta - analysis of 23 microsatellite linkage scans across ethnic groups that included both chicago and arkansas african american gennid families ( 23 ) , five regions of linkage overlapped with the current study : chromosomes 4 ( 135 cm ) , 6 ( 118 cm ) , 16 ( 56 cm ) , 20 ( 62 cm ) , and 22 ( 40 cm ) . despite the partial overlap in samples , the only possible overlap in linkage signals between our study and the african american subgroup from the meta - analysis was on chromosome 16 ( 3366 cm ) . other african / african american peaks from the meta - analysis ( chromosomes 4 , 6 , 10 , and 14 ) were not observed in the present study . simulation studies and comparisons of microsatellite and snp - based linkage scans have suggested that scans based on 10,000 snps provide superior information to the standard 10-cm microsatellite scans for diseases including prostate cancer ( 24 ) , rheumatoid arthritis ( 25,26 ) , and bipolar disorder ( 27 ) . each of the earlier studies used the 10,000-snp affymetrix platform , whereas we used the 5,914-snp illumina platform . we compared the information content between the illumina snp mapping panel and the earlier cidr microsatellite panel ( 8) using arkansas families that had been typed for both marker sets . the information content for all chromosomes was increased from 50 to 60% for the microsatellite scans to > 80% for the snp scan ( supplemental data and supplementary fig . this increased information content would be expected to generate a larger number of significant or suggestive linkage peaks , narrower and better defined peaks , and perhaps could explain the lack of overlap with earlier , generally smaller linkage scans in african american families . the typing of 5,914 snps at relatively even intervals allowed us to also map by admixture . in contrast to linkage , admixture mapping depends on the presence of extended haplotypes deriving from parental populations to map a disease such as type 2 diabetes that is more prevalent in the african than european populations . this approach has more power than linkage or genome scan approaches to identify genes that derive primarily or entirely from either the african or european populations when the markers also show large frequency differences between populations . admixture mapping would not be a powerful method to identify susceptibility loci that are common and of similar frequency in both ethnic groups ( 22 ) , and power would be reduced if marker frequencies are similar between parental populations . admixture mapping has recently been used successfully in mapping hypertension susceptibility genes ( 28,29 ) . the marker set in the current study was not specifically designed for admixture mapping , but the amount of data generated was sufficient to easily identify admixture . we found a strong negative correlation between european admixture and both type 2 diabetes risk and age of type 2 diabetes diagnosis . furthermore , our data easily demonstrated significant heterogeneity in the amount of admixture across centers . nonetheless , < 10% of the markers showed a minor allele frequency difference between yoruban and caucasian hapmap samples exceeding 0.5 . our strongest signal was at rs1565728 on chromosome 12 ( 90 cm ) . when we examined different combinations of markers in this region , we obtained p values from 0.00003 to 0.03 , suggesting that no single marker was responsible for the signal . the only gene in the immediate vicinity of this marker is the transcription factor , e2f7 , which is involved in cell cycle regulation but is expressed at low levels in most tissues ( 30 ) . however , admixture and linkage mapping peaks did not overlap , and no admixture signal achieved genome - wide significance ( supplementary table s2 ) . chromosome 1 marker rs1007460 , the second strongest admixture signal ( supplementary table s2 ) , was close to a well - replicated linkage signal on chromosome 1q21-q24 ( 31 ) . these findings will require follow - up with markers specifically selected for large differences in allele frequency between ancestral populations . we tested for family - based association with type 2 diabetes , bmi , and age of type 2 diabetes diagnosis , although recognizing the limitations of the relatively low - density snp linkage panel . no snp attained bonferroni - corrected significance ( supplementary table s5 , available in the online appendix ) ; but rs741923 on chromosome 19 ( p = 0.0001 ) was suggestive for association with type 2 diabetes ; and snps rs1459085 ( chromosome 5 ; p < 0.0002 ) , rs729958 ( chromosome 9 , p = 0.00005 ) , and rs1941487 ( chromosome 18 , p = 0.0002 ) were suggestive for association with diabetes age of type 2 diabetes diagnosis . snps on chromosomes 2 ( rs838715 , p = 0.0001 ) and 8 ( rs1483457 , p = 0.0001 ) showed suggestive associations with bmi . snps rs1459085 ( chromosome 5 ) and rs729958 ( chromosome 9 ) were associated with both type 2 diabetes and age of type 2 diabetes diagnosis . although association and linkage signals generally did not overlap , rs1051783 ( type 2 diabetes , p < 0.002 ) was within the chromosome 2q linkage peak and near gene mfsd9 , which is not an obvious candidate . several snps on chromosome 6q21 ( 113118 cm ) were modestly associated with type 2 diabetes ( p < 0.002 ) in a region of modest linkage ( lod 1.54 ) . among the known genes in this region are peptidylprolyl isomerase like 6 and zinc finger protein zbtb24 ; neither is an obvious diabetes candidate gene . snp rs2491222 ( p < 0.0005 with type 2 diabetes ) was under the chromosome 13 linkage peak for type 2 diabetes and age of type 2 diabetes diagnosis and within flt3 , which encodes a tyrosine kinase with known roles in hematopoiesis and leukemias . snp rs11901 ( chromosome 16 ) showed linkage to type 2 diabetes ( lod 1.76 ) and association with type 2 diabetes and age of type 2 diabetes diagnosis ( p < 0.002 ) and is upstream of the serine / threonine kinase , taok2 , which acts in the jun nh2-terminal kinase mitogen - activated protein ( map ) kinase pathway by activating upstream map kinases ( mkk ) mkk3 and mkk6 . 1 ) : chromosome 2 extending from 41 to 121 mb and encompassing 354 genes ( 498 unique transcripts ) , chromosome 13 from 18 to 31 mb and encompassing 73 genes ( 89 unique transcripts ) , and chromosome 18 from 27 to 64 mb and encompassing 130 genes ( 178 unique transcripts ) . within these 557 genes are many strong candidates for type 2 diabetes . on chromosome 2 , candidates include hexokinase 2 , transcription factor tcf7l1 , vesicular transport proteins vamp5 and vamp8 , liver fatty acid - binding protein fabp1 , dual - specificity phosphatase dusp2 , diabetes - related ankyrin repeat protein ankrd23 , and potential insulin signaling molecules map4k4 and inpp4a , as well as insig2 and steap3 . on chromosome 13 , the chromosome 18 peak included another tnf superfamily member , tnfrsf11a , as well as mc4r , tcf4 , phosphoinositide-3-kinase , class 3 , and phosphatidylinositol glycan anchor biosynthesis . only ipf1 was previously evaluated in african americans ( 32,33 ) but was not found to be associated with type 2 diabetes . in addition to the limitations noted above , our analysis of age of diagnosis was based on historical data . even when obtained by standardized questionnaire , historical age of type 2 diabetes diagnosis is inexact and likely not an accurate reflection of the true age of onset . nonetheless , this trait showed 60% heritability with similar values across centers and good correspondence with diabetes as a dichotomous trait . in summary , using a large multicenter collection of nuclear families , a snp - based linkage panel , and analytical methods based on variance component , admixture mapping , and ordered subsets , we have identified multiple regions of interest . phenotypes of type 2 diabetes and diabetes age of diagnosis frequently mapped to the same locations , lending additional confidence to these results . based on current understandings of complex disease pathogenesis , the majority of these regions probably represent collections of causative variants , rather than single snps or genes that can explain the linkage signal . dense maps suitable for narrowing linkage signals , further exploring admixture mapping , and testing family - based association using maps that tag all variants in known genes will be logical steps . with most genetic risk factors yet to be discovered , these studies will undoubtedly complement other genome - wide association efforts in identifying those genes that contribute to diabetes in african americans and other populations .

objective we used a single nucleotide polymorphism ( snp ) map in a large cohort of 580 african american families to identify regions linked to type 2 diabetes , age of type 2 diabetes diagnosis , and bmi.research design and methods after removing outliers and problematic samples , we conducted linkage analysis using 5,914 snps in 1,344 individuals from 530 families . linkage analysis was conducted using variance components for type 2 diabetes , age of type 2 diabetes diagnosis , and bmi and nonparametric linkage analyses . ordered subset analyses were conducted ranking on age of type 2 diabetes diagnosis , bmi , waist circumference , waist - to - hip ratio , and amount of european admixture . admixture mapping was conducted using 4,486 markers not in linkage disequilibrium.resultsthe strongest signal for type 2 diabetes ( logarithm of odds [ lod ] 4.53 ) was a broad peak on chromosome 2 , with weaker linkage to age of type 2 diabetes diagnosis ( lod 1.82 ) . type 2 diabetes and age of type 2 diabetes diagnosis were linked to chromosome 13p ( 322 cm ; lod 2.42 and 2.46 , respectively ) . age of type 2 diabetes diagnosis was linked to 18p ( 66 cm ; lod 2.96 ) . we replicated previous reports on chromosome 7p ( 79 cm ; lod 2.93 ) . ordered subset analysis did not overlap with linkage of unselected families . the best admixture score was on chromosome 12 ( 90 cm ; p = 0.0003).conclusions the linkage regions on chromosomes 7 ( 2778 cm ) and 18p overlap prior reports , whereas regions on 2p and 13p linkage are novel . among potential candidate genes implicated are tcf7l1 , vamp5 , vamp8 , cdk8 , insig2 , ipf1 , pax8 , il18r1 , members of the il1 and il1 receptor families , and map4k4 . these studies provide a complementary approach to genome - wide association scans to identify causative genes for african american diabetes .

RESEARCH DESIGN AND METHODS DNA preparation and genotyping. Quality control and error corrections. Statistical analysis. RESULTS DISCUSSION Supplementary Material

african american subjects were ascertained and tested using the protocols and procedures of the gennid project , as described previously ( 10 ) , from 10 sites : birmingham , al ; little rock , ar ; chicago ; denver , co ; los angeles ( two sites ) ; baltimore , md ; raleigh - durham , nc ; charleston , sc ; and st . age of diagnosis was obtained by history using a standardized questionnaire ( 10 ) . physical examination data and after removal of apparent sample discrepancies , the sample was reduced to 1,450 individuals , of whom 73 were the only members of the families and 9 were unrelated to any other pedigree member . additional samples were removed subsequently for missing diabetes status ( n = 2 ) , missing current age ( n = 1 ) , age of diagnosis reported < 15 years ( n = 36 ) , or bmi < 14 or > 55 kg / m ( n = 65 ) . for the analyses reported , we included 1,082 affected individuals for sibpair analyses and variance component analysis with age of type 2 diabetes diagnosis and 1,344 individuals for analysis of bmi and type 2 diabetes . genotyping was completed using the illumina golden gate assay to type illumina linkage panel ivb , comprising 6,008 snps , of which 5,958 were released . resulting corrections reduced the sample size to 1,450 members of 534 pedigrees , for which 4,004 genotypes ( 0.047% ) were missing . removal of these possibly poor - quality snps had minimal or no effect on logarithm of odds ( lod ) scores ( data not shown ) . we conducted linkage analysis using variance component likelihood analysis for the traits type 2 diabetes , type 2 diabetes age of diagnosis / onset , and bmi and nonparametric linkage ( npl ) analysis implemented in merlin ( 13 ) for type 2 diabetes . we report variance component linkage analysis using 5,914 markers and npl analyses using 5,870 markers . for variance component analysis sample size was 1,344 for type 2 diabetes and bmi and 1,082 for age of type 2 diabetes diagnosis . briefly , we modeled type 2 diabetes risk to account for age of type 2 diabetes diagnosis in affected pedigree members using a modification of the age at diagnosis regressive model described elsewhere ( 15 ) . we modeled the logit probability of type 2 diabetes on age , bmi , and sex . both age of type 2 diabetes diagnosis and bmi were modeled as a normal density with mean sd . to address this issue , snps were clustered using r thresholds of 0.25 , 0.50 , and 0.70 , as implemented in merlin , assuming no recombination within clusters and no linkage disequilibrium between clusters . using the three thresholds , we identified 338 snps in 147 clusters ( r > 0.7 ) , 462 snps in 202 clusters ( r > 0.5 ) , and 770 snps in 332 clusters ( r > 0.25 ) . ordered subset analysis was conducted using family - specific lod scores from the merlin output for both npl and s - pairs statistics . families were ranked from low to high and from high to low based on average - within - family values for age of type 2 diabetes diagnosis , bmi , waist circumference , waist - to - hip ratio ( whr ) , diabetes duration , and admixture . an approximate statistic was calculated as twice the natural logarithm of the likelihood ratio with the snp to the likelihood without the snp , and a 1 degree of freedom ( d.f . ) admixture mapping was conducted on 1,450 individuals using the program admixmap ( 17 ) to estimate the proportion of european admixture for each subject . two pairs of monozygotic twins were identified , 11 individuals were removed due to apparent sample mix - ups , and 89 families with nonpaternity or nonmaternity were identified and corrected . these steps removed 6,435 genotypes ( 0.075% of the total ) . for seven markers , an additional 37 snps showed deviations from hardy - weinberg equilibrium at p < 0.005 , and 31 showed deviations with 0.005 < p < 0.01 . removal of these possibly poor - quality snps had minimal or no effect on logarithm of odds ( lod ) scores ( data not shown ) . we conducted linkage analysis using variance component likelihood analysis for the traits type 2 diabetes , type 2 diabetes age of diagnosis / onset , and bmi and nonparametric linkage ( npl ) analysis implemented in merlin ( 13 ) for type 2 diabetes . we report variance component linkage analysis using 5,914 markers and npl analyses using 5,870 markers . for variance component analysis sample size was 1,344 for type 2 diabetes and bmi and 1,082 for age of type 2 diabetes diagnosis . briefly , we modeled type 2 diabetes risk to account for age of type 2 diabetes diagnosis in affected pedigree members using a modification of the age at diagnosis regressive model described elsewhere ( 15 ) . we modeled the logit probability of type 2 diabetes on age , bmi , and sex . both age of type 2 diabetes diagnosis and bmi were modeled as a normal density with mean sd . to address this issue , snps were clustered using r thresholds of 0.25 , 0.50 , and 0.70 , as implemented in merlin , assuming no recombination within clusters and no linkage disequilibrium between clusters . using the three thresholds , we identified 338 snps in 147 clusters ( r > 0.7 ) , 462 snps in 202 clusters ( r > 0.5 ) , and 770 snps in 332 clusters ( r > 0.25 ) . ordered subset analysis was conducted using family - specific lod scores from the merlin output for both npl and s - pairs statistics . families were ranked from low to high and from high to low based on average - within - family values for age of type 2 diabetes diagnosis , bmi , waist circumference , waist - to - hip ratio ( whr ) , diabetes duration , and admixture . an approximate statistic was calculated as twice the natural logarithm of the likelihood ratio with the snp to the likelihood without the snp , and a 1 degree of freedom ( d.f . ) admixture mapping was conducted on 1,450 individuals using the program admixmap ( 17 ) to estimate the proportion of european admixture for each subject . we first examined three traits , type 2 diabetes , age of type 2 diabetes diagnosis , and bmi , using variance components linkage in 5,914 markers for 1,344 members of 530 families . given the close association of bmi with type 2 diabetes , the likely role in modifying age of type 2 diabetes diagnosis , and as a surrogate for insulin resistance , our primary goal was to determine whether bmi mapped to type 2 diabetes or age of type 2 diabetes diagnosis ( diabesity loci ) . characteristics of the study population are shown in table 2 , and linkage results are shown in fig . type 2 diabetes was most strongly linked to chromosome 2 ( 124132 mb ) , with maxima of 4.53 at 127 cm and 2.98 at 108 cm , and this region showed suggestive linkage to age of type 2 diabetes diagnosis ( lod 1.82 at 84 cm and 1.66 at 115 cm ) . both type 2 diabetes and age of type 2 diabetes diagnosis showed suggestive linkage also to chromosome 13p ( 322 cm ; lod 2.42 for type 2 diabetes and 2.46 for age of type 2 diabetes diagnosis ) . the strongest linkage for age of type 2 diabetes diagnosis was on chromosome 18p ( lod 2.96 ; 5496 cm ) . type 2 diabetes also showed suggestive linkage to chromosome 7p ( 79 cm , 2.93 ) , a region implicated previously ( 18 ) . other suggestive type 2 diabetes peaks were on chromosome 4 ( 135 cm ; lod 2.26 ) , 11 ( 123 cm , 2.36 ) , and 16 ( 56 cm , 2.43 ) . the chromosome 18 linkage for age of type 2 diabetes diagnosis corresponded with previous reports in caucasians ( 19,20 ) . best linkages for bmi were on chromosomes 1 ( lod 2.30 , 69 cm ) , 18 ( 116 cm , at 2.45 ; telomeric to type 2 diabetes and age of type 2 diabetes diagnosis ) , and 21 ( 14 cm ) but did not overlap with type 2 diabetes or age of type 2 diabetes diagnosis linkages except on chromosome 1 ( 7090 cm and 123130 cm ; table 3 ; fig . as expected , npl analysis of type 2 diabetes gave overall smaller peaks and incomplete overlap with variance component scores . on chromosome 7 ( 27 cm ; 1.73 ) , type 2 diabetes linkage corresponded to a region identified in a preliminary report in african american gullah - speaking families ( 18 ) . other regions on chromosome 13 ( 20 cm ; lod 1.63 ) , 16 ( 51 cm ; 1.84 ) , and 22 ( rs815550 ; 1.19 ) were near variance component peaks ( fig . we performed ordered subset analysis on type 2 diabetes npl scores by ranking families ( both from low to high and high to low ) by mean age of type 2 diabetes diagnosis , bmi , waist , whr , diabetes duration , and admixture . locations with scores that increased significantly are shown in table 4 , with the most prominent regions in a subset of at least 100 families on chromosomes 1 ( 30 cm ; admixture ) , 3 ( 11 cm ; age of type 2 diabetes diagnosis ) , 7 ( 131 cm ; whr ) , and 14 ( 57 cm ; duration of diabetes ) . admixture mapping provides an alternative to family - based linkage to identify genes in recently admixed populations ( 21,22 ) . among the resulting 4,486 markers , the strongest association was for snp rs1565728 ( chromosome 12 , 90 cm ; p = 0.0003 ) . no other marker approached bonferroni - corrected experiment - wide significance ( supplemental data and supplementary table s3 , available in the online appendix ) , and no marker that achieved nominal uncorrected significance ( p < 0.05 ) mapped to a linkage peak . admixture proportion significantly affected both type 2 diabetes risk ( p = 0.01 ) and age of type 2 diabetes diagnosis ( p = 0.003 ) but had no effect on bmi ( p = 0.95 ) . considerable progress has been made in the identification of type 2 diabetes susceptibility genes for individuals of european ancestry . nonetheless , only a small portion of the genetic risk has been explained , and of the 18 reported susceptibility genes ( 4 ) , only tcf7l2 shows any evidence for a role in african - derived populations ( 5 ) . genome - wide association studies in populations of african descent may identify a subset of these genes , but a much larger number of snps will be required to overcome lower levels of linkage disequilibrium , with accompanying difficulties in separating real signals from noise in populations that are readily available . only the latter model is amenable to genome - wide association studies , but even for such variants , the additional information of linkage studies may be essential to select significant associations from the large number of nominally associated snps . as of this writing , no genome - wide association studies of african - based populations have been reported , and most european susceptibility loci do not appear to be important in african american populations ( 5 ) . the strongest signal from that study ( chromosome 6q , 163 cm ) is considerably telomeric to our strongest chromosome 6 peak ( lod 1.82 at 118 cm ) , whereas the second best signal from that study ( chromosome 22 , 32 cm , lod 1.33 ) is in the same location as a minor peak in our current study ( 33 cm , lod 1.68 ) . a microsatellite scan in west african families ( 9 ) reported signals on chromosome 20 ( lod 1.8 ; 6078 cm ) , which overlapped with a minor peak of similar size in our study at rs1406966 ( lod 1.77 ; 62 cm ) . a second minor peak on chromosome 4 from the west africa study ( lod 1.37 , 125 cm ) also overlapped with a peak on chromosome 4 ( 135 cm ; lod 2.26 ) in our study . ( 7 ) are entirely contained in the present multicenter study , and thus , any overlap in linkage signals would not be independent . ( 18 ) analyzed 471 individuals from the gullah - speaking african american population of south carolina for the same snp markers as the present study . chromosome 7 linkage to type 2 diabetes from that study overlaps with both npl and variance component peaks near 27 , 40 , and 78 cm reported here ( 18 ) ( m. sale , personal communication ) . because some of the gennid samples included gullah - speaking families from south carolina , we repeated our chromosome 7 analysis excluding all samples from south carolina . ( 18 ) found chromosome 7 to be most strongly implicated in early - onset type 2 diabetes , ordered subset analyses in our sample with families ranked by age of type 2 diabetes diagnosis implicated no peak in this region . in a recently published meta - analysis of 23 microsatellite linkage scans across ethnic groups that included both chicago and arkansas african american gennid families ( 23 ) , five regions of linkage overlapped with the current study : chromosomes 4 ( 135 cm ) , 6 ( 118 cm ) , 16 ( 56 cm ) , 20 ( 62 cm ) , and 22 ( 40 cm ) . despite the partial overlap in samples , the only possible overlap in linkage signals between our study and the african american subgroup from the meta - analysis was on chromosome 16 ( 3366 cm ) . other african / african american peaks from the meta - analysis ( chromosomes 4 , 6 , 10 , and 14 ) were not observed in the present study . each of the earlier studies used the 10,000-snp affymetrix platform , whereas we used the 5,914-snp illumina platform . this increased information content would be expected to generate a larger number of significant or suggestive linkage peaks , narrower and better defined peaks , and perhaps could explain the lack of overlap with earlier , generally smaller linkage scans in african american families . in contrast to linkage , admixture mapping depends on the presence of extended haplotypes deriving from parental populations to map a disease such as type 2 diabetes that is more prevalent in the african than european populations . admixture mapping would not be a powerful method to identify susceptibility loci that are common and of similar frequency in both ethnic groups ( 22 ) , and power would be reduced if marker frequencies are similar between parental populations . the marker set in the current study was not specifically designed for admixture mapping , but the amount of data generated was sufficient to easily identify admixture . we found a strong negative correlation between european admixture and both type 2 diabetes risk and age of type 2 diabetes diagnosis . our strongest signal was at rs1565728 on chromosome 12 ( 90 cm ) . however , admixture and linkage mapping peaks did not overlap , and no admixture signal achieved genome - wide significance ( supplementary table s2 ) . we tested for family - based association with type 2 diabetes , bmi , and age of type 2 diabetes diagnosis , although recognizing the limitations of the relatively low - density snp linkage panel . no snp attained bonferroni - corrected significance ( supplementary table s5 , available in the online appendix ) ; but rs741923 on chromosome 19 ( p = 0.0001 ) was suggestive for association with type 2 diabetes ; and snps rs1459085 ( chromosome 5 ; p < 0.0002 ) , rs729958 ( chromosome 9 , p = 0.00005 ) , and rs1941487 ( chromosome 18 , p = 0.0002 ) were suggestive for association with diabetes age of type 2 diabetes diagnosis . snps on chromosomes 2 ( rs838715 , p = 0.0001 ) and 8 ( rs1483457 , p = 0.0001 ) showed suggestive associations with bmi . snps rs1459085 ( chromosome 5 ) and rs729958 ( chromosome 9 ) were associated with both type 2 diabetes and age of type 2 diabetes diagnosis . although association and linkage signals generally did not overlap , rs1051783 ( type 2 diabetes , p < 0.002 ) was within the chromosome 2q linkage peak and near gene mfsd9 , which is not an obvious candidate . several snps on chromosome 6q21 ( 113118 cm ) were modestly associated with type 2 diabetes ( p < 0.002 ) in a region of modest linkage ( lod 1.54 ) . snp rs2491222 ( p < 0.0005 with type 2 diabetes ) was under the chromosome 13 linkage peak for type 2 diabetes and age of type 2 diabetes diagnosis and within flt3 , which encodes a tyrosine kinase with known roles in hematopoiesis and leukemias . snp rs11901 ( chromosome 16 ) showed linkage to type 2 diabetes ( lod 1.76 ) and association with type 2 diabetes and age of type 2 diabetes diagnosis ( p < 0.002 ) and is upstream of the serine / threonine kinase , taok2 , which acts in the jun nh2-terminal kinase mitogen - activated protein ( map ) kinase pathway by activating upstream map kinases ( mkk ) mkk3 and mkk6 . 1 ) : chromosome 2 extending from 41 to 121 mb and encompassing 354 genes ( 498 unique transcripts ) , chromosome 13 from 18 to 31 mb and encompassing 73 genes ( 89 unique transcripts ) , and chromosome 18 from 27 to 64 mb and encompassing 130 genes ( 178 unique transcripts ) . within these 557 genes are many strong candidates for type 2 diabetes . on chromosome 2 , candidates include hexokinase 2 , transcription factor tcf7l1 , vesicular transport proteins vamp5 and vamp8 , liver fatty acid - binding protein fabp1 , dual - specificity phosphatase dusp2 , diabetes - related ankyrin repeat protein ankrd23 , and potential insulin signaling molecules map4k4 and inpp4a , as well as insig2 and steap3 . on chromosome 13 , the chromosome 18 peak included another tnf superfamily member , tnfrsf11a , as well as mc4r , tcf4 , phosphoinositide-3-kinase , class 3 , and phosphatidylinositol glycan anchor biosynthesis . only ipf1 was previously evaluated in african americans ( 32,33 ) but was not found to be associated with type 2 diabetes . in addition to the limitations noted above , our analysis of age of diagnosis was based on historical data . even when obtained by standardized questionnaire , historical age of type 2 diabetes diagnosis is inexact and likely not an accurate reflection of the true age of onset . in summary , using a large multicenter collection of nuclear families , a snp - based linkage panel , and analytical methods based on variance component , admixture mapping , and ordered subsets , we have identified multiple regions of interest . phenotypes of type 2 diabetes and diabetes age of diagnosis frequently mapped to the same locations , lending additional confidence to these results . dense maps suitable for narrowing linkage signals , further exploring admixture mapping , and testing family - based association using maps that tag all variants in known genes will be logical steps . with most genetic risk factors yet to be discovered , these studies will undoubtedly complement other genome - wide association efforts in identifying those genes that contribute to diabetes in african americans and other populations .

oral cancer is a global health problem with considerably poor survival . despite advances in current treatment modalities , there has been minimal improvement in survival rates , emphasizing the need for novel strategies for managing this disease . head and neck cancers ( hncs ) develop in the oral cavity , the pharynx , the nasal cavity , and the larynx . most of these cancers are squamous cell carcinomas ( sccs ) that originate from the mucosal epithelium . several clinical trials have found pdt to be highly effective in the treatment of early and recurrent of hncs . patients have been treated with pdt using photofrin , hematoporphyrin derivative ( hpd ) , aminolevulinic acid ( ala ) and foscan ( temoporfin ) [ 510 ] . the use of conventional therapies does not preclude the use of pdt , and pdt does not compromise future surgical interventions or radiation therapy . originally , the aim of this study was to evaluate the photodynamic effects of phthalocyanines in oral cancer cells . however , because of the controversy regarding hela - contaminated kb cells , which have been wrongly identified as oral cancer cells , we compared hsc-3 oral cancer cells with hela cervical carcinoma cells . pdt utilizes light to activate a photosensitizing agent ( photosensitizer ) in the presence of oxygen . the exposure of the photosensitizer to light results in the formation of highly reactive oxygen species ( ros ) and free radicals , causing localized photodamage and cell death . the administration of the photosensitizer is followed by subsequent irradiation with red visible light to promote the excitation of the photosensitizer . mitochondria , lysosomes , cell membranes , and nuclei of tumor cells are considered potential targets . during light exposure , sensitizers that localize in mitochondria may induce apoptosis , while sensitizers that localize in lysosomes and cell membranes may cause necrosis . over the past few decades , liposomes have drawn considerable attention as carriers of therapeutic agents . due to their capability to incorporate hydrophilic and hydrophobic drugs , biocompatibility , low toxicity , and lack of immune system activation , active targeting of liposomes to tumor surface markers , e.g. , receptors of growth factors , transferrin , integrin , insulin and folate , has been studied extensively . the folate receptor ( fr ) , a glycosylphosphatidylinositol - anchored cell membrane protein , is upregulated in a variety of epithelial cancer cells , including ovarian , breast , kidney , lung , and colon cancers , and is rarely present in normal cells . the enhanced phototoxicity of several photosensitizers conjugated with folate or encapsulated in folate - targeted liposomes has been reported mostly in fr - positive kb cells , which were thought to be oral cancer cells , and in hela cells [ 1825 ] . in this study , we investigated the photodynamic effects of free and liposome - embedded zinc phthalocyanine ( znpc ) and aluminum phthalocyanine chloride ( alpc ) on the viability of hela cervical cancer cells and hsc-3 oral squamous cell carcinoma cells . we also examined the effect of fr - targeted liposomal znpc using highly fr - positive hela cells . we did not use kb cells , used often as a model for oral cancer cells and fr - positive cells [ 18,19,21,22,24,2729 ] . the nasopharyngeal kb cell line , originally isolated in 1955 from a human epidermoid carcinoma of the mouth , was subsequently found to be contaminated by hela cells ( cervical adenocarcinoma ) , based on isoenzyme analysis , hela marker chromosomes , and dna fingerprinting . therefore , the use of kb cells in any study that claims to represent an investigation of oral cancer phenotype cells is incorrect . we have used hela cells as an appropriate comparison with previous results obtained with kb cells . hela cervical adenocarcinoma cells were purchased from atcc ( crl-8798 , rockville , md , usa ) . hsc-3 cells , derived from squamous cell carcinoma ( scc ) of the tongue , were provided by dr . cells were incubated in tissue culture flasks ( falcon , becton dickinson labware , uk ) at 37c in a humidified atmosphere containing 5% co2 , and were passaged 1 : 6 twice a week . they were maintained in dulbecco s modified eagle s medium ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , penicillin ( 100 units / ml ) , streptomycin ( 100 g / ml ) , and l - glutamine ( 4 mm ) ( dmem/10 ) . all media , with or without phenol red , penicillin - streptomycin solution , l - glutamine , fbs , trypsin - edta ( ethylenediaminetetraacetic acid ) and phosphate buffered saline ( pbs ) , were obtained from the ucsf cell culture facility ( san francisco , ca ) . photosensitizers were solubilized in 100% dimethyl sulfoxide ( dmso ; sigma - aldrich , st . louis , mo , usa ) to a final concentration of 10 mm , and subsequently diluted in dmem ( without fbs and phenol red ) to a concentration of 50 m ( 0.5% dmso ) . the 50 m solutions were diluted in dmem to obtain the desirable concentration of photosensitizers used in the experiments . the cells were plated at a density of 1.810 cells per well , in 1 ml of dmem/10 in 48-well plates ( bd falcon , franklin lakes , nj , usa ) , and used after a 24 h incubation at 37c at approximately 80% confluence . subsequently , the cells were pre - washed twice with pbs ( 0.5 ml ) and 1 ml of medium without fbs and phenol red , containing photosensitizer at a given concentration , was added to each well except controls . the fbs - free media were used to avoid binding of photosensitizers to serum proteins . directly after light exposure , medium without fbs and phenol red was replaced with 1 ml of complete medium ( dmem/10 ) , and the cells were re - incubated for an additional 24 h at 37c . liposomes were prepared by hydration of dry lipid films in hepes - buffered saline , ph 7.4 , followed by extrusion through polycarbonate membranes . negatively charged liposomes were composed of palmitoyloleoylphosphatidylcholine ( popc ) : phosphatidylglycerol ( pg ) ( 1 : 1 ) ( avanti polar lipids , alabaster , al , usa ) . the lipids were dissolved in chloroform ( fisher scientific , fremont , ca , usa ) and the solvent was evaporated to dryness in a rotatory evaporator ( labconco , kansas city , mo , usa ) to deposit a lipid film on the walls of a test tube . to remove final traces of the solvent , the films were kept in a vacuum desiccator ( thermo scientific napco , fremont , ca , usa ) for 12 h. multilamellar vesicles were prepared by hydration of the dried lipid films under an argon atmosphere ( achieved by flushing argon over the buffer ) at a concentration of 10 mol lipid / ml of 140 mm nacl and 10 mm hepes buffer ( ph 7.4 ) , followed by vortexing for 10 min . liposomes were extruded 21 times through polycarbonate membranes of 100-nm pore diameter , using an avanti mini extruder , to achieve a uniform size distribution ( approx . the liposomes were not subjected to further purification to avoid exchange of the photosensitizers with gel filtration material . since the photosensitizers are hydrophobic and have very poor solubility in water , the hydrophobic interior of the liposome membrane provided an ideal solvent for these molecules . folate - conjugated liposomes contained 1,2-distearoyl - sn - glycero-3-phosphoethanolamine - n-[folate(polyethylene glycol)-2000 ] ( ammonium salt ) ( dspe - peg(2000 ) folate ; avanti polar lipids ) . phthalocyanine zinc salt ( znpc ) ( cas number 14320 - 04 - 8 , c32h16n8zn , mw 577.91 ) and aluminum phthalocyanine chloride ( chloro(29h,31h - phthalocyaninato)aluminum ( alpc ) ( cas number 14154 - 42 - 8 , c32h16alcln8 , mw 574.96 ) were purchased from sigma and used without further purification . liposomes were composed of popc : pg and contained in their membranes either znpc or alpc at a 5 : 5 : 0.1 molar ratio . although we did not compare liposomes of different composition , previous studies have shown that negatively charged liposomes are taken up more efficiently by cells than neutral liposomes . hela or hcs-3 cells were incubated with free or liposomal znpc or alpc , in the range 0.11 m , for 24 h at 37c . two light sources were used for irradiation , because of the differences in the photophysical properties of znpc and alpc . cells incubated with free or liposomal znpc were exposed to broadband visible light ( 350800 nm ) from a dura max light bulb ( 75w med 120v a19 cl / ll 20w ; philips electronics north america corporation , andover , ma , usa ) , at a distance of 10 cm to the plate , for 20 min at room temperature . the total spectral irradiance at the level of cells was 36 mw / cm ( light dose of 43.2 j / cm ) , as measured by an rd 0.2/2 radiometer with a td probe ( optel vision , quebec city , canada ) . these measurements indicated that the irradiance was constant over the small area occupied by the 48-well plates . infrared radiation was minimized using a 1 cm water filter between the cell plates and the light source . cells incubated with free or liposomal alpc were exposed to light ( 690 nm ) from a high - power led multi chip emitter ( 9.8v ; roithner lasertechnik , vienna , austria ) for 20 min . the light intensity at the surface of the plate was 3.0 mw / cm according to a thorlabs tm100a optical power meter ( thorlabs inc . cells treated with the photosensitizer but shielded from light were used for the evaluation of dark toxicity . cells incubated with medium alone , medium / liposomes , or medium/0.5% dmso served as controls . three independent experiments were performed in triplicate ( 3 wells ) for each condition ( dark and light toxicity of free or liposomal znpc or alpc , in the range 0.11.0 m ) , and controls . in the figures , each presented value is the mean standard deviation ( sd ) of 2 or 3 independent experiments performed in triplicate . the expression of the folate receptor 1 ( folr1 ) on the surface of hela and hsc-3 cells was examined by flow cytometry . the cells were maintained in dmem high - glucose medium ( thermofisher scientific , waltham , ma , usa ) supplemented with 10% fbs ( thermofisher ) and penicillin ( 100 units / ml)/streptomycin ( 100 g / ml ) ( ucsf cell culture ) . to evaluate the effect of folate on the folr1 expression , cells were cultured also for 1 week in dmem without folate ( ucsf cell culture ; custom made ) . after centrifugation for 3 min at 6000 rpm in a microcentrifuge ( eppendorf 5418 ; usa scientific , ocala , fl , usa ) , the cells were washed twice in pbs containing 2% fbs and 0.05% sodium azide ( sigma ) ( facs buffer ) and stained at 4c for 30 min with allophycocyanin ( apc)-conjugated anti - folr1 or the corresponding apc - conjugated isotype - matched control monoclonal antibody ( r&d systems , inc . , the cells were post - fixed with 1% paraformaldehyde in facs buffer and analyzed using a guava flow cytometer and incyte 2.7 software ( emd millipore , hayward , ca , usa ) . the number of viable cells used for the experiments was determined by the trypan blue exclusion assay . briefly , 1.0 ml of 10% ( v / v ) alamar blue dye ( life technologies , carlsbad , ca , usa ) in complete dmem/10 was added to each well . after incubation at 37c for 1.52 h , 200 l of the supernatant was assayed by measuring the absorbance at 570 nm and 600 nm in a molecular devices vmax microplate reader ( mountain view , ca , usa ) . cell viability ( as a percentage of control cells ) was calculated according to the formula [ ( a570a600 ) of test cells]100/[(a570a600 ) of control cells ] . statistical analysis was performed by the unpaired student s t - test , using statview software ( brainpower , inc . , calabasas , ca , usa ) . hela cervical adenocarcinoma cells were purchased from atcc ( crl-8798 , rockville , md , usa ) . hsc-3 cells , derived from squamous cell carcinoma ( scc ) of the tongue , were provided by dr . cells were incubated in tissue culture flasks ( falcon , becton dickinson labware , uk ) at 37c in a humidified atmosphere containing 5% co2 , and were passaged 1 : 6 twice a week . they were maintained in dulbecco s modified eagle s medium ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , penicillin ( 100 units / ml ) , streptomycin ( 100 g / ml ) , and l - glutamine ( 4 mm ) ( dmem/10 ) . all media , with or without phenol red , penicillin - streptomycin solution , l - glutamine , fbs , trypsin - edta ( ethylenediaminetetraacetic acid ) and phosphate buffered saline ( pbs ) , were obtained from the ucsf cell culture facility ( san francisco , ca ) . photosensitizers were solubilized in 100% dimethyl sulfoxide ( dmso ; sigma - aldrich , st . louis , mo , usa ) to a final concentration of 10 mm , and subsequently diluted in dmem ( without fbs and phenol red ) to a concentration of 50 m ( 0.5% dmso ) . the 50 m solutions were diluted in dmem to obtain the desirable concentration of photosensitizers used in the experiments . the cells were plated at a density of 1.810 cells per well , in 1 ml of dmem/10 in 48-well plates ( bd falcon , franklin lakes , nj , usa ) , and used after a 24 h incubation at 37c at approximately 80% confluence . subsequently , the cells were pre - washed twice with pbs ( 0.5 ml ) and 1 ml of medium without fbs and phenol red , containing photosensitizer at a given concentration , was added to each well except controls . the fbs - free media were used to avoid binding of photosensitizers to serum proteins . directly after light exposure , medium without fbs and phenol red was replaced with 1 ml of complete medium ( dmem/10 ) , and the cells were re - incubated for an additional 24 h at 37c . liposomes were prepared by hydration of dry lipid films in hepes - buffered saline , ph 7.4 , followed by extrusion through polycarbonate membranes . negatively charged liposomes were composed of palmitoyloleoylphosphatidylcholine ( popc ) : phosphatidylglycerol ( pg ) ( 1 : 1 ) ( avanti polar lipids , alabaster , al , usa ) . the lipids were dissolved in chloroform ( fisher scientific , fremont , ca , usa ) and the solvent was evaporated to dryness in a rotatory evaporator ( labconco , kansas city , mo , usa ) to deposit a lipid film on the walls of a test tube . to remove final traces of the solvent , the films were kept in a vacuum desiccator ( thermo scientific napco , fremont , ca , usa ) for 12 h. multilamellar vesicles were prepared by hydration of the dried lipid films under an argon atmosphere ( achieved by flushing argon over the buffer ) at a concentration of 10 mol lipid / ml of 140 mm nacl and 10 mm hepes buffer ( ph 7.4 ) , followed by vortexing for 10 min . liposomes were extruded 21 times through polycarbonate membranes of 100-nm pore diameter , using an avanti mini extruder , to achieve a uniform size distribution ( approx . the liposomes were not subjected to further purification to avoid exchange of the photosensitizers with gel filtration material . since the photosensitizers are hydrophobic and have very poor solubility in water , the hydrophobic interior of the liposome membrane provided an ideal solvent for these molecules . folate - conjugated liposomes contained 1,2-distearoyl - sn - glycero-3-phosphoethanolamine - n-[folate(polyethylene glycol)-2000 ] ( ammonium salt ) ( dspe - peg(2000 ) folate ; avanti polar lipids ) . phthalocyanine zinc salt ( znpc ) ( cas number 14320 - 04 - 8 , c32h16n8zn , mw 577.91 ) and aluminum phthalocyanine chloride ( chloro(29h,31h - phthalocyaninato)aluminum ( alpc ) ( cas number 14154 - 42 - 8 , c32h16alcln8 , mw 574.96 ) were purchased from sigma and used without further purification . liposomes were composed of popc : pg and contained in their membranes either znpc or alpc at a 5 : 5 : 0.1 molar ratio . although we did not compare liposomes of different composition , previous studies have shown that negatively charged liposomes are taken up more efficiently by cells than neutral liposomes . hela or hcs-3 cells were incubated with free or liposomal znpc or alpc , in the range 0.11 m , for 24 h at 37c . two light sources were used for irradiation , because of the differences in the photophysical properties of znpc and alpc . cells incubated with free or liposomal znpc were exposed to broadband visible light ( 350800 nm ) from a dura max light bulb ( 75w med 120v a19 cl / ll 20w ; philips electronics north america corporation , andover , ma , usa ) , at a distance of 10 cm to the plate , for 20 min at room temperature . the total spectral irradiance at the level of cells was 36 mw / cm ( light dose of 43.2 j / cm ) , as measured by an rd 0.2/2 radiometer with a td probe ( optel vision , quebec city , canada ) . these measurements indicated that the irradiance was constant over the small area occupied by the 48-well plates . infrared radiation was minimized using a 1 cm water filter between the cell plates and the light source . cells incubated with free or liposomal alpc were exposed to light ( 690 nm ) from a high - power led multi chip emitter ( 9.8v ; roithner lasertechnik , vienna , austria ) for 20 min . the light intensity at the surface of the plate was 3.0 mw / cm according to a thorlabs tm100a optical power meter ( thorlabs inc . cells treated with the photosensitizer but shielded from light were used for the evaluation of dark toxicity . cells incubated with medium alone , medium / liposomes , or medium/0.5% dmso served as controls . three independent experiments were performed in triplicate ( 3 wells ) for each condition ( dark and light toxicity of free or liposomal znpc or alpc , in the range 0.11.0 m ) , and controls . in the figures , each presented value is the mean standard deviation ( sd ) of 2 or 3 independent experiments performed in triplicate . the expression of the folate receptor 1 ( folr1 ) on the surface of hela and hsc-3 cells was examined by flow cytometry . the cells were maintained in dmem high - glucose medium ( thermofisher scientific , waltham , ma , usa ) supplemented with 10% fbs ( thermofisher ) and penicillin ( 100 units / ml)/streptomycin ( 100 g / ml ) ( ucsf cell culture ) . to evaluate the effect of folate on the folr1 expression , cells were cultured also for 1 week in dmem without folate ( ucsf cell culture ; custom made ) . after centrifugation for 3 min at 6000 rpm in a microcentrifuge ( eppendorf 5418 ; usa scientific , ocala , fl , usa ) , the cells were washed twice in pbs containing 2% fbs and 0.05% sodium azide ( sigma ) ( facs buffer ) and stained at 4c for 30 min with allophycocyanin ( apc)-conjugated anti - folr1 or the corresponding apc - conjugated isotype - matched control monoclonal antibody ( r&d systems , inc . , minneapolis , mn , usa ) . the cells were post - fixed with 1% paraformaldehyde in facs buffer and analyzed using a guava flow cytometer and incyte 2.7 software ( emd millipore , hayward , ca , usa ) . the number of viable cells used for the experiments was determined by the trypan blue exclusion assay . briefly , 1.0 ml of 10% ( v / v ) alamar blue dye ( life technologies , carlsbad , ca , usa ) in complete dmem/10 was added to each well . after incubation at 37c for 1.52 h , 200 l of the supernatant was assayed by measuring the absorbance at 570 nm and 600 nm in a molecular devices vmax microplate reader ( mountain view , ca , usa ) . cell viability ( as a percentage of control cells ) was calculated according to the formula [ ( a570a600 ) of test cells]100/[(a570a600 ) of control cells ] . statistical analysis was performed by the unpaired student s t - test , using statview software ( brainpower , inc . , cells treated with free or liposomal znpc and alpc , in the range 0.11.0 m , but shielded from light , were used for the evaluation of dark toxicity . the mean values of od570600 obtained for controls were considered as 100% and were used to calculate the percentage of control . the results obtained with 1.0 m free and liposomal phthalocyanines are shown in figure 1 . the treatment without exposure to light did not significantly affect the metabolic activity of hela and hsc-3 cells . thus , the effects of free and liposomal phthalocyanines ( vide infra ) were irradiation - dependent . figure 2 shows the percentage of cell viability of hela and hsc-3 cells incubated with free or liposomal znpc , in the range 0.11 m , and exposed to broadband visible light ( 350800 nm ) at a light dose of 43.2 j / cm . liposome - embedded znpc was much more effective than free znpc in reducing cell viability in both cell lines . free znpc , at 0.5 and 1 m , reduced hela cell viability to 87.013.6 and 52.72.1% , respectively , and hsc-3 cell viability to 58.19.9 and 22.12.8% , respectively . both cell lines were not sensitive to znpc - mediated pdt at a concentration of 0.1 m . liposomal znpc reduced hela cell viability to 68.08.6 and 15.19.9% , at 0.1 and 0.5 m , respectively , and hcs-3 cell viability to 7.22.0% at 0.1 m . a lethal photodynamic effect ( 100% of the cells are killed : ld100 ) of liposomal znpc was observed at 1 m in hela cells and at 0.5 and 1 m in hcs-3 cells . thus , hsc-3 cells were significantly more sensitive to the phototoxic activity of both free and liposomal znpc ( p<0.0005 ) than hela cells . figure 3 shows the percentage of cell viability of hela and hsc-3 cells incubated with free or liposomal alpc , in the range 0.11 m , and exposed to light ( 690 nm ) at a light dose of 3.6 j / cm . liposome - embedded alpc was much more effective than free alpc in reducing cell viability . at 0.1 , 0.5 , and 1 m , free alpc reduced hela cell viability to 78.29.9 , 55.111.9 , and 15.48.0% , respectively . hsc-3 cells were not sensitive to pdt mediated by free alpc at 0.1 m . at 0.5 and 1.0 m , however , hsc-3 cell viability was reduced to 76.71.5 and 56.68.6% , respectively . liposomal alpc , at 0.1 m , reduced the hela cell viability to 25.88.2% , while a lethal effect ( ld100 ) was achieved at 0.5 and 1 m . hsc-3 cells were significantly less susceptible to pdt mediated by liposomal alpc ( p<0.0005 ) than hela cells . at 0.1 , 0.5 , and 1 m , hsc-3 cells viability was reduced to 72.818.0 , 25.38.1 , and 21.30.3% , respectively . flow cytometric analysis of folate receptor 1 ( folr1 ) was performed for hela and hsc-3 cells cultured in complete dmem medium . deprivation of the cells from folate increased the percentage of cells displaying folr1 ( solid lane ) compared to cells cultured in medium containing folate . the viability of highly fr - positive hela cells treated with liposomal znpc at 0.1 m and irradiated decreased to ~70.0% ( figure 2 ) . to check whether targeting liposomal znpc to fr would enhance phototoxicity at the lower concentrations , we investigated the photodynamic activity of fr - targeted vs. non - targeted liposomal znpc in the range of 0.010.1 m . non - targeted liposomal znpc reduced hela cell viability to 89.83.0 , 88.96.4 , and 77.03.7% at 0.01 , 0.05 , and 0.1 m , respectively . however , targeting of liposomal znpc to fr was found to have no phototoxic effect on cell viability ( figure 5 ) . we did not examine the effect of targeting of liposome - embedded znpc using fr - negative hsc-3 cells . cells treated with free or liposomal znpc and alpc , in the range 0.11.0 m , but shielded from light , were used for the evaluation of dark toxicity . the mean values of od570600 obtained for controls were considered as 100% and were used to calculate the percentage of control . the results obtained with 1.0 m free and liposomal phthalocyanines are shown in figure 1 . the treatment without exposure to light did not significantly affect the metabolic activity of hela and hsc-3 cells . thus , the effects of free and liposomal phthalocyanines ( vide infra ) were irradiation - dependent . figure 2 shows the percentage of cell viability of hela and hsc-3 cells incubated with free or liposomal znpc , in the range 0.11 m , and exposed to broadband visible light ( 350800 nm ) at a light dose of 43.2 j / cm . liposome - embedded znpc was much more effective than free znpc in reducing cell viability in both cell lines . free znpc , at 0.5 and 1 m , reduced hela cell viability to 87.013.6 and 52.72.1% , respectively , and hsc-3 cell viability to 58.19.9 and 22.12.8% , respectively . both cell lines were not sensitive to znpc - mediated pdt at a concentration of 0.1 m . liposomal znpc reduced hela cell viability to 68.08.6 and 15.19.9% , at 0.1 and 0.5 m , respectively , and hcs-3 cell viability to 7.22.0% at 0.1 m . a lethal photodynamic effect ( 100% of the cells are killed : ld100 ) of liposomal znpc was observed at 1 m in hela cells and at 0.5 and 1 m in hcs-3 cells . thus , hsc-3 cells were significantly more sensitive to the phototoxic activity of both free and liposomal znpc ( p<0.0005 ) than hela cells . figure 3 shows the percentage of cell viability of hela and hsc-3 cells incubated with free or liposomal alpc , in the range 0.11 m , and exposed to light ( 690 nm ) at a light dose of 3.6 j / cm . liposome - embedded alpc was much more effective than free alpc in reducing cell viability . at 0.1 , 0.5 , and 1 m , free alpc reduced hela cell viability to 78.29.9 , 55.111.9 , and 15.48.0% , respectively . hsc-3 cells were not sensitive to pdt mediated by free alpc at 0.1 m . at 0.5 and 1.0 m , however , hsc-3 cell viability was reduced to 76.71.5 and 56.68.6% , respectively . liposomal alpc , at 0.1 m , reduced the hela cell viability to 25.88.2% , while a lethal effect ( ld100 ) was achieved at 0.5 and 1 m . hsc-3 cells were significantly less susceptible to pdt mediated by liposomal alpc ( p<0.0005 ) than hela cells . at 0.1 , 0.5 , and 1 m , hsc-3 cells viability was reduced to 72.818.0 , 25.38.1 , and 21.30.3% , respectively . flow cytometric analysis of folate receptor 1 ( folr1 ) was performed for hela and hsc-3 cells cultured in complete dmem medium . deprivation of the cells from folate increased the percentage of cells displaying folr1 ( solid lane ) compared to cells cultured in medium containing folate . in hsc-3 cells the viability of highly fr - positive hela cells treated with liposomal znpc at 0.1 m and irradiated decreased to ~70.0% ( figure 2 ) . to check whether targeting liposomal znpc to fr would enhance phototoxicity at the lower concentrations , we investigated the photodynamic activity of fr - targeted vs. non - targeted liposomal znpc in the range of 0.010.1 m . non - targeted liposomal znpc reduced hela cell viability to 89.83.0 , 88.96.4 , and 77.03.7% at 0.01 , 0.05 , and 0.1 m , respectively . however , targeting of liposomal znpc to fr was found to have no phototoxic effect on cell viability ( figure 5 ) . we did not examine the effect of targeting of liposome - embedded znpc using fr - negative hsc-3 cells . they are activated by light at longer wavelengths ( 650680 nm ) and exhibit a greater depth of tissue penetration , leading to a better pdt response . most of the phthalocyanine derivatives are , however , insoluble in water and tend to form aggregates in a hydrophilic environment . they are strongly hydrophobic and lipophilic , and are usually administered in liposomes . among the metal phthalocyanines , zn(ii ) and al(iii ) complexes ( znpc and alpc ) present the most favorable photophysical properties for application in pdt . oral squamous cell carcinoma ( oscc ) is the most frequent cancer of the head and neck region and the sixth leading cancer by incidence worldwide . despite advances in surgical , radiotherapeutic , and chemotherapeutic treatment , the 5-year survival rate of patients has not improved notably and is still about 4050% and searching for alternative treatment of these cancers is essential . in this work , we examined the phototoxicity of free or liposome - embedded znpc and alpc on the viability of hela cervical carcinoma cells and hsc-3 oscc cells . although it has been reported in 1967 that the kb cell line is a sub - line of hela cells and is not derived from oscc , kb cells have continued to be identified as being of oral cancer phenotype [ 18,21,22,24,2729 ] . we have used hela cells ( atcc ) as an appropriate comparison with previous results obtained with kb cells . as a model for oscc cells , we used hsc-3 cells derived from scc of the tongue that were used recently to evaluate the phototoxicity of novel zinc and magnesium phthalocyanine derivatives , and novel porphyrazines and their liposomal formulations . our studies showed that both free and liposomal phthalocyanines had not effect on cell viability without exposure to light and that the embedding of phthalocyanines in liposomes enhanced their phototoxicity . hela cells were more sensitive to alpc , whereas hsc-3 cells were more sensitive to znpc . only a few studies have evaluated free and liposomal phthalocyanine - mediated pdt against oral cancer cells . ketabchi et al . investigated the effect of aluminum disulfonated phthalocyanine ( als2pc ) at 25 g / ml ( 34 m ) on the viability of oscc - derived h376 cells and human hpv16-transformed epidermal keratinocytes . human dysplastic oral keratinocytes ( premalignant dok cells ) established from oral scc and standardized by the european collection ( ecacc no.94122104 ) were incubated with di- or tetra - sulfonated alpc ( als24pc ) at 24 m for 24 h and irradiated with a he - ne laser source ( =632.8 nm ) . the cell viability was reduced by 70% by apoptosis , as determined by protein microarray analysis . pdt with pyropheophorbide - a methyl ester ( mppa ) enhanced apoptosis and reduced the mitochondrial membrane potential in cne2 nasopharyngeal carcinoma cells . in kb cells , pdt mediated by 5 m alpc encapsulated in dimyristoyl phosphatidylcholine ( dmpc ) liposomes reduced cell viability by 95% , causing morphologic alterations and necrosis . in hela cells , pdt mediated by 1 m znpc encapsulated in dipalmitoyl phosphatidylcholine ( dppc ) liposomes showed a decrease in survival close to 100% . the effect of non - liposomal alpc and znpc was not reported in these studies . pdt efficacy of the symmetric derivative zn(ii)pc 1 and asymmetrically substituted znpc(ii ) 2 have been investigated in mouse mammary carcinoma emt6/p cells and hela cervical adenocarcinoma cells . zn(ii)pc 1 was photocytotoxic in emt6/p cells ( ic50 ~3.14 m ) but not in hela cells , whereas zn(ii)pc 2 was not phototoxic in emt6/p cells but was photoactive in hela cells ( ic50 ~3.13 m at 30 j / cm ) . these results are in agreement with our observations that the phototoxicity of free and liposomal znpc and alpc is dependent on cell type . a variety of epithelial cancer cells overexpress fr . the high affinity of folate for its receptor provides a novel approach for specific delivery of photosensitizers encapsulated in fr - targeted liposomes . we evaluated the expression of folr1 on the surface of hela and hsc-3 cells used in our study . our results showed that pdt mediated by fr - targeted liposomal znpc , in the range of 0.010.1 m , did not reduced the viability of fr - positive hela cells . investigated the phototoxicity of 5,10,15,20-tetraphenyl-21h,23h - porphine zinc ( zntpp ) encapsulated in non - targeted popc : dops ( 1,2-dioleoyl - sn - glycero-3-phospho - l - serine ) liposomes and fr - targeted liposomes popc / dops / fa - peg - dspe ( 1,2-distearoyl - sn - glycero-3-phosphoethanolamine - n [ folate(polyethylene glycol)-2000 ] ) . in hela cells incubated with 1 m zntpp for 24 h and irradiated with 10 j cm , non - targeted liposomes cause 655% cell death , while fr - targeted liposomes increase cell mortality to 945% . screened 10 human oscc cell lines and kb cells for folate binding protein - alfa ( fbp- ) , using the quantitative real - time polymerase chain reaction . seven cell lines were obtained from the university of michigan hnc spore ( um - scc-1 , 6 , 11 , 14a , 17b , 22b , 81b ) and 4 cell lines from atcc ( fadu , scc-25 , scc-15 , kb ) . interestingly , already in 1995 , orr et al . reported that um - scc-38 cells express limited amounts of fr antigen , which does not bind folic acid or 5-methyltetrahydrofolic acid . a tissue microarray with tumor and tumor - free tissue from 22 patients with hnscc demonstrated that scc cells do not express fr . fr was expressed in tissue samples obtained from nasopharyngeal and laryngeal carcinomas , but not in nasopharyngeal carcinoma cells . saba et al . detected the fr in 45% of primary hnscc and in 40% of corresponding lymph node metastases . these results emphasize that the kb cell line should not be regarded as a model for highly fr - positive oral cancer cells . because of the confusing reports regarding the fr expression in oral cancer cells , alternative ligands could be explored , e.g. , the epidermal growth factor receptor ( egfr ) , which is overexpressed on oral carcinomas . further studies are planned to evaluate liposomal phthalocyanine - mediated pdt against several oscc cells ( e.g. , cal27 , fadu , h357 ) and non - tumor gingival epithelial cells ( gecs ) , and the effect of targeting liposomes encapsulating photosensitizers to oral cancer cells . the present study shows that free and liposomal znpc and alpc , in the range 0.11.0 m , did not cause dark toxicity , the embedding of phthalocyanines in negatively charged popc : pg ( 1 : 1 ) liposomes enhance their phototoxicity , and that this effect is dependent on cell type . scc hsc-3 cells were more sensitive to free and liposomal znpc , while hela cervical adenocarcinoma cells were more sensitive to alpc . in hsc-3 cells , there was essentially no expression of folate receptor ( folr1 ) ; therefore , alternative ligands should be explored for targeting oscc cells . it is likely that targeted pdt will minimize the effects of the treatment on normal cells in the vicinity of the malignancy .

backgroundphotodynamic therapy ( pdt ) utilizes light to activate a photosensitizer in the presence of oxygen , and leads to local photodamage by the generation of highly reactive oxygen species ( ros ) . liposomal delivery of photosensitizers is adaptable to the treatment of cancers . we examined the phototoxicity of free or liposome - embedded phthalocyanine photosensitizers using hela cervical carcinoma and hsc-3 oral squamous cell carcinoma cells.material/methodsliposomes were composed of palmitoyloleoyphosphatidylcholine ( popc ) : phosphatidylglycerol ( pg ) , and contained either zinc phthalocyanine ( znpc ) or aluminum phthalocyanine chloride ( alpc ) . free or liposomal znpc and alpc were incubated with cells for 24 h at 37c . cells incubated with znpc were exposed to broadband visible light ( 350800 nm ; light dose 43.2 j / cm2 ) , whereas cells treated with alpc were exposed to light at 690 nm ( light dose 3.6 j / cm2 ) . the effect of folate receptor - targeted liposomal znpc was evaluated with hela cells . cytotoxicity was analyzed by the alamar blue assay.resultscell viability , expressed as a percentage of control cells , was calculated according to the formula [ ( a570a600 ) of test cells]100/[(a570a600 ) of control cells ] . the relative percentage changes then defined the phototoxic efficacy of the experimental conditions . in hela cells , 1 m free znpc and alpc , reduced cell viability to 52.72.1 and 15.48.0% , respectively . liposomal phthalocyanines , at 0.1 , 0.5 , and 1.0 m , reduced the viability to 68.08.6 , 15.19.9 and 0% ( znpc ) , and to 25.88.2 , 0 and 0% ( alpc ) , respectively . in hsc-3 cells , 1 m free znpc and alpc , reduced cell viability to 22.12.8 and 56.68.6% , respectively . with 1 m liposomal znpc and alpc , the viability was reduced to 0 and 21.30.3% , respectively.conclusionsthe embedding of phthalocyanines in liposomes enhanced their phototoxicity and this effect was dependent on cell type .

Background Material and Methods Cell culture Liposome preparation Photodynamic treatment Flow cytometric analysis Cell viability Results Dark toxicity of free and liposomal phthalocyanines Phototoxicity of free and liposome-embedded ZnPc Phototoxicity of free and liposome-embedded AlPc The expression of folate receptor 1 (FOLR1) on the surface of HeLa and HSC-3 cells Phototoxicity of liposomal ZnPc and folate-embedded liposomal ZnPc Discussion Conclusions

however , because of the controversy regarding hela - contaminated kb cells , which have been wrongly identified as oral cancer cells , we compared hsc-3 oral cancer cells with hela cervical carcinoma cells . pdt utilizes light to activate a photosensitizing agent ( photosensitizer ) in the presence of oxygen . the exposure of the photosensitizer to light results in the formation of highly reactive oxygen species ( ros ) and free radicals , causing localized photodamage and cell death . the enhanced phototoxicity of several photosensitizers conjugated with folate or encapsulated in folate - targeted liposomes has been reported mostly in fr - positive kb cells , which were thought to be oral cancer cells , and in hela cells [ 1825 ] . in this study , we investigated the photodynamic effects of free and liposome - embedded zinc phthalocyanine ( znpc ) and aluminum phthalocyanine chloride ( alpc ) on the viability of hela cervical cancer cells and hsc-3 oral squamous cell carcinoma cells . we also examined the effect of fr - targeted liposomal znpc using highly fr - positive hela cells . the nasopharyngeal kb cell line , originally isolated in 1955 from a human epidermoid carcinoma of the mouth , was subsequently found to be contaminated by hela cells ( cervical adenocarcinoma ) , based on isoenzyme analysis , hela marker chromosomes , and dna fingerprinting . hsc-3 cells , derived from squamous cell carcinoma ( scc ) of the tongue , were provided by dr . subsequently , the cells were pre - washed twice with pbs ( 0.5 ml ) and 1 ml of medium without fbs and phenol red , containing photosensitizer at a given concentration , was added to each well except controls . directly after light exposure , medium without fbs and phenol red was replaced with 1 ml of complete medium ( dmem/10 ) , and the cells were re - incubated for an additional 24 h at 37c . negatively charged liposomes were composed of palmitoyloleoylphosphatidylcholine ( popc ) : phosphatidylglycerol ( pg ) ( 1 : 1 ) ( avanti polar lipids , alabaster , al , usa ) . phthalocyanine zinc salt ( znpc ) ( cas number 14320 - 04 - 8 , c32h16n8zn , mw 577.91 ) and aluminum phthalocyanine chloride ( chloro(29h,31h - phthalocyaninato)aluminum ( alpc ) ( cas number 14154 - 42 - 8 , c32h16alcln8 , mw 574.96 ) were purchased from sigma and used without further purification . liposomes were composed of popc : pg and contained in their membranes either znpc or alpc at a 5 : 5 : 0.1 molar ratio . hela or hcs-3 cells were incubated with free or liposomal znpc or alpc , in the range 0.11 m , for 24 h at 37c . two light sources were used for irradiation , because of the differences in the photophysical properties of znpc and alpc . cells incubated with free or liposomal znpc were exposed to broadband visible light ( 350800 nm ) from a dura max light bulb ( 75w med 120v a19 cl / ll 20w ; philips electronics north america corporation , andover , ma , usa ) , at a distance of 10 cm to the plate , for 20 min at room temperature . the total spectral irradiance at the level of cells was 36 mw / cm ( light dose of 43.2 j / cm ) , as measured by an rd 0.2/2 radiometer with a td probe ( optel vision , quebec city , canada ) . cells incubated with free or liposomal alpc were exposed to light ( 690 nm ) from a high - power led multi chip emitter ( 9.8v ; roithner lasertechnik , vienna , austria ) for 20 min . three independent experiments were performed in triplicate ( 3 wells ) for each condition ( dark and light toxicity of free or liposomal znpc or alpc , in the range 0.11.0 m ) , and controls . cell viability ( as a percentage of control cells ) was calculated according to the formula [ ( a570a600 ) of test cells]100/[(a570a600 ) of control cells ] . hsc-3 cells , derived from squamous cell carcinoma ( scc ) of the tongue , were provided by dr . the cells were plated at a density of 1.810 cells per well , in 1 ml of dmem/10 in 48-well plates ( bd falcon , franklin lakes , nj , usa ) , and used after a 24 h incubation at 37c at approximately 80% confluence . directly after light exposure , medium without fbs and phenol red was replaced with 1 ml of complete medium ( dmem/10 ) , and the cells were re - incubated for an additional 24 h at 37c . negatively charged liposomes were composed of palmitoyloleoylphosphatidylcholine ( popc ) : phosphatidylglycerol ( pg ) ( 1 : 1 ) ( avanti polar lipids , alabaster , al , usa ) . phthalocyanine zinc salt ( znpc ) ( cas number 14320 - 04 - 8 , c32h16n8zn , mw 577.91 ) and aluminum phthalocyanine chloride ( chloro(29h,31h - phthalocyaninato)aluminum ( alpc ) ( cas number 14154 - 42 - 8 , c32h16alcln8 , mw 574.96 ) were purchased from sigma and used without further purification . liposomes were composed of popc : pg and contained in their membranes either znpc or alpc at a 5 : 5 : 0.1 molar ratio . hela or hcs-3 cells were incubated with free or liposomal znpc or alpc , in the range 0.11 m , for 24 h at 37c . two light sources were used for irradiation , because of the differences in the photophysical properties of znpc and alpc . cells incubated with free or liposomal znpc were exposed to broadband visible light ( 350800 nm ) from a dura max light bulb ( 75w med 120v a19 cl / ll 20w ; philips electronics north america corporation , andover , ma , usa ) , at a distance of 10 cm to the plate , for 20 min at room temperature . the total spectral irradiance at the level of cells was 36 mw / cm ( light dose of 43.2 j / cm ) , as measured by an rd 0.2/2 radiometer with a td probe ( optel vision , quebec city , canada ) . cells incubated with free or liposomal alpc were exposed to light ( 690 nm ) from a high - power led multi chip emitter ( 9.8v ; roithner lasertechnik , vienna , austria ) for 20 min . three independent experiments were performed in triplicate ( 3 wells ) for each condition ( dark and light toxicity of free or liposomal znpc or alpc , in the range 0.11.0 m ) , and controls . the expression of the folate receptor 1 ( folr1 ) on the surface of hela and hsc-3 cells was examined by flow cytometry . cell viability ( as a percentage of control cells ) was calculated according to the formula [ ( a570a600 ) of test cells]100/[(a570a600 ) of control cells ] . , cells treated with free or liposomal znpc and alpc , in the range 0.11.0 m , but shielded from light , were used for the evaluation of dark toxicity . the results obtained with 1.0 m free and liposomal phthalocyanines are shown in figure 1 . the treatment without exposure to light did not significantly affect the metabolic activity of hela and hsc-3 cells . figure 2 shows the percentage of cell viability of hela and hsc-3 cells incubated with free or liposomal znpc , in the range 0.11 m , and exposed to broadband visible light ( 350800 nm ) at a light dose of 43.2 j / cm . liposome - embedded znpc was much more effective than free znpc in reducing cell viability in both cell lines . free znpc , at 0.5 and 1 m , reduced hela cell viability to 87.013.6 and 52.72.1% , respectively , and hsc-3 cell viability to 58.19.9 and 22.12.8% , respectively . liposomal znpc reduced hela cell viability to 68.08.6 and 15.19.9% , at 0.1 and 0.5 m , respectively , and hcs-3 cell viability to 7.22.0% at 0.1 m . a lethal photodynamic effect ( 100% of the cells are killed : ld100 ) of liposomal znpc was observed at 1 m in hela cells and at 0.5 and 1 m in hcs-3 cells . thus , hsc-3 cells were significantly more sensitive to the phototoxic activity of both free and liposomal znpc ( p<0.0005 ) than hela cells . figure 3 shows the percentage of cell viability of hela and hsc-3 cells incubated with free or liposomal alpc , in the range 0.11 m , and exposed to light ( 690 nm ) at a light dose of 3.6 j / cm . at 0.1 , 0.5 , and 1 m , free alpc reduced hela cell viability to 78.29.9 , 55.111.9 , and 15.48.0% , respectively . hsc-3 cells were not sensitive to pdt mediated by free alpc at 0.1 m . at 0.5 and 1.0 m , however , hsc-3 cell viability was reduced to 76.71.5 and 56.68.6% , respectively . liposomal alpc , at 0.1 m , reduced the hela cell viability to 25.88.2% , while a lethal effect ( ld100 ) was achieved at 0.5 and 1 m . at 0.1 , 0.5 , and 1 m , hsc-3 cells viability was reduced to 72.818.0 , 25.38.1 , and 21.30.3% , respectively . flow cytometric analysis of folate receptor 1 ( folr1 ) was performed for hela and hsc-3 cells cultured in complete dmem medium . the viability of highly fr - positive hela cells treated with liposomal znpc at 0.1 m and irradiated decreased to ~70.0% ( figure 2 ) . to check whether targeting liposomal znpc to fr would enhance phototoxicity at the lower concentrations , we investigated the photodynamic activity of fr - targeted vs. non - targeted liposomal znpc in the range of 0.010.1 m . non - targeted liposomal znpc reduced hela cell viability to 89.83.0 , 88.96.4 , and 77.03.7% at 0.01 , 0.05 , and 0.1 m , respectively . we did not examine the effect of targeting of liposome - embedded znpc using fr - negative hsc-3 cells . cells treated with free or liposomal znpc and alpc , in the range 0.11.0 m , but shielded from light , were used for the evaluation of dark toxicity . the results obtained with 1.0 m free and liposomal phthalocyanines are shown in figure 1 . the treatment without exposure to light did not significantly affect the metabolic activity of hela and hsc-3 cells . figure 2 shows the percentage of cell viability of hela and hsc-3 cells incubated with free or liposomal znpc , in the range 0.11 m , and exposed to broadband visible light ( 350800 nm ) at a light dose of 43.2 j / cm . liposome - embedded znpc was much more effective than free znpc in reducing cell viability in both cell lines . free znpc , at 0.5 and 1 m , reduced hela cell viability to 87.013.6 and 52.72.1% , respectively , and hsc-3 cell viability to 58.19.9 and 22.12.8% , respectively . liposomal znpc reduced hela cell viability to 68.08.6 and 15.19.9% , at 0.1 and 0.5 m , respectively , and hcs-3 cell viability to 7.22.0% at 0.1 m . a lethal photodynamic effect ( 100% of the cells are killed : ld100 ) of liposomal znpc was observed at 1 m in hela cells and at 0.5 and 1 m in hcs-3 cells . thus , hsc-3 cells were significantly more sensitive to the phototoxic activity of both free and liposomal znpc ( p<0.0005 ) than hela cells . figure 3 shows the percentage of cell viability of hela and hsc-3 cells incubated with free or liposomal alpc , in the range 0.11 m , and exposed to light ( 690 nm ) at a light dose of 3.6 j / cm . at 0.1 , 0.5 , and 1 m , free alpc reduced hela cell viability to 78.29.9 , 55.111.9 , and 15.48.0% , respectively . hsc-3 cells were not sensitive to pdt mediated by free alpc at 0.1 m . at 0.5 and 1.0 m , however , hsc-3 cell viability was reduced to 76.71.5 and 56.68.6% , respectively . liposomal alpc , at 0.1 m , reduced the hela cell viability to 25.88.2% , while a lethal effect ( ld100 ) was achieved at 0.5 and 1 m . at 0.1 , 0.5 , and 1 m , hsc-3 cells viability was reduced to 72.818.0 , 25.38.1 , and 21.30.3% , respectively . flow cytometric analysis of folate receptor 1 ( folr1 ) was performed for hela and hsc-3 cells cultured in complete dmem medium . in hsc-3 cells the viability of highly fr - positive hela cells treated with liposomal znpc at 0.1 m and irradiated decreased to ~70.0% ( figure 2 ) . to check whether targeting liposomal znpc to fr would enhance phototoxicity at the lower concentrations , we investigated the photodynamic activity of fr - targeted vs. non - targeted liposomal znpc in the range of 0.010.1 m . non - targeted liposomal znpc reduced hela cell viability to 89.83.0 , 88.96.4 , and 77.03.7% at 0.01 , 0.05 , and 0.1 m , respectively . we did not examine the effect of targeting of liposome - embedded znpc using fr - negative hsc-3 cells . among the metal phthalocyanines , zn(ii ) and al(iii ) complexes ( znpc and alpc ) present the most favorable photophysical properties for application in pdt . oral squamous cell carcinoma ( oscc ) is the most frequent cancer of the head and neck region and the sixth leading cancer by incidence worldwide . in this work , we examined the phototoxicity of free or liposome - embedded znpc and alpc on the viability of hela cervical carcinoma cells and hsc-3 oscc cells . as a model for oscc cells , we used hsc-3 cells derived from scc of the tongue that were used recently to evaluate the phototoxicity of novel zinc and magnesium phthalocyanine derivatives , and novel porphyrazines and their liposomal formulations . our studies showed that both free and liposomal phthalocyanines had not effect on cell viability without exposure to light and that the embedding of phthalocyanines in liposomes enhanced their phototoxicity . hela cells were more sensitive to alpc , whereas hsc-3 cells were more sensitive to znpc . human dysplastic oral keratinocytes ( premalignant dok cells ) established from oral scc and standardized by the european collection ( ecacc no.94122104 ) were incubated with di- or tetra - sulfonated alpc ( als24pc ) at 24 m for 24 h and irradiated with a he - ne laser source ( =632.8 nm ) . in hela cells , pdt mediated by 1 m znpc encapsulated in dipalmitoyl phosphatidylcholine ( dppc ) liposomes showed a decrease in survival close to 100% . zn(ii)pc 1 was photocytotoxic in emt6/p cells ( ic50 ~3.14 m ) but not in hela cells , whereas zn(ii)pc 2 was not phototoxic in emt6/p cells but was photoactive in hela cells ( ic50 ~3.13 m at 30 j / cm ) . these results are in agreement with our observations that the phototoxicity of free and liposomal znpc and alpc is dependent on cell type . the high affinity of folate for its receptor provides a novel approach for specific delivery of photosensitizers encapsulated in fr - targeted liposomes . our results showed that pdt mediated by fr - targeted liposomal znpc , in the range of 0.010.1 m , did not reduced the viability of fr - positive hela cells . in hela cells incubated with 1 m zntpp for 24 h and irradiated with 10 j cm , non - targeted liposomes cause 655% cell death , while fr - targeted liposomes increase cell mortality to 945% . , cal27 , fadu , h357 ) and non - tumor gingival epithelial cells ( gecs ) , and the effect of targeting liposomes encapsulating photosensitizers to oral cancer cells . the present study shows that free and liposomal znpc and alpc , in the range 0.11.0 m , did not cause dark toxicity , the embedding of phthalocyanines in negatively charged popc : pg ( 1 : 1 ) liposomes enhance their phototoxicity , and that this effect is dependent on cell type . in hsc-3 cells , there was essentially no expression of folate receptor ( folr1 ) ; therefore , alternative ligands should be explored for targeting oscc cells .

in preliminary work generalizing our earlier synthesis of n - acylated unnatural amino acid amides 1 to procedures compatible with a wide variety of r groups , an unexpected r - dependent hydrolytic instability was encountered ( scheme 1 ) . during the trifluoroacetic acid ( tfa ) cleavage of the final rink resin - bound intermediate 2 to give 1 , varying amounts of carboxylic it was of concern that the instability of 1 ( r = h in the encountered case ) could be a more general phenomenon and potentially affect the synthesis of n - acylated natural and unnatural amino acid amides , peptides , and proteins that are targets of organic and peptide chemists . because of these wider implications , we sought to understand the origins and nature of this instability , to develop predictive tools to anticipate it , and to establish reaction and workup conditions to minimize it . amide bonds such as those in n - acylated amino acid amides 1 are generally stable to conditions that would readily cause hydrolysis of an analogous ester bond . strong acids such as tfa , with varying amounts of water or triethylsilane ( tes ) , are often used to cleave peptides from solid - phase resins without significant hydrolysis of amide bonds . the rink resin was specifically designed to permit tfa - promoted cleavage of the resin link ( such as that in 2 ) to provide peptide primary amides 1 ( r = h ) with both the internal amide(s ) and c - terminal primary amide remaining intact . in general , when amide bond cleavage does occur , it is usually under more vigorous conditions or by virtue of a unique structural feature that permits intramolecular - catalyzed hydrolysis . for example , hydrolysis of a peptide into its constituent amino acids requires 6 n hcl at 100 c , and cleavage of the n - terminal amino acid from a polypeptide via the classic edman degradation is promoted by an intramolecular reaction to form a key cyclic intermediate . several examples have appeared in the literature in which specific peptidyl amino acid sequences and n - acylated peptides undergo facile amide cleavage under mildly acidic conditions . during acid deprotection of a peptide containing consecutive pipecolic acid residues , ruzza and co - workers observed unexpected amide bond hydrolysis and provided experimental evidence supporting the intermediacy of an oxazolinium ion . this hydrolytic sensitivity of peptidyl pipecolic acid residues was exploited by zajdel , subra , and co - workers in the design of pipecolic resin linkers for solid - phase applications . the closest precedent for the observations reported in this work comes from goodman and co - workers , who observed amide hydrolysis under mild conditions in a series of n - benzoyl - n - methylaib - phe - ome analogues ( 4 ; scheme 2 ) . it was demonstrated that this instability is a function of the remote benzoyl groups on the n - terminal amino acid of this unusual dipeptide and that the rate of hydrolysis was correlated with the hammett constants of the benzoyl substituents , g. analysis of the data established the existence of a linear free - energy relationship and supported a mechanism involving the intermediacy of an oxazolinium ion that proceeded from the tertiary amide . to our knowledge , there are no reports of mild hydrolyses of the simple n - acylated primary amides or peptides represented by 1 . we were surprised , then , in our earlier published work synthesizing 1 ( scheme 3 , r = h ) from the benzophenone imine of rink mbha glycine amide 7 to observe small amounts of amide bond hydrolysis products 3 under tfa cleavage conditions . this occurred when r was naphthyl but not when it was an fmoc group . our focus in that report was to develop chemistry to incorporate unnatural side chains , r , into the -position of glycine with the acyl group , r , being used only to enable isolation and quantification of 1 ( r = h ) produced in the key alkylation step . therefore , r was limited to the fmoc group for all subsequent work quantifying the conditions and success of the alkylation chemistry , and the hydrolysis of the primary amide in the naphthyl case was mentioned only parenthetically . however , when recently seeking to develop scheme 3 further into a robust distributed drug discovery ( d3 ) lab that would permit the synthesis of large numbers of new compounds through widespread variation of both r and r , it became apparent that the amide hydrolysis observed earlier was not an anomaly but a clear function of the nature of the r group and that it would be important to understand the factors involved in this r - dependent hydrolytic instability so that its occurrence could be anticipated , predicted , and minimized . creating d3 laboratories involves developing robust , detailed procedures that enable students , in the course of their regular undergraduate organic chemistry lab , to reproducibly synthesize many new biomimetic molecules as potential drug leads for neglected diseases . we were developing a new d3 laboratory , the combinatorial synthesis of n - acylated unnatural amino acid amides , based on the published reaction sequence shown in scheme 3 . it would be compatible with a wide variety of r groups obtained from carboxylic acids rco2h . for this adaptation to be successful , it would soon become apparent that understanding and addressing our earlier , parenthetical observation of r - dependent hydrolytic instability would be critically important . this became clear when , in the course of this adaptation , two students conducted independent replicated syntheses of six new molecules , 1a f ( scheme 3 and table 1 ) . the molecules were made through combinatorial reactions carried out in a 2 3 combinatorial grid that utilized two different carboxylic acids , rco2h , in rows a and b and three different alkylating agents , rx , in columns 13 . the replicated and combinatorial nature of this study provided a concise set of compelling observations , revealing an r - dependent primary amide instability . if any one of the results for row a ( a1a3 ) of this grid had been obtained through a single , isolated experiment , then the presence of acid 3 might have been viewed as an anomaly . the fact that every reaction in row a gave significant amounts of carboxylic acids 3a c as side products , that none of the reaction mixtures in row b contained hydrolyzed material , and that these results were independently repeated by two researchers suggested the presence of a systematic effect worthy of further inquiry . in addition , the combinatorial nature of these six experiments made it possible to narrow the important variable quickly to the acylating group r ( row a vs row b rather than columns 13 ) and to narrow further the common determining variable to the nature of the para substituent on the aromatic acylating agent . on the basis of these observations , it was hypothesized that hydrolysis was a function of the electronic properties of the remote aromatic acyl group , r , and that electron - donating groups would facilitate the hydrolysis , whereas electron - withdrawing substituents would suppress it . accordingly , follow - up experiments were designed to explore this assumption systematically . to undertake a systematic investigation of the r - dependent hydrolytic instability , compounds 10a g , featuring a representative range of hammett constants at the para position of the aromatic benzoyl group , were prepared on rink amide mbha resins ( 2a g ) , cleaved , and studied in solution ( scheme 4 ) . after subjection to the original cleavage cocktail ( 95:5 tfa / water , 0.5 h , rt ) , all samples were filtered from resin so that subsequent time points ( 2 , 4 , 8 , and 24 h ) would reflect the instability of 10 solely in solution . the study was designed to follow the initial formation of hydrolysis products 11a g ( either directly from 2a g or through 10a g during the 0.5 h cleavage ) along with continuing formation of 11a g from 10a g in solution at increasing times postcleavage and postfiltration ( 2 , 4 , 8 , and 24 h ) . the results from all time points are shown in table 2 and are reported as the percent of acid 11 present . that the amount of acid increases with time of exposure to the cleavage cocktail after cleavage and filtration from the resin ( all time points after 0.5 h ) indicates that hydrolysis proceeds primarily in solution and comes directly from 10 . linear regression analysis assigns a rho value , , of 2.3 to this reaction ; see the supporting information . the data shows that electron - withdrawing substituents ( 10a d , high values ) suppress hydrolysis , whereas electron - donating substituents with the lowest hammett values ( 10e g ) are associated with the most rapid hydrolyses to acids . this is readily apparent at all time points and mirrors the results obtained from the acylated n - methylaib - phe - ome dipeptides studied by goodman and co - workers ( scheme 2 ) . plotting the percent acid present versus the hammett constant for the data obtained at 24 h exposure revealed a linear relationship , which allowed qualitative predictions for other substituents to be made ( see the supporting information ) . this study was expanded ( table 3 ) to include various alkyl groups represented by r. resin precursor 12 was prepared and cleaved to 13 , and time - dependent hydrolysis was studied . the results support those of our initial study : electron donation from r enhances production of n - acylated phenylalanines 14 . comparison of the results from 13d , 13e , 13h , and 13i illustrates how hydrolysis increases in proceeding from primary to secondary to tertiary alkyl . the rate - enhancing substituent effect of the p - methoxy group can be blocked or retained by carbonyl group insulation ( see 13b : r = 4-meo - c6h4ch2 vs 13j : r = 4-meo - c6h4 ) or conjugation ( see 13 g : r = 4-meo - c6h4ch = ch vs 13j : r = 4-meo - c6h4 ) , respectively . having demonstrated that r - dependent amide hydrolysis can occur under typical resin - cleavage conditions , various cleavage methods were investigated to identify conditions that would maximize compound yield while minimizing subsequent hydrolysis . in the earlier study of the hammett constant - dependent hydrolysis of 10 to 11 ( table 2 ) , we had already determined that if solutions of 10 were immediately stored at 20 c after the 0.5 h cleavage time at room temperature then subsequent hydrolysis to 11 was completely suppressed ( see the supporting information ) . however , for a robust and uncomplicated d3 procedure , it was necessary to develop optimized cleavage methods that would not require the use of low temperatures . the cleavage of 12k to 13k and hydrolysis to 14k ( table 4 ) was used as the test case in evaluating a series of cleavage methods . the percent yield of 13k was determined in each case by quantitative liquid chromatography calibrated with authentic 13k ( qlc , expressed as the percent yield of 13k ) , and the ratio of 13k to hydrolysis product 14k was determined by proton nmr . included in the list of methods are mixtures composed of lower percentages of tfa , shorter reaction times , and mixtures in which water was replaced by triethylsilane as a carbocation scavenger . hydrolysis was substantially reduced ( higher 13k:14k ratios ) by using cleavage cocktails consisting of 65 or 35% tfa or by elimination of water from the cocktail ( entries 3 and 4 ) . however , reducing the percentage of tfa ( method e , entries 9 and 10 ) or reducing the time of cleavage ( 30 min vs 1 or 2 h ) runs the risk of incomplete cleavage from the resin . methods : ( a ) 95:5 tfa / h2o ; ( b ) 95:5 tfa / et3sih ; ( c ) 65:30:5 tfa / ch2cl2/et3sih ; ( d ) 35:60:5 tfa / ch2cl2/h2o ; and ( e ) 35:60:5 tfa / ch2cl2/et3sih . the data indicates that reducing cleavage time to 30 min results , without exception , in lower yields of 13k . the use of 35:60:5 tfa / dcm / water ( method d , entries 7 and 8) resulted in the lowest yields , but , interestingly , when triethylsilane was substituted for water ( method e , entries 9 and 10 ) , this composition performed significantly better . the improved yields associated with triethylsilane may reflect its ability to irreversibly scavenge the benzhydryl carbocation derived from the resin . scavenging by water to give the diarylcarbinol would be reversible under the tfa conditions , potentially resulting in reattachment of the cleaved product to the resin via the primary amide or by way of friedel the combination of a 1 h cleavage , replacement of water with triethylsilane , and use of a lower percentage of tfa represents the preferred methods that are described by c and e ( entries 6 and 10 ) . our observation and analysis of the mild hydrolysis promoted by remote aromatic acyl groups r on the n - terminus of simple n - acylated amino acid amides 1 ( r = h ) prompted an additional experiment documenting its implications for n - acylated peptide instability . rink amide mbha and wang n - acylated dipeptide resins 15 and 16 , respectively , were prepared and subjected to cleavage ( 95:5 tfa / water , 0.5 h , rt , scheme 5 ) . analysis by lc / ms was performed after 0.5 , 6 , and 24 h ( table 5 ) . although negligible hydrolysis occurred in the initial 30 min exposure , by 6 h , 1015% of the resin - free mixture was composed of hydrolysis product 14j , increasing to nearly 50% by 24 h. resin still present at this time point . observations from the above studies are consistent with hydrolysis proceeding through a rate - limiting step in which a positively charged transition state is stabilized by an electron - donating r group . initial protonation at the c - terminal amide carbonyl to give 19 is followed by cyclization to the tetrahedral intermediate 20 . collapse of the tetrahedral intermediate affords an oxazolinium ion ( 21 ) of the type originally proposed by urban et al . and subsequently put forward by others . hammett substituent effects arising from r ( alkyl or aryl ) might be expected to be operative in the cyclization of 19 to 20 or in the acid - catalyzed ring opening of 21 to 3 . in the future , the substituent - dependent amide hydrolytic lability we have observed could be advantageously used . with the appropriate choice of the aryl group , a peptide temporarily acylated with arco - aa - oh ( ar = electron - rich aryl group , aa = amino acid residue ) could be converted back to the unacylated parent peptide under mild , perhaps even physiological , conditions . for example , if the peptide s physical properties require modification to improve its pharmacological profile , then a highly electron - rich ar group might permit arco - aa - oh to be temporarily attached and then removed in a prodrug strategy . this report shows that amide bonds in simple n - acylated amino acid amide or peptide derivatives can be surprisingly unstable . when an electron - rich aromatic carboxylic acid is acylated to the amino nitrogen of a simple amino acid carboxamide or the n - terminus of a peptide , room - temperature trifluoroacetic acid / water mixtures result in hydrolysis of the amide bond four bonds away from the nh - acyl group . even simple aromatic acyl derivatives , when appropriately substituted with electron - donating substituents , can promote facile remote amide cleavage . in particular , this hydrolysis has been found to be sensitive to the substituent effects of r located on a remote n - terminal aromatic acyl group . in the case of n - benzoyl amides , the relative rate of hydrolysis can be predicted from the substituent s hammett value . after 24 h at room temperature , compounds having benzoyl acyl substituents with negative values are almost completely hydrolyzed , those with values between 0 and 0.60 are moderately hydrolyzed , and those with values 0.60 show minimal amide cleavage . acyl groups derived from secondary or tertiary alkyls are electron - rich and also promote substantial hydrolysis after 6 h at room temperature . this acyl group - dependent acid - catalyzed lability occurs under acidic conditions commonly employed when cleaving products from solid - phase resins . when these products are n - acylated peptides , the hydrolysis reaction could become problematic . to address this issue , a systematic evaluation of cleavage conditions was conducted . it was found that even with hydrolysis - promoting substituents postcleavage hydrolysis can be minimized by the use of cleavage cocktails containing 65% or less tfa , replacing water with triethylsilane . it is important to note that in the current study n - capped dipeptides are also hydrolyzed under typical cleavage conditions . this suggests that unwanted peptide truncation may occur during synthesis or storage when dipeptides or longer peptides are acylated with electron - rich aromatic groups on the n - terminus . the combined ability to predict hydrolytic instability based on acyl group hammett values with the availability of cleavage conditions to minimize the predicted hydrolysis should help chemists working in this field to design appropriate synthetic strategies to n - acylated amino acid amides and peptides . fmoc - protected amino acid - bound rink amide mbha and wang resins were purchased from peptides international ( louisville , ky ) . boc - protected amino acid - bound merrifield resins were purchased from novabiochem ( emd millipore ) or polymer laboratories ( now agilent ) . combinatorial and parallel synthetic sequences and resin cleavage / hydrolysis experiments were performed in custom - made 3.5 ml glass reaction vessels containing a sintered glass frit and screw - cap ends fitted with poly(tetrafluoroethylene ) ( ptfe ) septa all purchased from chemglass ( vineland , nj ) . the reaction vessels were secured in polypropylene bill - boards consisting of a 2 3 or 4 6 array of position holes fitted with rubber o - rings and were supplied by leads metal ( indianapolis , in ) . synthetic sequences for the preparation of advanced intermediate resins were performed in 25 or 50 ml solid - phase peptide synthesis vessels ( spps ) purchased from chemglass . rotation of bill - boards was accomplished using a spit rod fitted with three 6-brackett metal assemblies custom - made by leads metal and was driven by a jenn - air rotisserie motor or using brackets to fit 2 3 or 4 6 bill - boards held in a rotovap assembly . to the resin contained in a 150 ml beaker with stirrer bar was added 60 ml of nmp followed by 30 ml of dcm . if resin showed a tendency to accumulate downward , then dcm was added until there was no overall movement upward or downward . if resin showed a tendency to rise , then nmp was added until neutral buoyancy was achieved . distribution of equal quantities of resin to multiple 3.5 ml glass reaction vessels in a bill - board was accomplished with the use of an eppendorf repeater pipet fitted with a 50 ml tip . an aliquot of 2 ml was dispensed to each vessel followed by an aliquot of 1 ml to each vessel . this procedure was repeated until the number of milliliters of isopycnic suspension remaining in the beaker was judged to be less than the number of vessels , n ( the smallest volume needed to dispense 1 ml into all vessels in a single pass ) . isopycnic solvent was added to the beaker to give a volume of ( 2n + 10 ) ml . isopycnic solvent was added to the beaker again to give a volume of ( 2n + 10 ) ml . the resin in each vessel was then washed with 4 2 ml of the solvent to be used in the next step . the dcm - washed resins , contained in 3.5 ml reaction vessels , were treated with 1.5 to 2 ml of cleavage reagent . the vessels were rotated at room temperature for the required time period ( 0.5 , 1 , or 2 h ) and were then drained into 5-dram , tooled - neck vials . each resin was then washed with 2 ml of the cleavage cocktail followed by 2 ml of dcm . aliquots of 100 l were removed and immediately evaporated to dryness under a stream of nitrogen , and the residue was dissolved in acetonitrile for lc / ms analysis . analyses were performed using an agilent eclipse xdb - c18 5 m column , 4.6 150 mm length , with 5 l injections and a flow rate of 1.0 ml / min . a linear gradient from 20% 1:1 mecn / meoh ( 5 mm nh4oac ) and 80% water ( 5 mm nh4oac ) to 100% 1:1 mecn / meoh ( 5 mm nh4oac ) over 10 min was used . percent amide and/or percent acid were calculated from the reported peak areas observed at 254 nm unless otherwise noted . a 25 ml spps vessel was charged with 1.36 g ( 0.598 mmol , 0.44 mmol / g ) of fmoc - gly - nh - rink mbha resin . the resin was washed with 3 15 ml of nmp and was then treated with 5 10 ml washes of 20% ( v / v ) piperidine in nmp delivered over a 35 min period . the deprotected resin was then washed with 3 15 ml of nmp and was treated with 1.09 g ( 6.01 mmol , 10.0 equiv ) of benzophenone imine in 9 ml of nmp followed by 0.313 g ( 5.22 mmol , 8.72 equiv ) of acetic acid in 1 ml of nmp . the vessel was rocked on an orbital shaker for 22 h and then drained , and the resin was washed with 3 10 ml each of nmp , thf , 3:1 thf / h2o , thf , and dcm to give resin 7 . the resin was transferred to a 150 ml beaker using nmp , was made into an isopycnic suspension , and was equally distributed to twelve 3.5 ml reaction vessels contained in two 2 3 bill - boards to provide 50 mol to each vessel ( see the general procedure for the preparation and distribution of isopycnic suspensions of resins ) . each bill - board was then treated identically as follows : phosphazene base tert - butylimino - tri(pyrrolidino)phosphorane ( btpp , 1.0 m , 0.50 ml , 0.50 mmol , 10 equiv ) in nmp was added to each vessel and allowed to stand for 5 min . 4-methylbenzyl bromide , ethyl iodide , and allyl bromide ( 0.50 ml of 1.0 m solutions in nmp , 0.50 mmol , 10 equiv ) were added , respectively , to column 1 vessels a1/b1 , column 2 vessels a2/b2 , and column 3 vessels a3/b3 . the bill - boards were rotated for 22 h , the vessels were drained , and the resins were washed with 3 2 ml each of nmp , dcm , and thf to give resins 8 ( scheme 3 , r = 4-methylbenzyl , ethyl , or allyl ) . to each vessel was added 2 ml of 1.0 n hcl / thf ( 1:2 ) , and the bill - boards were rotated for 20 min , the vessels were drained , and the resins were washed with 3 ml of thf , 2 2.5 ml of 0.2 m diisopropylethylamine in nmp , and 2 2.5 ml of nmp to give resins 9 ( scheme 3 , r = 4-methylbenzyl , ethyl , allyl ) . 4-methylbenzoic acid and 4-trifluoromethylbenzoic acid ( 0.25 m , 1.0 ml , 0.25 mmol , 5.0 equiv ) in 0.25 m 1-hydroxybenzotriazole ( hobt ) in nmp were added , respectively , to row a vessels a1a3 and row b vessels b1b3 . each vessel was then treated with 0.50 ml ( 0.25 mmol , 5.0 equiv ) of 0.50 m diisopropylcarbodiimide in nmp , and the bill - boards were rotated for 22 h. the vessels were drained , and the resins were washed with 2 3 ml each of nmp , thf , and 3 3 ml of dcm to afford resins 2 ( scheme 3 ) . cleavage of each resin with 95:5 tfa / water for 2 h was performed as described above ( general procedure of resin cleavage ) . cleavage of 2 gave the crude products in table 1 ( see general procedure of resin cleavage ) . analytical data for crude products 1 ( z = nh2 ) and 3 ( z = oh ) include lc / ms and diagnostic peaks in the h nmr . for 1a : h nmr ( 500.13 mhz , cdcl3 ) : 2.30 ( s ) , 2.38 ( s ) , 3.19 ( dd ) , 3.29 ( dd ) , 5.01 ( dd ) , 7.55 ( d ) ; lc / ms ( esi ) : 8.70 min , m / z 297 [ m + h ] . for 3a : h nmr ( 500.13 mhz , cdcl3 ) : 3.13 ( 2dd ) , 4.94 ( dd ) , 7.59 ( d ) ; lc / ms ( esi ) : 6.36 min , m / z 298 [ m + h ] . for 1b : h nmr ( 500.13 mhz , cdcl3 ) : 1.02 ( t ) , 1.90 ( septet ) , 2.08 ( septet ) , 4.75 ( dd ) , 7.68 ( d ) ; lc / ms ( esi ) : 6.08 min , m / z 221 [ m + h ] . for 3b : h nmr ( 500.13 mhz , cdcl3 ) : 1.83 ( septet ) , 1.99 ( septet ) , 4.68 ( dd ) ; lc / ms ( esi ) : 3.64 min , m / z 222 [ m + h ] . for 1c : h nmr ( 500.13 mhz , cdcl3 ) : 2.40 ( s ) , 2.602.78 ( m ) , 4.81 ( dd ) , 5.185.23 ( m ) , 5.745.82 ( m ) , 7.24 ( d ) , 7.65 ( d ) ; lc / ms ( esi ) : 6.66 min , m / z 233 [ m + h ] . for 3c : h nmr ( 500.13 mhz , cdcl3 ) : 4.77 ( dd ) ; lc / ms ( esi ) : 4.02 min , m / z 234 [ m + h ] . h nmr ( 500.13 mhz , cdcl3 ) : 2.32 ( s ) , 3.10 ( dd ) , 3.19 ( dd ) , 4.85 ( m ) , 7.70 ( d ) , 7.86 ( d ) ; lc / ms ( esi ) : 9.39 min , m / z 351 [ m + h ] . h nmr ( 500.13 mhz , cdcl3 ) : 1.02 ( t ) , 1.80 ( septet ) , 2.02 ( septet ) , 4.62 ( m ) , 7.72 ( d ) , 7.93 ( d ) ; lc / ms ( esi ) : 7.39 min , m / z 275 [ m + h ] . h nmr ( 500.13 mhz , cdcl3 ) : 2.64 ( m ) , 4.74 ( m ) , 5.195.23 ( m ) , 5.785.85 ( m ) , 7.71 ( d ) , 7.90 ( d ) ; lc / ms ( esi ) : 7.85 min , m / z 287 [ m + h ] . a 50 ml spps vessel was charged with 3.43 g ( 1.75 mmol , 0.51 mmol / g ) of fmoc - gly - nh - rink mbha resin . the resin was washed with 3 15 ml of nmp and was then treated with 5 20 ml washes of 20% ( v / v ) piperidine in nmp delivered over a 45 min period . the deprotected resin was then washed with 4 15 ml of nmp and was treated with 3.16 g ( 17.4 mmol , 9.94 equiv ) of benzophenone imine in 15 ml of nmp followed by 910 mg ( 15.2 mmol , 8.70 equiv ) of acetic acid . the vessel was rocked on an orbital shaker for 20 h and then drained , and the resin was washed with 3 20 ml each of nmp , thf , 3:1 thf / h2o , thf , and dcm . resin 7 was dried under a gentle stream of nitrogen gas for 2 h and then under vacuum for 3 h. resin 7 ( 1.75 mmol ) was swelled in the spps vessel with 30 ml of nmp for 30 min . the vessel was drained , and the resin was treated with 5.49 g ( 17.6 mmol , 10.0 equiv ) of phosphazene base tert - butylimino - tri(pyrrolidino)phosphorane ( btpp ) in 9.5 ml of nmp . the contents were gently agitated by hand to affect mixing , and the mixture was treated with 3.26 g ( 17.6 mmol , 10.0 equiv ) of 4-methylbenzyl bromide in 14.3 ml of nmp . the vessel was rocked overnight for 24 h and then drained , and the resin was washed with 3 20 ml each of nmp , dcm , and thf . resin 8 was dried under a slow stream of argon for 45 min and was then stored overnight . n hcl , and the vessel was rocked for 40 min and then drained . the resin was washed with 3 20 ml of thf , neutralized with 3 20 ml washes of 0.2 m diisopropylethylamine ( diea ) in nmp , and then washed with 3 20 ml of nmp to give resin 9 . resin 9 was then quantitatively transferred to a 400 ml beaker using 100 ml of nmp to facilitate the transfer . dichloromethane ( 50 ml ) was then added , and the composition was adjusted to achieve neutral buoyancy . a final volume of 200250 ml was then equally dispensed 2 ml at a time into a 5 column 7 row array of reaction vessels with uncapped bottoms held in two 4 6 bill - boards ( see general procedure for the preparation and distribution of isopycnic suspensions of resins ) . each vessel was then washed with 3 2 ml of nmp and then capped . the array of 35 vessels each containing 50.0 mol of resin 9 was treated in the following manner : 1.0 ml of a 0.25 m solution of row 1 carboxylic acid rco2h ( 250 mol , 5.0 equiv ) dissolved in 0.25 m 1-hydroxybenzotriazole ( 250 mol , 5.0 equiv , hobt ) in nmp was added to each of the five vessels in row 1 followed by 0.5 ml of a 0.50 m solution of diisopropylcarbodiimide ( 250 mol , 5.0 equiv , dic ) in nmp . the five vessels in each of the remaining six rows were treated in an identical manner with its assigned carboxylic acid , hobt , and dic . the bill - board was rotated for 2 days , and the vessels were drained and then washed with 3 1.5 ml each of nmp , thf , and dcm to give resins 2a g . each of the 35 vessels in the 5 7 array ( 50 mol each ) was treated with 1.5 ml of 95:5 tfa / h2o and was rotated at room temperature for 30 min . all vessels were then drained into collection vials , and the resins were washed with 1 ml of 95:5 tfa / h2o . a 90 l aliquot from the combined filtrates of each acylated amide 10a g in column 1 ( representing the 0.5 h exposure ) was removed and immediately evaporated to dryness under a stream of nitrogen . the remainder of each filtrate in column 1 ( 0.5 h exposure ) was then stored at 20 c and assayed by lc / ms again after 48 h. these column 1 filtrates , stored at 20 c , would be worked up for the purpose of isolating primary amides 10a g . filtrates in columns 25 were allowed to stand at room temperature for 1.5 , 3.5 , 7.5 , and 23.5 h , respectively , and were then assayed by lc / ms ( table 2 ) . g ( x = h , me , and ome ) , for the purpose of isolation and characterization , was accomplished by allowing their column 5 filtrates to stand at room temperature for 5 days . after storage at 20 c for 2 days , each solution from column 1 was added to 100 ml of cold 1.0 n sodium hydroxide . the combined extracts were dried over na2so4 and were concentrated to give crude amides 10a g as amorphous solids . yield 7.6 mg ( 49% ) , mp 235238 c ; h nmr ( 500.13 mhz , dmso - d6 ) : 2.22 ( s , 3h ) , 2.93 ( dd , j = 13.6 and 10.9 hz , 1h ) , 3.09 ( dd , j = 13.7 and 3.9 hz , 1h ) , 4.62 ( ddd , j = 10.8 , 8.5 , and 4.1 hz , 1h ) , 7.05 ( d , j = 7.8 hz , 2h ) , 7.13 ( br s , 1h ) , 7.22 ( d , j = 8.0 hz , 2h ) , 7.61 ( br s , 1h ) , 7.94 ( s , 4h ) , 8.80 ( d , j = 8.5 hz , 1h ) ; c nmr ( 125.77 mhz , dmso - d6 ) : 20.5 , 36.7 , 55.0 , 113.5 , 118.2 , 128.1 , 128.6 , 128.9 , 132.2 , 135.0 , 135.2 , 138.0 , 164.7 , 173.0 ; hrms ( esi - tof ) : m / z [ m + na ] calcd for c18h17n3o2na , 330.1219 ; found , 330.1215 . yield 4.8 mg ( 32% ) , mp 208211 c ( meoh ) ; h nmr ( 500.13 mhz , dmso - d6 ) : 2.22 ( s , 3h ) , 2.92 ( dd , j = 13.6 and 10.4 hz , 1h ) , 3.01 ( dd , j = 13.7 and 4.2 hz , 1h ) , 4.53 ( ddd , j = 10.2 , 8.5 , and 4.2 hz , 1h ) , 5.60 ( br s , 2h ) , 6.51 ( d , j = 8.6 hz , 2h ) , 7.02 ( br s , 1h ) , 7.04 ( d , j = 7.9 hz , 2h ) , 7.18 ( d , j = 8.0 hz , 2h ) , 7.41 ( br s , 1h ) , 7.53 ( d , j = 8.6 hz , 2h ) , 7.90 ( d , j = 8.4 hz , 1h ) ; c nmr ( 125.77 mhz , dmso - d6 ) : 20.5 , 36.8 , 54.5 , 112.3 , 120.6 , 128.5 , 128.8 , 128.9 , 134.8 , 135.5 , 151.6 , 165.9 , 173.7 ; hrms ( esi - tof ) m / z : [ m + na ] calcd for c17h19n3o2na , 320.1375 ; found , 320.1386 . yield 4.1 mg ( 23% ) , mp 220224 c ; h nmr ( 500.13 mhz , dmso - d6 ) : 2.22 ( s , 3h ) , 2.94 ( dd , j = 13.6 and 10.9 hz , 1h ) , 3.09 ( dd , j = 13.7 and 3.9 hz , 1h ) , 4.63 ( ddd , j = 10.9 , 8.2 , and 4.1 hz , 1h ) , 7.05 ( d , j = 7.8 hz , 2h ) , 7.12 ( br s , 1h ) , 7.22 ( d , j = 7.9 hz , 2h ) , 7.58 ( br s , 1h ) , 7.83 ( d , j = 8.3 hz , 2h ) , 7.99 ( d , j = 8.1 hz , 2h ) , 8.75 ( d , j = 8.4 hz , 1h ) ; c nmr ( 125.77 mhz , dmso - d6 ) : 20.5 , 36.7 , 54.9 , 123.8 ( q , jcf = 272.4 hz ) , 125.1 ( q , jcf = 3.6 hz ) , 128.2 , 128.5 , 128.9 , 131.1 ( q , jcf = 31.7 hz ) , 135.0 , 135.2 , 137.8 , 164.9 , 173.0 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c18h18f3n2o2 , 351.1315 ; found , 351.1317 . yield 7.5 mg ( 47% ) , 225.5227 c ( etoh ) ; h nmr ( 500.13 mhz , dmso - d6 ) : 2.22 ( s , 3h ) , 2.92 ( dd , j = 13.7 and 10.8 hz , 1h ) , 3.06 ( dd , j = 13.7 and 4.0 hz , 1h ) , 4.59 ( ddd , j = 10.8 , 8.4 , and 4.1 hz , 1h ) , 7.05 ( d , j = 7.8 hz , 2h ) , 7.10 ( br s , 1h ) , 7.20 ( d , j = 8.0 hz , 2h ) , 7.52 ( d , j = 8.6 hz , 2h ) , 7.56 ( br s , 1h ) , 7.82 ( d , j = 8.7 hz , 2h ) , 8.58 ( d , j = 8.4 hz , 1h ) ; c nmr ( 125.77 mhz , dmso - d6 ) : 20.5 , 36.7 , 54.8 , 128.1 , 128.5 , 128.9 , 129.2 , 132.8 , 135.0 , 135.3 , 135.9 , 165.0 , 173.1 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c17h18cln2o2 , 317.1051 ; found , 317.1050 . yield 8.2 mg ( 58% ) , mp 216220 c ; h nmr ( 500.13 mhz , dmso - d6 ) : 2.22 ( s , 3h ) , 2.93 ( dd , j = 13.6 and 10.6 hz , 1h ) , 3.06 ( dd , j = 13.7 and 4.1 hz , 1h ) , 4.61 ( ddd , j = 10.6 , 8.4 , and 4.1 hz , 1h ) , 7.05 ( d , j = 7.8 hz , 2h ) , 7.10 ( br s , 1h ) , 7.22 ( d , j = 8.0 hz , 2h ) , 7.44 ( t , j = 7.5 hz , 2h ) , 7.51 ( tt , j = 7.4 and 1.3 hz , 1h ) , 7.54 ( br s , 1h ) , 7.80 ( d , j = 7.1 hz , 2h ) , 8.46 ( d , j = 8.4 hz , 1h ) ; c nmr ( 125.77 mhz , dmso - d6 ) : 20.5 , 36.7 , 54.8 , 127.3 , 128.0 , 128.5 , 128.9 , 131.1 , 134.0 , 134.9 , 135.3 , 166.0 , 173.3 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c17h19n2o2 , 283.1441 ; found , 283.1440 . yield 8.0 mg ( 54% ) , mp 203206 c ; h nmr ( 500.13 mhz , cdcl3 ) : 2.32 ( s , 3h ) , 2.40 ( s , 3h ) , 3.07 ( dd , j = 13.9 and 8.0 hz , 1h ) , 3.24 ( dd , j = 13.8 and 5.8 hz , 1h ) , 4.83 ( m , 1h ) , 5.32 ( br s , 1h ) , 5.73 ( br s , 1h ) , 6.77 ( d , j = 7.1 hz , 1h ) , 7.12 ( d , j = 7.8 hz , 2h ) , 7.19 ( d , j = 8.0 hz , 2h ) , 7.23 ( d , j = 8.0 hz , 2h ) , 7.63 ( d , j = 8.2 hz , 2h ) ; c nmr ( 125.77 mhz , dmso - d6 ) : 20.6 , 20.9 , 36.8 , 54.8 , 127.4 , 128.58 , 128.60 , 129.0 , 131.3 , 135.0 , 135.4 , 141.0 , 165.9 , 173.4 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c18h21n2o2 , 297.1598 ; found , 297.1595 . yield 6.6 mg ( 42% ) , mp 210212 c ; h nmr ( 500.13 mhz , dmso - d6 ) : 2.21 ( s , 3h ) , 2.93 ( dd , j = 13.6 and 10.6 hz , 1h ) , 3.03 ( dd , j = 13.7 and 4.0 hz , 1h ) , 3.70 ( s , 3h ) , 4.57 ( ddd , j = 10.5 , 8.4 , and 4.1 hz , 1h ) , 6.97 ( d , j = 7.8 hz , 2h ) , 7.04 ( d , j = 7.9 hz , 2h ) , 7.06 ( br s , 1h ) , 7.21 ( d , j = 8.0 hz , 2h ) , 7.49 ( br s , 1h ) , 7.79 ( d , j = 8.9 hz , 2h ) , 8.29 ( d , j = 8.4 hz , 1h ) ; c nmr ( 125.77 mhz , dmso - d6 ) : 20.5 , 36.7 , 54.7 , 55.2 , 113.2 , 126.2 , 128.5 , 128.9 , 129.1 , 134.9 , 135.4 , 161.4 , 165.4 , 173.4 ; hrms ( esi - tof ) m / z : [ m + na ] calcd for c18h20n2o3na , 335.1372 ; found , 335.1362 . the benzoyl substituents present in the corresponding amides 10e g facilitate hydrolysis at rates high enough to result in complete conversion to the acids over time , rendering convenient isolation and purification . after standing at room temperature for 5 additional days , the filtrate from the 24 h postcleavage exposure was evaporated under a stream of nitrogen gas to give 14.4 mg of a residue that was chromatographed on a prepacked 500 mg column of hypersep cn ( cyanosilica , thermo scientific ) using step - gradient elution with hexanes ( 3 3 ml ) , 8:2 hexanes / acetone ( 3 ml ) , and eluting the desired material with 7:3 hexanes / acetone to give 6.9 mg ( 51% ) 11e as an amorphous solid , mp 171172 c ; h nmr ( 500.13 mhz , cdcl3 ) : 2.32 ( s , 3h ) , 3.23 ( dd , j = 14.1 and 6.0 hz , 1h ) , 3.32 ( dd , j = 14.1 and 5.6 hz , 1h ) , 5.03 ( m , 1h ) , 6.51 ( br d , j = 7.0 hz , 1h ) , 7.11 ( m , 4h ) , 7.42 ( t , j = 7.6 hz , 2h ) , 7.52 ( t , j = 7.4 hz , 1h ) , 7.68 ( d , j = 7.9 hz , 2h ) ; c nmr ( 125.77 mhz , cd3od ) : 21.1 , 37.8 , 55.7 , 128.4 , 129.5 , 130.1 , 130.2 , 132.8 , 135.4 , 135.6 , 137.5 , 170.2 , 174.9 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c17h18no3 , 284.1281 ; found , 284.1285 . after standing at room temperature for 5 additional days , the filtrate from the 24 h postcleavage exposure was evaporated under a stream of nitrogen gas to give 16.5 mg of a residue that was chromatographed on a dynamax microsorb c-18 , 5 m column ( 21.4 250 mm ) using the isochratic system of 50/50 1:1 mecn / meoh ( 5 mm ammonium acetate)/water ( 5 mm ammonium acetate ) at a 5 ml / min flow rate to give 8.6 mg ( 61% ) 11f as an amorphous solid after evaporation , mp 184187 c ; h nmr ( 500.13 mhz , cd3od ) : 2.26 ( s , 3h ) , 2.37 ( s , 3h ) , 3.07 ( dd , j = 13.8 and 8.3 hz , 1h ) , 3.27 ( dd , j = 13.8 and 4.9 hz , 1h ) , 4.75 ( dd , j = 8.2 and 5.0 hz , 1h ) , 7.05 ( d , j = 7.9 hz , 2h ) , 7.12 ( d , j = 8.0 hz , 2h ) , 7.23 ( d , j = 8.0 hz , 2h ) , 7.61 ( d , j = 8.2 hz , 2h ) ; c nmr ( 125.77 mhz , cd3od ) : 21.1 , 21.4 , 38.2 , 56.5 , 128.3 , 129.9 , 130.1 , 130.3 , 132.7 , 135.9 , 137.2 , 143.4 , 169.7 , 176.1 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c18h20no3 , 298.1438 ; found , 298.1441 . after standing an additional 3 days at 20 c , the filtrate from the 24 h postcleavage exposure was evaporated under a stream of nitrogen gas to give 17.1 mg of a residue that was chromatographed on a prepacked 500 mg column of hypersep cn ( cyanosilica , thermo scientific ) using a step - gradient elution with hexanes ( 3 ml ) , 8:2 hexanes / acetone ( 3 ml ) , and eluting the desired material with 7:3 hexanes / acetone to give 5.3 mg ( 36% ) 11 g as an amorphous solid , mp 16872 c ; h nmr ( 500.13 mhz , cd3od ) : 2.27 ( s , 3h ) , 3.07 ( dd , j = 13.9 and 9.3 hz , 1h ) , 3.27 ( dd , j = 13.9 and 5.0 hz , 1h ) , 3.83 ( s , 3h ) , 4.80 ( dd , j = 9.4 and 5.1 hz , 1h ) , 6.94 ( d , j = 8.9 hz , 2h ) , 7.08 ( d , j = 7.9 hz , 2h ) , 7.14 ( d , j = 8.0 hz , 2h ) , 7.71 ( d , j = 8.9 hz , 2h ) ; c nmr ( 125.77 mhz , cd3od ) : 21.1 , 37.8 , 55.7 , 55.9 , 114.7 , 127.4 , 130.1 , 130.2 , 130.3 , 135.6 , 137.4 , 164.1 , 169.7 , 175.1 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c18h20no4 , 314.1387 ; found , 314.1390 . a 50 ml spps vessel was charged with 780940 mg ( 250300 mol ) of fmoc - phe - nh - rink amide mbha resin ( 0.32 mmol / g , peptides international ) and treated with 20 ml of 20% ( v / v ) piperidine in n - methylpyrolidinone ( nmp ) . the mixture was rocked for 45 min at room temperature and was then drained . the resin was washed with 4 15 ml of nmp and was then diluted with 55:45 nmp / dcm to make an isopycnic mixture ( neutral buoyancy ) . the mixture was then equally distributed to 3.5 ml reaction vessels using a repeater pipet ( see general procedure for the preparation and distribution of isopycnic suspensions of resins ) . each resin was washed with 4 1.5 ml of nmp and was then treated with a 0.25 m solution of the required carboxylic acid ( 5.00 equiv ) in 0.25 m n , n , n,n-tetramethyl - o-(1h - benzotriazol-1-yl)uronium hexafluorophosphate ( 5.00 equivalents , hbtu ) in dmf followed by 10.0 equiv of a 0.50 m solution of diisopropylethylamine in dmf . the vessels were rotated at room temperature for 48 h and were then drained . each resin was washed with 5 1.5 ml of dmf and 6 1.5 ml of dcm and was then stored overnight in its capped vessel . each vessel containing 50 mol of resin 12a j was treated with 1.5 ml of 95:5 tfa / h2o and was rotated at room temperature for 0.5 h. all vessels were then drained into collection vials , and the resins were washed with 1.5 ml of 95:5 tfa / h2o followed by 1.5 ml of dcm . three 200 l aliquots from each vial were removed and placed in sets of three vials representing the three time exposures of 0.5 , 6 , and 23 h , at which point they were evaporated under a stream of nitrogen gas and analyzed by lc / ms ( 214 nm ) as solutions in acetonitrile . to prevent further hydrolysis of primary amides 13 after 30 min exposure to the tfa solution , the vials containing the bulk of the filtrates were stored at 20 c . the filtrate was evaporated to dryness under a stream of nitrogen to give 6.6 mg of crude material . trituration under diethyl ether afforded 6.1 mg ( 33% ) of 13a as an amorphous solid , mp 192194 c ( dec ) ; h nmr ( 500.13 mhz , dmso - d6 ) : 2.78 ( dd , j = 13.5 and 10.6 hz , 1h ) , 3.00 ( dd , j = 13.6 and 4.0 hz , 1h ) , 4.164.19 ( m , 4h ) , 7.08 ( br s , 1h ) , 7.18 ( t , j = 7.1 hz , 1h ) , 7.247.33 ( m , 6h ) , 7.397.43 ( m , 2h ) , 7.45 ( br s , 1h ) , 7.53 ( d , j = 8.8 hz , 1h ) , 7.63 ( d , j = 7.6 hz , 1h ) , 7.65 ( d , j = 7.6 hz , 1h ) , 7.88 ( d , j = 7.5 hz , 2h ) ; c nmr ( 125.77 mhz , dmso - d6 ) : 37.4 , 46.4 , 55.9 , 65.5 , 120.0 , 125.17 , 125.25 , 126.1 , 126.9 , 127.5 , 127.9 , 129.1 , 138.2 , 140.5 , 143.6 , 143.7 , 155.7 , 173.3 ( note that two extra aromatic signals are present , indicative of the diastereotopic nature of the aromatic carbons , which display identical atom connectivity . two extra signals are also present in the c nmr of commercial fmoc - phe - oh ) ; hrms ( esi - tof ) m / z : [ m + na ] calcd for c24h22n2o3na , 409.1528 ; found , 409.1535 . the filtrate was added carefully to 25 ml of cold 2 m naoh , and the mixture was extracted with 2 20 ml of dcm . the combined extracts were dried ( na2so4 ) and concentrated to give 7.7 mg of crude material . trituration under 2 ml of diethyl ether afforded 5.7 mg ( 36% ) of 13b as an amorphous solid , mp 16970 c ; h nmr ( 500.13 mhz , dmso - d6 ) : 2.75 ( dd , j = 13.7 and 9.6 hz , 1h ) , 2.98 ( dd , j = 13.7 and 4.7 hz , 1h ) , 3.28 ( d , j = 14.0 hz , 1h ) , 3.34 ( d , j = 13.9 hz , 1h ) , 3.71 ( s , 3h ) , 4.43 ( ddd , j = 9.3 , 8.5 , and 4.7 hz , 1h ) , 6.77 ( d , j = 8.7 hz , 2h ) , 7.00 ( d , j = 8.6 hz , 2h ) , 7.05 ( br s , 1h ) , 7.187.24 ( m , 5h ) , 7.45 ( br s , 1h ) , 8.11 ( br d , j = 8.5 hz , 1h ) ; c nmr ( 125.77 mhz , dmso - d6 ) : 39.2 , 39.6 , 53.6 , 54.9 , 113.4 , 126.0 , 127.9 , 128.1 , 129.1 , 129.8 , 137.9 , 157.6 , 170.0 , 173.0 ; hrms ( esi - tof ) m / z : [ m + na ] calcd for c18h20n2o3na , 335.1372 ; found , 335.1375 . lc / ms ( 210 nm / esi ) : 33.7 min , m / z 207 [ m + h ] . , 3 m , 0.75 ml / min , 10% 50:50 acetonitrile / methanol with 5 mm ammonium acetate/90% water with 5 mm ammonium acetate . the filtrate was added carefully to 25 ml of cold 2 m naoh and the mixture was extracted with 2 20 ml of dcm . the combined extracts were dried ( na2so4 ) and concentrated to give 5.7 mg of crude material . trituration under 2 ml of diethyl ether afforded 4.0 mg ( 32% ) of 13d as an amorphous solid , mp 182183 c ; h nmr ( 500.13 mhz , dmso - d6 ) : 0.67 ( d , j = 6.5 hz , 3h ) , 0.75 ( d , j = 6.5 hz , 3h ) , 1.83 ( septet , j = 6.6 hz , 1h ) , 1.90 ( d , j = 6.3 hz , 2h ) , 2.72 ( dd , j = 13.7 and 10.1 hz , 1h ) , 2.99 ( dd , j = 13.7 and 4.5 hz , 1h ) , 4.46 ( ddd , j = 10.1 , 8.7 , and 4.5 hz , 1h ) , 7.01 ( br s , 1h ) , 7.157.18 ( m , 1h ) , 7.227.24 ( m , 4h ) , 7.38 ( br s , 1h ) , 7.91 ( br d , j = 8.6 hz , 1h ) ; c nmr ( 125.77 mhz , dmso - d6 ) : 22.0 , 22.1 , 25.3 , 37.5 , 44.4 , 53.5 , 126.0 , 127.8 , 129.0 , 138.1 , 171.2 , 173.3 ; hrms ( esi - tof ) m / z : [ m + na ] calcd for c14h20n2o2na , 271.1422 ; found , 271.1427 . the filtrate was added carefully to 25 ml of cold 2 m naoh , and the mixture was extracted with 2 20 ml of dcm . the combined extracts were dried ( na2so4 ) and concentrated to give 6.6 mg of crude material . trituration under 2 ml of diethyl ether gave 4.1 mg ( 35% ) of 13e as an amorphous solid , mp 200202 c ; h nmr ( 500.13 mhz , dmso - d6 ) : 0.530.60 ( m , 4h ) , 1.601.63 ( m , 1h ) , 2.76 ( dd , j = 13.7 and 9.5 hz , 1h ) , 2.96 ( dd , j = 13.7 and 5.0 hz , 1h ) , 4.45 ( ddd , j = 9.2 , 8.5 , and 5.0 hz , 1h ) , 7.00 ( br s , 1h ) , 7.177.20 ( m , 1h ) , 7.237.28 ( m , 4h ) , 7.42 ( br s , 1h ) , 8.22 ( br d , j = 8.5 hz , 1h ) ; c nmr ( 125.77 mhz , dmso - d6 ) : 6.1 , 6.2 , 13.3 , 37.7 , 53.7 , 126.0 , 127.9 , 129.0 , 138.0 , 172.3 , 173.1 ; hrms ( esi - tof ) m / z : [ m + na ] calcd for c13h16n2o2na , 255.1109 ; found , 255.1102 . the filtrate was added carefully to 25 ml of cold 2 m naoh and the mixture was extracted with 2 20 ml of dcm . the combined extracts were dried ( na2so4 ) and concentrated to give 7.3 mg of crude material . trituration under 2 ml of diethyl ether gave 5.2 mg ( 33% ) of 13f as an amorphous solid , mp 203205 c ; h nmr ( 500.13 mhz , dmso - d6 ) : 3.03 ( dd , j = 13.7 and 10.7 hz , 1h ) , 3.15 ( dd , j = 13.7 and 4.1 hz , 1h ) , 4.72 ( ddd , j = 10.7 , 8.6 , and 4.3 hz , 1h ) , 7.147.15 ( m , 2h ) , 7.26 ( t , j = 7.6 hz , 2h ) , 7.37 ( d , j = 7.2 hz , 2h ) , 7.587.62 ( m , 3h ) , 7.88 ( dd , j = 8.6 and 1.7 hz , 1h ) , 7.96 ( d , j = 8.3 hz , 2h ) , 8.01 ( d , j = 7.6 hz , 1h ) , 8.42 ( br s , 1h ) , 8.66 ( br d , j = 8.4 hz , 1h ) ; c nmr ( 125.77 , dmso - d6 ) : 37.2 , 54.7 , 124.2 , 126.1 , 126.6 , 127.48 , 127.55 , 127.6 , 127.9 , 128.7 , 129.0 , 131.3 , 131.9 , 134.0 , 138.5 , 166.0 , 173.2 ( 18 c signals are predicted , but only 17 were observed ) ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c20h19n2o2 , 319.1441 ; found , 319.1447 . the filtrate was immediately evaporated to dryness with a stream of nitrogen gas to give 10.5 mg of crude material that was purified by reverse - phase hplc on a dynamax microsorb c-18 , 5 m column ( 21.4 250 mm ) eluting isochratically with 60:40 1:1 mecn / meoh ( with 5 mm ammonium acetate)/water ( with 5 mm ammonium acetate ) at 5 ml / min and detection at 254 nm to afford 3.5 mg ( 21% ) of 13 g ( 95:5 mixture of e / z isomers ) as an amorphous solid , mp 191201 c ( dec ) ; h nmr ( 500.13 mhz , cdcl3 ) : 3.10 ( dd , j = 13.8 and 8.0 hz , 1h ) , 3.22 ( dd , j = 13.8 and 5.9 hz , 1h ) , 3.83 ( s , 3h ) , 4.82 ( dd , j = 13.8 and 7.3 hz , 1h ) , 5.33 ( br s , 1h ) , 5.77 ( br s , 1h ) , 6.23 ( br s , 1h ) , 6.26 ( d , j = 15.5 hz , 1h ) , 6.88 ( d , j = 8.5 hz , 2h ) , 7.267.34 ( m , 5h ) , 7.44 ( d , j = 8.6 hz , 2h ) , 7.58 ( d , j = 15.5 hz , 1h ) ; h nmr ( 500.13 mhz , cd3od ) : 2.99 ( dd , j = 13.9 and 8.9 hz , 1h ) , 3.22 ( dd , j = 13.9 and 5.8 hz , 1h ) , 3.84 ( s , 3h ) , 4.77 ( dd , j = 8.9 and 5.8 hz , 1h ) , 6.52 ( d , j = 15.8 hz , 1h ) , 6.95 ( d , j = 8.7 hz , 2h ) , 7.227.24 ( m , 1h ) , 7.307.31 ( m , 4h ) , 7.46 ( d , j = 15.7 hz , 1h ) , 7.51 ( d , j = 8.7 hz , 2h ) ; c nmr ( 125.77 mhz , cd3od ) : 39.2 , 55.87 , 55.94 , 115.4 , 118.8 , 127.8 , 128.8 , 129.5 , 130.3 , 130.6 , 138.6 , 142.1 , 162.8 , 168.8 , 176.3 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c19h21n2o3 , 325.1547 ; found , 325.1552 . the filtrate was immediately evaporated to dryness with a stream of nitrogen gas to give crude material that was purified by reverse - phase hplc on a dynamax microsorb c-18 , 5 m column ( 21.4 250 mm ) eluting isochratically with 60:40 1:1 mecn / meoh ( with 5 mm ammonium acetate)/water ( with 5 mm ammonium acetate ) at 5 ml / min and detection at 254 nm to afford 8.0 mg ( 43% ) of 13h as an amorphous solid , mp 9496 c ; h nmr ( 500.13 mhz , cdcl3 ) : 1.12 ( s , 9h ) , 3.09 ( m , 2h ) , 4.69 ( dd , j = 14.4 and 7.2 hz , 1h ) , 5.59 ( br s , 1h ) , 6.12 ( br s , 1h ) , 6.30 ( br d , j = 7.2 hz , 1h ) , 7.227.25 ( m , 3h ) , 7.287.31 ( m , 2h ) ; c nmr ( 125.77 mhz , cdcl3 ) : 27.3 , 38.0 , 38.7 , 53.8 , 127.1 , 128.7 , 129.3 , 136.6 , 173.4 , 178.7 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c14h21n2o2 , 249.1598 ; found , 249.1597 . the filtrate was immediately evaporated to dryness with a stream of nitrogen gas to give crude material that was purified by reverse - phase hplc on a dynamax microsorb c-18 , 5 m column ( 21.4 250 mm ) eluting isochratically with 75:25 1:1 mecn / meoh ( with 5 mm ammonium acetate)/water ( with 5 mm ammonium acetate ) at 5 ml / min and detection at 254 nm to afford 8.1 mg ( 33% ) of 13i as a glass : h nmr ( 500.13 mhz , cdcl3 ) : 1.641.67 ( m , 3h ) , 1.711.74 ( m , 3h ) , 1.76 ( m , 6h ) , 2.01 ( br s , 3h ) , 3.08 ( m , 2h ) , 4.69 ( dd , j = 14.2 and 6.9 hz , 1h ) , 5.58 ( br s , 1h ) , 6.06 ( br s , 1h ) , 6.24 ( br d , j = 7.1 hz , 1h ) , 7.227.25 ( m , 3h ) , 7.287.31 ( m , 2h ) ; c nmr ( 125.77 mhz , cdcl3 ) : 28.0 , 36.4 , 38.0 , 39.0 , 40.6 , 53.6 , 127.1 , 128.7 , 129.3 , 136.6 , 173.5 , 178.2 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c20h27n2o2 , 327.2067 ; found 327.2064 . in a 25 ml solid - phase peptide synthesis ( spps ) vessel , fmoc - phe - nh - rink amide mbha resin ( 576 mg , 190 mol , 0.33 mmol / g , peptides international ) was deprotected using 30% piperidine in nmp . the amine resin was then treated with 3.77 ml of a 0.25 m solution of 4-methoxybenzoic acid ( 143 mg , 942 mol , 4.96 equiv ) in 0.25 m n , n , n,n-tetramethyl - o-(1h - benzotriazol-1-yl)uronium hexafluorophosphate ( hbtu ) ( 361 mg , 952 mol , 5.01 equiv ) in nmp followed by 3.77 ml of a 0.50 m solution of diisopropylethylamine ( 1880 mol , 9.91 equiv ) in nmp . the vessel was rocked at room temperature on an orbital shaker for 22 h and was then drained . the resin gave a negative kaiser test and was then washed with 4 10 ml 2 min each successively of nmp , thf , and dcm . acylated resin 12j was treated with 6 ml of 95:5 ( v / v ) tfa / h2o , and the vessel was rocked for 30 min at room temperature . the filtrate was collected , and the resin was washed once with 6 ml of the cleavage cocktail and once with 6 ml of dcm . the filtrate was allowed to stand at room temperature for 5 h to allow for hydrolysis to proceed partially and was then evaporated under a stream of nitrogen gas over a 3 h period ( effluent gases were scrubbed with a solution of sodium hydroxide ) to give 64 mg of a sticky white solid that was passed through a 500 mg cartridge of cyanosilica gel using 7:3 hexanes / acetone to elute the amide / acid mixture . after solvent removal by evaporation , the mixture was dissolved in dcm and was washed with 2 10 ml of 1.0 n naoh and once with 10 ml of water and was then dried ( na2so4 ) . concentration gave 19.6 mg ( 35% ) of 13j as an amorphous solid , mp 201204 c ; h nmr ( 500.13 mhz , dmso - d6 ) : 2.97 ( dd , j = 13.6 and 10.7 hz , 1h ) , 3.10 ( dd , j = 13.6 and 4.0 hz , 1h ) , 3.79 ( s , 3h ) , 4.61 ( ddd , j = 10.8 , 8.6 , and 4.2 hz , 1h ) , 6.97 ( d , j = 8.9 hz , 2h ) , 7.08 ( br s , 1h ) , 7.15 ( t , j = 7.4 hz , 1h ) , 7.24 ( t , j = 7.5 hz , 2h ) , 7.33 ( d , j = 7.1 hz , 2h ) , 7.51 ( br s , 1h ) , 7.78 ( d , j = 8.9 hz , 2h ) , 8.32 ( d , j = 8.5 hz , 1h ) ; c nmr ( 125.77 mhz , dmso - d6 ) : 37.1 , 54.6 , 55.2 , 113.2 , 126.0 , 126.3 , 127.9 , 129.0 , 129.1 , 138.5 , 161.5 , 165.5 , 173.4 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c17h19n2o3 , 299.1390 ; found , 299.1392 . the combined basic extracts were adjusted to ph 1 with 3 n hcl and were extracted with 2 25 ml of dcm . the combined extracts were dried ( na2so4 ) and concentrated to give 12.1 mg ( 21% ) of 14j as an amorphous solid , mp 164167 c ; h nmr ( 500.13 mhz , cd3od ) : 3.11 ( dd , j = 13.9 and 9.4 hz , 1h ) , 3.32 ( dd , j = 5.0 hz , 1h ) , 3.83 ( s , 3h ) , 4.83 ( dd , j = 9.4 and 4.9 hz , 1h ) , 6.94 ( d , j = 8.9 hz , 2h ) , 7.19 ( m , 1h ) , 7.247.28 ( m , 4h ) , 7.70 ( d , j = 8.9 hz , 2h ) ; c nmr ( 125.77 mhz , cd3od ) : 38.2 , 55.6 , 55.9 , 114.7 , 127.4 , 127.8 , 129.4 , 130.3 , 138.8 , 164.1 , 169.7 , 175.0 ( only 12 of 13 lines were observed ) ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c17h18no4 , 300.1230 ; found , 300.1229 . a 25 ml spps vessel was charged with 1.857 g ( 0.5478 mmol , 0.295 mmol / g ) of fmoc - phe - nh - rink amide mbha resin . the resin was swelled by washing with 3 10 ml of nmp followed by 10 ml of 20% ( v / v ) piperidine in nmp . the resin the vessel was rocked on an orbital shaker for 40 min and was then drained , and the resin was washed with 5 10 ml of nmp . to the resin was then added a solution of 0.374 g ( 2.75 mmol , 5.02 equiv ) of 4-methylbenzoic acid and 0.418 g ( 2.73 mmol , 4.98 equiv ) of 1-hydroxybenzotriazole in 10.9 ml of nmp followed by a solution of 0.346 g ( 2.74 mmol , 5.00 equiv ) of diisopropylcarbodiimide in 1 ml of nmp . the vessel was rocked on an orbital shaker for 6 days and was then drained , and the resin was washed with 3 10 ml each of nmp and thf and then with 5 10 ml of dcm to give 12k , which was prepared as an isopycnic mixture using 2:1 nmp / dcm and was equally distributed to eleven 3.5 ml reaction vessels to provide 50 mol per vessel ( see general procedure for the preparation and distribution of isopycnic suspensions of resins ) . each resin was then washed with 5 2 ml of dcm . compound 13k was synthesized in solution phase using the method of uchida et al . and was purified for use as the authentic standard in the quantitative lc measurements ( optimization of cleavage conditions , table 4 ) . a 25 ml , three - necked , round - bottomed flask fitted with a stirrer bar , thermometer , and rubber septum was charged with 0.105 g ( 1.25 mmol ) of sodium bicarbonate , 2.0 ml of water , and 2.0 ml of acetone . the contents were cooled with an ice bath , and 0.200 g ( 1.22 mmol ) of l - phenylalanineamide ( chem - impex , int . ) 4-methylbenzoyl chloride ( 0.208 g , 1.34 mmol , 1.10 equiv ) , dissolved in 0.5 ml of acetone , was added dropwise via syringe . the contents were allowed to gradually warm to room temperature and were stirred for 18 h. the contents were cooled with an ice bath , and the solid was collected by suction filtration washing with a few milliliters of cold 1:1 acetone / water to afford 97 mg of crude 13k , which was triturated under hot methanol to give 29.5 mg ( 9% ) of the qlc calibration sample . h nmr ( 500.13 mhz , dmso - d6 ) : 2.33 ( s , 3h ) , 2.98 ( dd , j = 13.6 and 10.7 hz , 1h ) , 3.10 ( dd , j = 13.6 and 4.0 hz , 1h ) , 4.62 ( ddd , j = 10.7 , 8.6 , and 4.2 hz , 1h ) , 7.09 ( br s , 1h ) , 7.15 ( t , j = 7.3 hz , 1h ) , 7.24 ( m , 4h ) , 7.32 ( d , j = 7.5 hz , 2h ) , 7.52 ( br s , 1h ) , 7.70 ( d , j = 8.2 hz , 2h ) , 8.37 ( d , j = 8.4 hz , 1h ) ; c nmr ( 125.77 mhz , dmso - d6 ) : 20.8 . 37.1 , 54.6 , 126.0 , 127.3 , 127.9 , 128.6 , 129.0 , 131.2 , 138.5 , 141.0 , 165.9 , 173.3 . lc / ms ( esi ) : 8.19 min , m / z 283 [ m + h ] . two 50 ml spps vessels were charged , respectively , with 566 mg ( 300 mol ) of fmoc - ala - o wang resin ( 0.53 mmol / g , peptides international ) and 698 mg ( 300 mol ) of fmoc - ala - nh - rink mbha resin ( 0.43 mmol / g , peptides international ) . to each vessel was added 20 ml of 20% ( v / v ) piperidine in nmp . the vessels were rocked at room temperature for 45 min and were then drained . each resin was washed with 4 10 ml of nmp and was treated with 3.0 ml ( 750 mmol , 2.5 equiv ) of 0.25 m hbtu in nmp , 3.0 ml ( 750 mmol , 2.5 equiv ) of 0.25 m fmoc - phe - oh in nmp , and 3.0 ml ( 1500 mol , 5.0 equiv ) of 0.50 m diisopropylethylamine in nmp . the vessels were rocked overnight at room temperature . to each vessel was again added 3.0 ml ( 750 mmol , 2.5 equiv ) of 0.25 m hbtu in nmp , 3.0 ml ( 750 mmol , 2.5 equiv ) of 0.25 m fmoc - phe - oh in nmp , and 3.0 ml ( 1500 mol , 5.0 equiv ) of 0.50 m diisopropylethylamine in nmp . each resin was then washed with 4 10 ml of nmp followed by 4 10 ml of dcm and then 3 10 ml of nmp . piperidine ( 20% in nmp , 20 ml ) was added to each resin , and the vessels were rocked for 45 min . the resins were washed with 4 10 ml of nmp and were then treated with 6.0 ml of 0.25 m ( 1500 mol , 5.0 equiv ) hbtu in nmp , 6.0 ml of 0.25 m ( 1500 mol , 5.0 equiv ) 4-methoxybenzoic acid in nmp , and 6.0 ml ( 3000 mol , 10.0 equiv ) of 0.50 m diisopropylethylamine in nmp . the vessels were drained , and the resins were washed with 3 10 ml of nmp , 3 10 ml of thf , and 3 10 ml of dcm and then capped for storage for 3 days . isopycnic suspensions of rink resin 15 ( 300 mol ) and wang resin 16 ( 300 mol ) were each equally distributed , respectively , to six 3.5 ml reaction vessels in rows a and b of two 2 3 bill - boards ( 50 mol of resin per vessel ) . all vessels of one bill - board were then treated with 1.5 ml of 95:5 tfa / water and were rotated for 30 min at room temperature . the vessels were drained into 4-dram , tooled - neck collection vials , and each resin was rinsed with 2 ml of 95:5 tfa / water . filtrates from a1 and b1 were sampled and evaporated to dryness and analyzed ( 0.5 h time point ) , filtrates from a2 and b2 were allowed to stand at room temperature for 6 h and were then analyzed by lc / ms , and then the filtrates from a3 and b3 , which had stood for 24 h , were analyzed . each vial assayed at 30 min was immediately stored at 20 c overnight and was concentrated under a stream of nitrogen gas to afford 10.9 mg of crude 17 and 15.2 mg of crude 18 . the crude material ( 9.3 mg ) was purified by cyanosilica gel chromatography ( 500 mg prepacked column , eluting with 1:1 and then 4:6 hexanes / acetone ) followed by reverse - phase hplc on a dynamax microsorb c-18 , 5 m column ( 21.4 250 mm ) using the isochratic system of 55:45 1:1 mecn / meoh ( 5 mm ammonium acetate)/water ( 5 mm ammonium acetate ) at a 5 ml / min flow rate to give 2.1 mg ( 11% ) of 17 as an amorphous solid , mp 229231 c ; h nmr ( 500.13 mhz , cd3od ) : 1.36 ( d , j = 7.2 hz , 3h ) , 3.06 ( dd , j = 13.8 and 9.2 hz , 1h ) , 3.26 ( dd , j = 13.9 and 6.0 hz , 1h ) , 3.84 ( s , 3h ) , 4.34 ( q , j = 7.2 hz , 1h ) , 4.79 ( dd , j = 9.1 and 5.9 hz , 1h ) , 6.95 ( d , j = 8.8 hz , 2h ) , 7.20 ( m , 1h ) , 7.267.32 ( m , 4h ) , 7.72 ( d , j = 8.9 hz , 2h ) ; c nmr ( 125.77 mhz , cd3od ) : 18.2 , 38.6 , 50.1 , 55.9 , 56.7 , 114.7 , 127.2 , 127.9 , 129.5 , 130.37 , 130.39 , 138.6 , 164.1 , 169.9 , 173.6 , 177.3 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c20h24n3o4 , 370.1761 ; found , 370.1767 . the crude material ( 12.9 mg ) was chromatographed using a 500 mg prepacked cyanosilica cartridge ( thermo scientific ) and eluted with hexanes / acetone ( 7:3 and 6:4 ) to afford 4.8 mg ( 26% ) of 18 as an amorphous solid , mp 181184 c ( dec ) ; h nmr ( 500.13 mhz , cd3od ) : 1.42 ( d , j = 7.3 hz , 3h ) , 3.03 ( dd , j = 14.0 and 9.7 hz , 1h ) , 3.29 ( dd , j = 14.4 and 4.9 hz , 1h ) , 3.83 ( s , 3h ) , 4.39 ( q , j = 7.2 hz , 1h ) , 4.86 ( dd , j = 9.8 and 4.9 hz , 1h ) , 6.94 ( d , j = 8.9 hz , 2h ) , 7.18 ( t , j = 7.3 hz , 1h ) , 7.25 ( t , j = 7.5 hz , 2h ) , 7.31 ( d , j = 7.1 hz , 2h ) , 7.69 ( d , j = 8.9 hz , 2h ) ; c nmr ( 125.77 mhz , cd3od ) : 18.0 , 38.9 , 49.9 , 55.9 , 56.3 , 114.7 , 127.3 , 127.7 , 129.4 , 130.3 , 130.4 , 138.7 , 164.1 , 169.7 , 173.5 , 176.3 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c20h23n2o5 371.1601 ; found 371.1604 .

remote amide bonds in simple n - acyl amino acid amide or peptide derivatives 1 can be surprisingly unstable hydrolytically , affording , in solution , variable amounts of 3 under mild acidic conditions , such as trifluoroacetic acid / water mixtures at room temperature . this observation has important implications for the synthesis of this class of compounds , which includes n - terminal - acylated peptides . we describe the factors contributing to this instability and how to predict and control it . the instability is a function of the remote acyl group , r2co , four bonds away from the site of hydrolysis . electron - rich acyl r2 groups accelerate this reaction . in the case of acyl groups derived from substituted aromatic carboxylic acids , the acceleration is predictable from the substituent s hammett value . n - acyl dipeptides are also hydrolyzed under typical cleavage conditions . this suggests that unwanted peptide truncation may occur during synthesis or prolonged standing in solution when dipeptides or longer peptides are acylated on the n - terminus with electron - rich aromatic groups . when amide hydrolysis is an undesired secondary reaction , as can be the case in the trifluoroacetic acid - catalyzed cleavage of amino acid amide or peptide derivatives 1 from solid - phase resins , conditions are provided to minimize that hydrolysis .

Introduction Results and Discussion Conclusions Experimental Section

in preliminary work generalizing our earlier synthesis of n - acylated unnatural amino acid amides 1 to procedures compatible with a wide variety of r groups , an unexpected r - dependent hydrolytic instability was encountered ( scheme 1 ) . during the trifluoroacetic acid ( tfa ) cleavage of the final rink resin - bound intermediate 2 to give 1 , varying amounts of carboxylic it was of concern that the instability of 1 ( r = h in the encountered case ) could be a more general phenomenon and potentially affect the synthesis of n - acylated natural and unnatural amino acid amides , peptides , and proteins that are targets of organic and peptide chemists . because of these wider implications , we sought to understand the origins and nature of this instability , to develop predictive tools to anticipate it , and to establish reaction and workup conditions to minimize it . amide bonds such as those in n - acylated amino acid amides 1 are generally stable to conditions that would readily cause hydrolysis of an analogous ester bond . strong acids such as tfa , with varying amounts of water or triethylsilane ( tes ) , are often used to cleave peptides from solid - phase resins without significant hydrolysis of amide bonds . the rink resin was specifically designed to permit tfa - promoted cleavage of the resin link ( such as that in 2 ) to provide peptide primary amides 1 ( r = h ) with both the internal amide(s ) and c - terminal primary amide remaining intact . in general , when amide bond cleavage does occur , it is usually under more vigorous conditions or by virtue of a unique structural feature that permits intramolecular - catalyzed hydrolysis . for example , hydrolysis of a peptide into its constituent amino acids requires 6 n hcl at 100 c , and cleavage of the n - terminal amino acid from a polypeptide via the classic edman degradation is promoted by an intramolecular reaction to form a key cyclic intermediate . several examples have appeared in the literature in which specific peptidyl amino acid sequences and n - acylated peptides undergo facile amide cleavage under mildly acidic conditions . this hydrolytic sensitivity of peptidyl pipecolic acid residues was exploited by zajdel , subra , and co - workers in the design of pipecolic resin linkers for solid - phase applications . the closest precedent for the observations reported in this work comes from goodman and co - workers , who observed amide hydrolysis under mild conditions in a series of n - benzoyl - n - methylaib - phe - ome analogues ( 4 ; scheme 2 ) . it was demonstrated that this instability is a function of the remote benzoyl groups on the n - terminal amino acid of this unusual dipeptide and that the rate of hydrolysis was correlated with the hammett constants of the benzoyl substituents , g. analysis of the data established the existence of a linear free - energy relationship and supported a mechanism involving the intermediacy of an oxazolinium ion that proceeded from the tertiary amide . to our knowledge , there are no reports of mild hydrolyses of the simple n - acylated primary amides or peptides represented by 1 . we were surprised , then , in our earlier published work synthesizing 1 ( scheme 3 , r = h ) from the benzophenone imine of rink mbha glycine amide 7 to observe small amounts of amide bond hydrolysis products 3 under tfa cleavage conditions . our focus in that report was to develop chemistry to incorporate unnatural side chains , r , into the -position of glycine with the acyl group , r , being used only to enable isolation and quantification of 1 ( r = h ) produced in the key alkylation step . however , when recently seeking to develop scheme 3 further into a robust distributed drug discovery ( d3 ) lab that would permit the synthesis of large numbers of new compounds through widespread variation of both r and r , it became apparent that the amide hydrolysis observed earlier was not an anomaly but a clear function of the nature of the r group and that it would be important to understand the factors involved in this r - dependent hydrolytic instability so that its occurrence could be anticipated , predicted , and minimized . we were developing a new d3 laboratory , the combinatorial synthesis of n - acylated unnatural amino acid amides , based on the published reaction sequence shown in scheme 3 . this became clear when , in the course of this adaptation , two students conducted independent replicated syntheses of six new molecules , 1a f ( scheme 3 and table 1 ) . the molecules were made through combinatorial reactions carried out in a 2 3 combinatorial grid that utilized two different carboxylic acids , rco2h , in rows a and b and three different alkylating agents , rx , in columns 13 . the fact that every reaction in row a gave significant amounts of carboxylic acids 3a c as side products , that none of the reaction mixtures in row b contained hydrolyzed material , and that these results were independently repeated by two researchers suggested the presence of a systematic effect worthy of further inquiry . in addition , the combinatorial nature of these six experiments made it possible to narrow the important variable quickly to the acylating group r ( row a vs row b rather than columns 13 ) and to narrow further the common determining variable to the nature of the para substituent on the aromatic acylating agent . on the basis of these observations , it was hypothesized that hydrolysis was a function of the electronic properties of the remote aromatic acyl group , r , and that electron - donating groups would facilitate the hydrolysis , whereas electron - withdrawing substituents would suppress it . to undertake a systematic investigation of the r - dependent hydrolytic instability , compounds 10a g , featuring a representative range of hammett constants at the para position of the aromatic benzoyl group , were prepared on rink amide mbha resins ( 2a g ) , cleaved , and studied in solution ( scheme 4 ) . after subjection to the original cleavage cocktail ( 95:5 tfa / water , 0.5 h , rt ) , all samples were filtered from resin so that subsequent time points ( 2 , 4 , 8 , and 24 h ) would reflect the instability of 10 solely in solution . that the amount of acid increases with time of exposure to the cleavage cocktail after cleavage and filtration from the resin ( all time points after 0.5 h ) indicates that hydrolysis proceeds primarily in solution and comes directly from 10 . having demonstrated that r - dependent amide hydrolysis can occur under typical resin - cleavage conditions , various cleavage methods were investigated to identify conditions that would maximize compound yield while minimizing subsequent hydrolysis . in the earlier study of the hammett constant - dependent hydrolysis of 10 to 11 ( table 2 ) , we had already determined that if solutions of 10 were immediately stored at 20 c after the 0.5 h cleavage time at room temperature then subsequent hydrolysis to 11 was completely suppressed ( see the supporting information ) . scavenging by water to give the diarylcarbinol would be reversible under the tfa conditions , potentially resulting in reattachment of the cleaved product to the resin via the primary amide or by way of friedel the combination of a 1 h cleavage , replacement of water with triethylsilane , and use of a lower percentage of tfa represents the preferred methods that are described by c and e ( entries 6 and 10 ) . our observation and analysis of the mild hydrolysis promoted by remote aromatic acyl groups r on the n - terminus of simple n - acylated amino acid amides 1 ( r = h ) prompted an additional experiment documenting its implications for n - acylated peptide instability . rink amide mbha and wang n - acylated dipeptide resins 15 and 16 , respectively , were prepared and subjected to cleavage ( 95:5 tfa / water , 0.5 h , rt , scheme 5 ) . although negligible hydrolysis occurred in the initial 30 min exposure , by 6 h , 1015% of the resin - free mixture was composed of hydrolysis product 14j , increasing to nearly 50% by 24 h. resin still present at this time point . observations from the above studies are consistent with hydrolysis proceeding through a rate - limiting step in which a positively charged transition state is stabilized by an electron - donating r group . hammett substituent effects arising from r ( alkyl or aryl ) might be expected to be operative in the cyclization of 19 to 20 or in the acid - catalyzed ring opening of 21 to 3 . in the future , the substituent - dependent amide hydrolytic lability we have observed could be advantageously used . with the appropriate choice of the aryl group , a peptide temporarily acylated with arco - aa - oh ( ar = electron - rich aryl group , aa = amino acid residue ) could be converted back to the unacylated parent peptide under mild , perhaps even physiological , conditions . this report shows that amide bonds in simple n - acylated amino acid amide or peptide derivatives can be surprisingly unstable . when an electron - rich aromatic carboxylic acid is acylated to the amino nitrogen of a simple amino acid carboxamide or the n - terminus of a peptide , room - temperature trifluoroacetic acid / water mixtures result in hydrolysis of the amide bond four bonds away from the nh - acyl group . even simple aromatic acyl derivatives , when appropriately substituted with electron - donating substituents , can promote facile remote amide cleavage . in particular , this hydrolysis has been found to be sensitive to the substituent effects of r located on a remote n - terminal aromatic acyl group . in the case of n - benzoyl amides , the relative rate of hydrolysis can be predicted from the substituent s hammett value . acyl groups derived from secondary or tertiary alkyls are electron - rich and also promote substantial hydrolysis after 6 h at room temperature . this acyl group - dependent acid - catalyzed lability occurs under acidic conditions commonly employed when cleaving products from solid - phase resins . when these products are n - acylated peptides , the hydrolysis reaction could become problematic . it is important to note that in the current study n - capped dipeptides are also hydrolyzed under typical cleavage conditions . this suggests that unwanted peptide truncation may occur during synthesis or storage when dipeptides or longer peptides are acylated with electron - rich aromatic groups on the n - terminus . the combined ability to predict hydrolytic instability based on acyl group hammett values with the availability of cleavage conditions to minimize the predicted hydrolysis should help chemists working in this field to design appropriate synthetic strategies to n - acylated amino acid amides and peptides . fmoc - protected amino acid - bound rink amide mbha and wang resins were purchased from peptides international ( louisville , ky ) . synthetic sequences for the preparation of advanced intermediate resins were performed in 25 or 50 ml solid - phase peptide synthesis vessels ( spps ) purchased from chemglass . the vessels were rotated at room temperature for the required time period ( 0.5 , 1 , or 2 h ) and were then drained into 5-dram , tooled - neck vials . each of the 35 vessels in the 5 7 array ( 50 mol each ) was treated with 1.5 ml of 95:5 tfa / h2o and was rotated at room temperature for 30 min . g ( x = h , me , and ome ) , for the purpose of isolation and characterization , was accomplished by allowing their column 5 filtrates to stand at room temperature for 5 days . after standing at room temperature for 5 additional days , the filtrate from the 24 h postcleavage exposure was evaporated under a stream of nitrogen gas to give 14.4 mg of a residue that was chromatographed on a prepacked 500 mg column of hypersep cn ( cyanosilica , thermo scientific ) using step - gradient elution with hexanes ( 3 3 ml ) , 8:2 hexanes / acetone ( 3 ml ) , and eluting the desired material with 7:3 hexanes / acetone to give 6.9 mg ( 51% ) 11e as an amorphous solid , mp 171172 c ; h nmr ( 500.13 mhz , cdcl3 ) : 2.32 ( s , 3h ) , 3.23 ( dd , j = 14.1 and 6.0 hz , 1h ) , 3.32 ( dd , j = 14.1 and 5.6 hz , 1h ) , 5.03 ( m , 1h ) , 6.51 ( br d , j = 7.0 hz , 1h ) , 7.11 ( m , 4h ) , 7.42 ( t , j = 7.6 hz , 2h ) , 7.52 ( t , j = 7.4 hz , 1h ) , 7.68 ( d , j = 7.9 hz , 2h ) ; c nmr ( 125.77 mhz , cd3od ) : 21.1 , 37.8 , 55.7 , 128.4 , 129.5 , 130.1 , 130.2 , 132.8 , 135.4 , 135.6 , 137.5 , 170.2 , 174.9 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c17h18no3 , 284.1281 ; found , 284.1285 . after standing at room temperature for 5 additional days , the filtrate from the 24 h postcleavage exposure was evaporated under a stream of nitrogen gas to give 16.5 mg of a residue that was chromatographed on a dynamax microsorb c-18 , 5 m column ( 21.4 250 mm ) using the isochratic system of 50/50 1:1 mecn / meoh ( 5 mm ammonium acetate)/water ( 5 mm ammonium acetate ) at a 5 ml / min flow rate to give 8.6 mg ( 61% ) 11f as an amorphous solid after evaporation , mp 184187 c ; h nmr ( 500.13 mhz , cd3od ) : 2.26 ( s , 3h ) , 2.37 ( s , 3h ) , 3.07 ( dd , j = 13.8 and 8.3 hz , 1h ) , 3.27 ( dd , j = 13.8 and 4.9 hz , 1h ) , 4.75 ( dd , j = 8.2 and 5.0 hz , 1h ) , 7.05 ( d , j = 7.9 hz , 2h ) , 7.12 ( d , j = 8.0 hz , 2h ) , 7.23 ( d , j = 8.0 hz , 2h ) , 7.61 ( d , j = 8.2 hz , 2h ) ; c nmr ( 125.77 mhz , cd3od ) : 21.1 , 21.4 , 38.2 , 56.5 , 128.3 , 129.9 , 130.1 , 130.3 , 132.7 , 135.9 , 137.2 , 143.4 , 169.7 , 176.1 ; hrms ( esi - tof ) m / z : [ m + h ] calcd for c18h20no3 , 298.1438 ; found , 298.1441 . to prevent further hydrolysis of primary amides 13 after 30 min exposure to the tfa solution , the vials containing the bulk of the filtrates were stored at 20 c . two extra signals are also present in the c nmr of commercial fmoc - phe - oh ) ; hrms ( esi - tof ) m / z : [ m + na ] calcd for c24h22n2o3na , 409.1528 ; found , 409.1535 . and was purified for use as the authentic standard in the quantitative lc measurements ( optimization of cleavage conditions , table 4 ) . the contents were allowed to gradually warm to room temperature and were stirred for 18 h. the contents were cooled with an ice bath , and the solid was collected by suction filtration washing with a few milliliters of cold 1:1 acetone / water to afford 97 mg of crude 13k , which was triturated under hot methanol to give 29.5 mg ( 9% ) of the qlc calibration sample . all vessels of one bill - board were then treated with 1.5 ml of 95:5 tfa / water and were rotated for 30 min at room temperature . filtrates from a1 and b1 were sampled and evaporated to dryness and analyzed ( 0.5 h time point ) , filtrates from a2 and b2 were allowed to stand at room temperature for 6 h and were then analyzed by lc / ms , and then the filtrates from a3 and b3 , which had stood for 24 h , were analyzed .

in many developed countries , osteoporosis is now recognised as one of the most serious problems in public health [ 13 ] . for a 50-year - old white woman , the life - time risk of suffering a fragility fracture is estimated to be 3040% , which compares with the figures for breast cancer and cardiovascular disease of 912% and 3040% respectively . the increased recognition of the impact of osteoporosis on the lives of elderly people and the consequent costs of healthcare has led to the development of a variety of new treatments for preventing fractures [ 47 ] . scans to measure bone mineral density ( bmd ) using the technique of dual x - ray absorptiometry ( dxa ) are widely believed to be the most effective way of identifying patients at risk of fracture and targeting these treatments appropriately . conventionally , the hip and spine are regarded as the most important measurement sites because fractures at these sites have the greatest impact on quality of life , morbidity and mortality of patients . there is rapid skeletal growth until peak bone mass ( pbm ) is reached at around age 30 , after which there is minimal change until the menopause in women . following menopause pbm occurs in the third decade of life , but an inadequate nutritional intake of calcium can prevent optimum peak bone mass being achieved . low levels of physical activity during puberty can also lend to suboptimal bone density in later life . certain medical conditions and therapies can also affect bone metabolism and thus adversely affect bmd . long - term corticosteroid use is one such example , causing changes to trabecular bone structure , as well as reducing bmd . other factors known to compromise bmd include a previous atraumatic fracture or maternal hip fracture , a previous report of x - ray osteopenia , a positive family history of osteoporosis , a body mass index ( bmi ) of less than 19kg / m2 , smoking and suffering from rheumatoid arthritis . sex hormone deficiency , most notably oestrogen in females , is a well established contributor to the pathogenesis of osteoporosis . menopause results in oestrogen deficiency , which stimulates bone resorption and is associated with substantial bone loss continuing into old age . however , approximately 1% of women develop premature ovarian failure ( pof ) and go through an early menopause by the age of 40 . although pof patients commonly receive hormone replacement therapy ( hrt ) , their bmd remains significantly lower than that of age - matched controls . with such a variety of risk factors for a reduced bmd and osteoporosis , there is a need to identify patients who are at increased risk of sustaining fractures . the royal college of physicians in the united kingdom have issued guidelines for referring patients for bone densitometry investigations based on clinical risk factors . similar guidelines have also been published by the european foundation of osteoporosis and bone disease , the national osteoporosis foundation and the international society for clinical densitometry . all of the aforementioned guidelines include early menopause as a recognised risk factor for osteoporosis and as a basis to refer patients for a bone density examination . the most widely accepted method of performing bmd scans to establish a diagnosis of osteoporosis is the dxa technique . dxa involves scanning the lumbar spine and hip , measurement sites chosen because they are the most prone to osteoporotic fractures . however , dxa scanners are relatively expensive pieces of equipment and their availability is generally restricted to major hospitals . if the diagnostic benefits of bone densitometry are to be fully realised , smaller , cheaper devices are required . one possibility is the introduction of small dxa scanners designed to scan only the forearm . measurements of broadband ultrasonic attenuation ( bua ) in the calcaneus can discriminate elderly women with hip fractures and there is a consensus that qus has a potentially valuable role to assess fracture risk . the attraction to qus devices is that as well as being cheap and portable , they do not use ionising radiation . since fractures may be present or absent in patients with similar bmd , bone strength can not depend exclusively on bone density , but also on bone architecture . use of ultrasound in fracture risk assessment is thus advantageous as it seems to provide structural information , e.g. data on trabecular orientation , and can also reflect the mechanical properties of bone in addition to estimation of its density . the calcaneus is an easily accessible , weight - bearing site , rich in trabecular bone and has been the most extensively studied site with qus devices . qus measurements at this site have shown the ability to detect changes associated with age and menopause . measurements have also demonstrated the ability to differentiate healthy subjects from those with fractures and also identifying those who are at an increased risk of fracture . variations in temperature ( both ambient and of the patient s limb ) are also believed to have an adverse effect on measurements . an alternative , non - weight - bearing site for the assessment of bone mass using qus is at the phalanges . the phalanges are composed of predominantly cortical bone and the regions of interest are easily accessible . the dbm sonic bone profiler ( igea , italy ) has been designed to transmit a single ultrasound burst through the distal metaphyses of the proximal phalanges . bone resorption results in enlargement of the medullary canal and other structural changes , decreasing the ultrasound velocity and altering the characteristics of the signal arriving at the receiver probe . the hand is very sensitive to these changes and is thus ideal for such assessment . previous studies using the dbm sonic device have reported good precision with a coefficient of variation of 0.34% . there is also evidence suggesting that phalangeal qus measurements may be more sensitive than calcaneal measurements in identifying trends due to ageing and menopause . the purpose of the present study was to examine the correlation between qus parameters as measured at the proximal phalanges using the dbm sonic bone profiler and conventional dxa measurements of bmd at the spine and hip in caucasian women with pof . the study population was readily available to us from a local menopause clinic and hence women with pof were chosen as a specific group in which to assess the correlation . it was not the aim of the present study to determine the fracture discrimination capability of the dbm sonic device . thirteen caucasian women ( mean age 35.7 yrs , range 30.5 40.6 yrs ) , suffering from pof , were recruited from the menopause clinic at chelsea and westminster hospital ( london , uk ) . the control group consisted of 19 age - matched healthy caucasian women ( mean age 32.9 yrs , range 20.4 40.1 yrs ) and were recruited with advertisements placed around the hospital sites . the study was approved by the charing cross hospital research ethics committee and all participants gave written informed consent . health assessment was carried out using a detailed questionnaire which included questions relating to known risk factors for osteoporosis , detailed medical history of pof , and use of hrt . women on hrt were not excluded from the study . however , those with known risk factors for osteoporosis were excluded . these included women with a previous atraumatic fracture , and those on therapies or with conditions known to affect bmd ( including thyroid conditions , malabsorption and use of corticosteroids ) . bmd was measured with a lunar prodigy ( ge healthcare , madison , wi ) dxa scanner . measurements were performed at the lumbar spine ( l1l4 ) , hip ( total and neck of femur ) . in addition to recording bmd at these sites , we also recorded the t - score ( a measure of how a subjects bmd value compares to those of a normal young adult at pbm defined in terms of the standard deviation of young normal subjects ) and z - score ( a measure of how a subjects bmd compares to an age - matched population defined in terms of the standard deviation of the age - matched population ) . bmd measurement of the spine could not be obtained in one subject due to a navel ring , which could not be removed . thirteen caucasian women ( mean age 35.7 yrs , range 30.5 40.6 yrs ) , suffering from pof , were recruited from the menopause clinic at chelsea and westminster hospital ( london , uk ) . the control group consisted of 19 age - matched healthy caucasian women ( mean age 32.9 yrs , range 20.4 40.1 yrs ) and were recruited with advertisements placed around the hospital sites . the study was approved by the charing cross hospital research ethics committee and all participants gave written informed consent . health assessment was carried out using a detailed questionnaire which included questions relating to known risk factors for osteoporosis , detailed medical history of pof , and use of hrt . women on hrt were not excluded from the study . however , those with known risk factors for osteoporosis were excluded . these included women with a previous atraumatic fracture , and those on therapies or with conditions known to affect bmd ( including thyroid conditions , malabsorption and use of corticosteroids ) . bmd was measured with a lunar prodigy ( ge healthcare , madison , wi ) dxa scanner . measurements were performed at the lumbar spine ( l1l4 ) , hip ( total and neck of femur ) . in addition to recording bmd at these sites , we also recorded the t - score ( a measure of how a subjects bmd value compares to those of a normal young adult at pbm defined in terms of the standard deviation of young normal subjects ) and z - score ( a measure of how a subjects bmd compares to an age - matched population defined in terms of the standard deviation of the age - matched population ) . bmd measurement of the spine could not be obtained in one subject due to a navel ring , which could not be removed . qus measurements were performed on all subjects using the dbm sonic bone profiler ( igea , carpi , italy ) . a calliper was used to position the two probes ( transmitter and receiver ) laterally on either side of the metaphysis of the proximal phalanx , with ultrasound gel to achieve coupling . an ultrasound signal of 1.2mhz frequency is transmitted through the finger and an amplitude - dependent speed of sound ( ad - sos ) parameter is generated , which depends on both the amplitude and the velocity of the signal received . finger thickness and ad - sos measurements were obtained for digits ii v of the non - dominant hand . further measurement parameters included the ultrasound bone profile index ( ubpi ) , a number between 0 and 1 describing the fracture risk , and the bone transmission time ( btt ) - the interval between the first received signal and the received signal that is propagated through soft tissue only . t - scores and z - scores based on the ad - sos values are also automatically generated . duplicate measurements were performed for each subject to estimate precision , which was expressed as the coefficient of variation ( cv ) . the mean values of the two measurements for each parameter were recorded and used for analysis . ambient room temperature was kept constant with an air conditioning system as previous reports have suggested that qus measurements are temperature - dependent . daily quality control measurements to ensure consistency of calibration were performed with a phantom supplied by the manufacturer . student s t - test was used to compare differences in demographic variables and in qus and dxa parameters between the two groups . linear regression analysis was carried out to determine the correlation between qus and dxa parameters in ( i ) patients only ( n=13 ) , ( ii ) control group only ( n=19 ) ( iii ) all participating subjects ( n=32 ) . the following comparisons were made : spine bmd v ad - sos ; spine bmd v btt ; spine bmd v qus z - score ; spine z - score v ad - sos ; spine z - score v btt ; spine z - score v qus z - score . data analysis was performed using microsoft excel ( seattle , wa , usa ) . student s t - test was used to compare differences in demographic variables and in qus and dxa parameters between the two groups . linear regression analysis was carried out to determine the correlation between qus and dxa parameters in ( i ) patients only ( n=13 ) , ( ii ) control group only ( n=19 ) ( iii ) all participating subjects ( n=32 ) . the following comparisons were made : spine bmd v ad - sos ; spine bmd v btt ; spine bmd v qus z - score ; spine z - score v ad - sos ; spine z - score v btt ; spine z - score v qus z - score . duplicate qus measurements with repositioning on all 32 patients were combined to give short - term precision ( cv% ) of 0.37% , 3.36% 1.67% for ad - sos , ubpi and btt respectively . table 1 gives a summary of the demographic data , bmd parameters and qus parameters for the patient group and the controls . student s t - test showed no significant differences in any of the demographic or measurement parameters between the two groups . the correlation between remaining qus parameters and spine bmd are shown in table 2 . a significant correlation ( r=0.66 ) this relationship remained significant ( r=0.48 ) when all 32 subjects were included in the analysis ( figure 1 ) . all qus variables ( ad - sos , btt and z - score ) showed significant correlations ( r=0.47 to 0.60 ) with spine z - scores ( table 2 , figures 24 ) . the t - score is a linear function of ad - sos ( for qus measurements ) and bmd for ( dxa measurements ) . t - score correlations therefore provide no additional information and were excluded from the analysis . qus correlations with dxa measurements at the hip ( total hip and femoral neck sites ) were not significant ( table 2 ) . normalising the qus parameters by age and bmi did not improve the strength of correlation between any of above comparisons . as well as providing the mean ad - sos values , the bone profiler displays data for the thickness and ad - sos for each of the four phalanges measured . correlations between the ad - sos values for individual fingers and the dxa parameters were again moderate ( r= 0.44 0.63 ) but only significant when compared to spine z - scores ( table 3 ) . our results show a reasonably strong inverse correlation between all 3 qus parameters ( ad - sos , ubpi and btt ) and bmi ( table 4 ) . the strongest correlation was with ubpi in the pof patient group ( r = 0.78 ) ( figure 5 ) . similar inverse correlations were observed ( r = 0.48 to 0.76 ) between qus parameters and subjects weight . similar values were also observed when comparing weight and bmi with ad - sos values from individual fingers . the correlation between height and bmi was also established for dxa measurements at the spine and hip . moderate but significant positive correlations were observed with spine and hip bmd ( r = 0.37 0.62 ) . finally , the correlation of ad - sos values between the digits was established and found to be highly significant ( p<0.0001 ) . the strongest correlation ( r=0.96 ) student s t - test demonstrated significant differences in ad - sos and thickness values between fingers . the ad - sos values were greatest in phalanx iii , followed by iv , ii and v ( p < 0.02 ) . because of the growing demand for bone densitometry services , there is a need for cheap , safe and portable devices that can be used in a primary care setting to assess bmd and fracture risk . the ability of a device to independently assess fracture risk is best established through prospective fracture studies which can be expensive and time consuming . when evaluating new bone density scanners it is of interest to examine the correlation with dxa measurements at the spine and hip the established gold standard bone density measurements . in this study , we investigated the correlation between qus parameters obtained using the dbm sonic bone profiler and spine and hip measurements acquired on a lunar prodigy system . the precision for the prodigy dxa device has been previously quoted as 1.0% for the spine ( l1l4 ) , 0.9% for hip ( total ) and 1.5% for femoral neck . linear regression analysis between dxa and qus parameters showed a significant correlation of 0.66 ( p=0.01 ) between the btt value and spine bmd in women with pof , although this was not the case for other qus parameters . btt is a measure of the time delay between the two signals arriving at the receiver probe , with one having passed through bone and the other through the soft tissue phase . the significant correlation observed in our study could indicate that an improved bone structure at the phalanx correlates with higher bone density at the spine , and thus allows a more rapid transmission of the ultrasound signal through bone compared to soft tissue . although much of the literature currently focuses on the ad - sos values provided by the qus device , montagnani et al have presented data highlighting the importance of btt as the parameter most comparable to dxa and with the greatest ability to predict osteoporotic fractures . significant correlations of r=0.5 to 0.6 were observed between spine z - scores and ad - sos , as well as with btt . qus and dxa z - scores showed a moderate correlation when the data from patients and controls was pooled ( r=0.51 ) . although z - scores are not used in the diagnosis of osteoporosis our results suggest that this relationship between z - scores merits further investigation . if qus z - scores can predict dxa z - scores , it may suggest a role for qus measurements as a screening tool . it has previously been suggested that phalangeal qus z - scores may be a useful tool in the screening of bone disturbances in young patients with type i diabetes mellitus . it is possible that phalangeal qus may also have a role in other disease states affecting bone . regression analysis demonstrated that ad - sos , ubpi and qus z - score parameters are all inversely related with both bmi and weight . although the bone profiler accounts for soft tissue content , weak but significant correlations ( r = 0.30 to 0.32 ) between ad - sos and bmi have been reported previously . interestingly , btt does not show a similar correlation but was the only qus parameter to show a positive correlation with height in the pof group . a similarly significant correlation was seen between spine bmd and height ( r = 0.56 ) , which strengthens the argument for further research into the potential value of btt as a predictor of spine bmd . currently however , the association between qus and bmi remains unclear with some showing evidence of a significant correlation , whilst other data remains inconclusive . postulate qus may depend on body weight and bmi as the soft tissue surrounding the phalanges influences both acoustical contact and velocity . repeated student s t - test analyses for the differences in mean thickness between phalanges ii to v showed a significant , progressive increase in width from index to small fingers . however , the mean ad - sos values were significantly greater in the middle finger , followed by the ring , index and small fingers , implying that the qus ad - sos parameter is indeed not simply a function of the distance between the probes , but can identify quantitative differences between the structures of the digits . the potential of qus measurement to reflect bone structure , as well as bone density , has in fact been one of the driving forces for qus densitometry . qus measurements may have a role in assessing fracture risk in patients on corticosteroids for example , which are known to affect the structure of trabecular bone and have a detrimental effect over and beyond that reflected in a dxa measurement of low bone mass alone . analysis of the collected data has shown significant correlations between a number of qus and dxa device parameters , as well as upon comparison with demographics . perhaps the most promising of these is the correlation of spinal bmd with btt and , with an r2 value of 0.44 in the pof group , offers the best predictive ability of bmd compared to the other qus parameters . however , other studies have highlighted the use of alternative qus parameters for the estimation of bmd . alexandersen et al demonstrated weak but significant correlations between ad - sos and bmd at the spine and hip ( femoral neck ) ( r = 0.21 for both sites ) , whereas wuster et al obtained higher correlations ( r = 0.46 and r=0.36 spine and hip bmd , respectively ) . the lack of concordance with our study may lie in the differences between populations from denmark , scandinavia and the uk , respectively . furthermore , the bone profiler is a device with a higher sensitivity to skeletal changes occurring in the early postmenopausal period ( in the range 5065 ) , which encompasses the population used by wuster et al and thus providing the strongest correlations . the mean age in the alexandersen study was 69.9 years , at which age postmenopausal changes can still be identified . however , our study had a mean age for all subjects of 34.1 years , with one group having been through an early menopause and the other group acting as controls . thus , differences in the population age and inconsistencies of menopausal state of the subjects may account for the discrepancies . however , the unique patient group we selected has not been assessed by others in this manner . an unlikely alternative explanation for the discrepancies between studies is user - dependence which was highlighted as significant factor affecting the accuracy of measurements in a study by krieg et al who carried out measurements using the dbm sonic on over 7000 women . one advantage of assessing the human phalanges is their sensitivity to early changes in bone mass , and work originally based on animal studies has supported the argument that phalangeal ultrasound can identify architectural characteristics of bone . however , unlike the calcaneus , phalangeal bone is not subject to weight - bearing activity and thus provides weaker correlations compared to peripheral weight - bearing sites . furthermore , inaccurate positioning of the calliper on the phalanx can lead to errors , as the joint itself has greater trabecular bone content than the metaphysis and can therefore contribute to operator errors . qus values are also dependent on the temperature of the limb , which can be difficult to control in variable climatic conditions . however , in the present study room temperature was controlled using an air conditioning system . one further source of measurement error that has been reported is that the phalangeal joints are common sites for osteoarthritis , which could interfere with qus measurements . the stronger correlation of our results occurring between qus and dxa at the spine , as opposed to at the hip , may partly be explained by the fact that trabecular rich vertebrae are more sensitive in identifying changes occurring with age or therapy compared to the hip . as well as the relatively small sample size , there were further limitations to our study . hrt is known to significantly reduce the impact of the menopause on bone loss but its duration of use in the present study ( 4 months to 7 yrs ) varied widely between patients . uygur et al have suggested that current hrt regimes , designed for women undergoing natural menopause , may be inappropriate for some women with pof and thus cause further variations in the effects of hrt in pof groups . the potential benefits for phalangeal qus to be adopted as a screening device could extend beyond its correlation with dxa . other studies have shown the ability of the phalangeal qus device to identify changes associated with age and with the menopause , but also to predict fracture risk [ 5659 ] . although qus can not provide a direct measurement of bmd itself , its potential for detecting structural changes may be more beneficial than established areal bmd measurements . our study suggests that there may be a need to move away from focusing on the ad - sos parameter in place of the btt , and even a greater role for z - scores . btt has shown stronger correlation with dxa values and stronger predictive r2 ability compared to other qus parameters . whilst some authors have described a combination of clinical risk factors and qus values in the form of a nomogram as the ideal method of screening for increased fracture risk , others believe the complex soft tissue bone ultrasound interactions and the resultant waveform they produce should be analysed . in either case , the aim is to identify patients at risk of fracture before the fracture occurs . possible further roles for phalangeal qus may include measurements in children on long - term corticosteroids . all this is only possible through studies such as this being able to identify and harness the great expectations with which these devices were introduced , and allow early preventative interventions to be taken in high - risk groups . the strongest correlation was observed between btt and spine bmd , as well as between the z - scores from the two devices . these correlations were seen within the pof group , control group and when data for all patients was pooled . our data support further studies to evaluate the ability of phalangeal qus measurements to independently assess fracture risk in patients with and without different risk factors . such studies will help establish whether phalangeal qus can be used as a screening tool in the primary care setting . currently , the best measurement sites for qus and the ideal parameters with which to form guidelines still have to be established .

objectiveswith the growing demand for bone densitometry services there is a need for simple , cost - effective and ideally mobile devices which can identify individuals who are at risk of osteoporotic fracture . when new devices are evaluated , it is useful to examine the correlation with the established gold standard technique of dual x - ray absorptiometry ( dxa ) . this study examined the correlation between quantitative ultrasound ( qus ) measurements performed at the phalanges and conventional dxa measurements of the spine and hip in women with premature ovarian failure a known risk factor for osteoporosis.methodsthirteen white caucasian women suffering from premature ovarian failure and 19 age- and sex - matched controls were recruited into the study . dxa measurements were performed at the spine and hip , followed by quantitative ultrasonography at phalanges ii v of the non - dominant hand.resultssignificant correlations were observed between the bone transit time ( btt ) value from the bone profiler and bone mineral density measured at the spine ( r=0.66 ) . the spine z - scores also correlated with many of the ultrasound values ( r=0.44 0.63 ) . significant inverse correlations were observed between bmi , weight and ultrasound parameters ( r = 0.48 to 0.78).conclusionwe have reported moderate but significant correlations between phalangeal qus and dxa parameters . the strongest correlation was observed between btt and spine bmd , as well as between the z - scores from the two devices . qus parameters also demonstrated an inverse correlation with weight and bmi .

INTRODUCTION METHODS Study Population Dual X-ray Absorptiometery QUS Statistical Analysis RESULTS DISCUSSION CONCLUSION

in many developed countries , osteoporosis is now recognised as one of the most serious problems in public health [ 13 ] . for a 50-year - old white woman , the life - time risk of suffering a fragility fracture is estimated to be 3040% , which compares with the figures for breast cancer and cardiovascular disease of 912% and 3040% respectively . scans to measure bone mineral density ( bmd ) using the technique of dual x - ray absorptiometry ( dxa ) are widely believed to be the most effective way of identifying patients at risk of fracture and targeting these treatments appropriately . there is rapid skeletal growth until peak bone mass ( pbm ) is reached at around age 30 , after which there is minimal change until the menopause in women . long - term corticosteroid use is one such example , causing changes to trabecular bone structure , as well as reducing bmd . other factors known to compromise bmd include a previous atraumatic fracture or maternal hip fracture , a previous report of x - ray osteopenia , a positive family history of osteoporosis , a body mass index ( bmi ) of less than 19kg / m2 , smoking and suffering from rheumatoid arthritis . sex hormone deficiency , most notably oestrogen in females , is a well established contributor to the pathogenesis of osteoporosis . however , approximately 1% of women develop premature ovarian failure ( pof ) and go through an early menopause by the age of 40 . although pof patients commonly receive hormone replacement therapy ( hrt ) , their bmd remains significantly lower than that of age - matched controls . with such a variety of risk factors for a reduced bmd and osteoporosis , there is a need to identify patients who are at increased risk of sustaining fractures . the royal college of physicians in the united kingdom have issued guidelines for referring patients for bone densitometry investigations based on clinical risk factors . all of the aforementioned guidelines include early menopause as a recognised risk factor for osteoporosis and as a basis to refer patients for a bone density examination . dxa involves scanning the lumbar spine and hip , measurement sites chosen because they are the most prone to osteoporotic fractures . if the diagnostic benefits of bone densitometry are to be fully realised , smaller , cheaper devices are required . measurements of broadband ultrasonic attenuation ( bua ) in the calcaneus can discriminate elderly women with hip fractures and there is a consensus that qus has a potentially valuable role to assess fracture risk . the attraction to qus devices is that as well as being cheap and portable , they do not use ionising radiation . since fractures may be present or absent in patients with similar bmd , bone strength can not depend exclusively on bone density , but also on bone architecture . measurements have also demonstrated the ability to differentiate healthy subjects from those with fractures and also identifying those who are at an increased risk of fracture . an alternative , non - weight - bearing site for the assessment of bone mass using qus is at the phalanges . the phalanges are composed of predominantly cortical bone and the regions of interest are easily accessible . the dbm sonic bone profiler ( igea , italy ) has been designed to transmit a single ultrasound burst through the distal metaphyses of the proximal phalanges . bone resorption results in enlargement of the medullary canal and other structural changes , decreasing the ultrasound velocity and altering the characteristics of the signal arriving at the receiver probe . previous studies using the dbm sonic device have reported good precision with a coefficient of variation of 0.34% . there is also evidence suggesting that phalangeal qus measurements may be more sensitive than calcaneal measurements in identifying trends due to ageing and menopause . the purpose of the present study was to examine the correlation between qus parameters as measured at the proximal phalanges using the dbm sonic bone profiler and conventional dxa measurements of bmd at the spine and hip in caucasian women with pof . the study population was readily available to us from a local menopause clinic and hence women with pof were chosen as a specific group in which to assess the correlation . it was not the aim of the present study to determine the fracture discrimination capability of the dbm sonic device . thirteen caucasian women ( mean age 35.7 yrs , range 30.5 40.6 yrs ) , suffering from pof , were recruited from the menopause clinic at chelsea and westminster hospital ( london , uk ) . the control group consisted of 19 age - matched healthy caucasian women ( mean age 32.9 yrs , range 20.4 40.1 yrs ) and were recruited with advertisements placed around the hospital sites . the study was approved by the charing cross hospital research ethics committee and all participants gave written informed consent . health assessment was carried out using a detailed questionnaire which included questions relating to known risk factors for osteoporosis , detailed medical history of pof , and use of hrt . women on hrt were not excluded from the study . however , those with known risk factors for osteoporosis were excluded . measurements were performed at the lumbar spine ( l1l4 ) , hip ( total and neck of femur ) . in addition to recording bmd at these sites , we also recorded the t - score ( a measure of how a subjects bmd value compares to those of a normal young adult at pbm defined in terms of the standard deviation of young normal subjects ) and z - score ( a measure of how a subjects bmd compares to an age - matched population defined in terms of the standard deviation of the age - matched population ) . bmd measurement of the spine could not be obtained in one subject due to a navel ring , which could not be removed . thirteen caucasian women ( mean age 35.7 yrs , range 30.5 40.6 yrs ) , suffering from pof , were recruited from the menopause clinic at chelsea and westminster hospital ( london , uk ) . the control group consisted of 19 age - matched healthy caucasian women ( mean age 32.9 yrs , range 20.4 40.1 yrs ) and were recruited with advertisements placed around the hospital sites . the study was approved by the charing cross hospital research ethics committee and all participants gave written informed consent . health assessment was carried out using a detailed questionnaire which included questions relating to known risk factors for osteoporosis , detailed medical history of pof , and use of hrt . women on hrt were not excluded from the study . however , those with known risk factors for osteoporosis were excluded . measurements were performed at the lumbar spine ( l1l4 ) , hip ( total and neck of femur ) . in addition to recording bmd at these sites , we also recorded the t - score ( a measure of how a subjects bmd value compares to those of a normal young adult at pbm defined in terms of the standard deviation of young normal subjects ) and z - score ( a measure of how a subjects bmd compares to an age - matched population defined in terms of the standard deviation of the age - matched population ) . bmd measurement of the spine could not be obtained in one subject due to a navel ring , which could not be removed . qus measurements were performed on all subjects using the dbm sonic bone profiler ( igea , carpi , italy ) . a calliper was used to position the two probes ( transmitter and receiver ) laterally on either side of the metaphysis of the proximal phalanx , with ultrasound gel to achieve coupling . an ultrasound signal of 1.2mhz frequency is transmitted through the finger and an amplitude - dependent speed of sound ( ad - sos ) parameter is generated , which depends on both the amplitude and the velocity of the signal received . finger thickness and ad - sos measurements were obtained for digits ii v of the non - dominant hand . further measurement parameters included the ultrasound bone profile index ( ubpi ) , a number between 0 and 1 describing the fracture risk , and the bone transmission time ( btt ) - the interval between the first received signal and the received signal that is propagated through soft tissue only . t - scores and z - scores based on the ad - sos values are also automatically generated . duplicate measurements were performed for each subject to estimate precision , which was expressed as the coefficient of variation ( cv ) . the mean values of the two measurements for each parameter were recorded and used for analysis . daily quality control measurements to ensure consistency of calibration were performed with a phantom supplied by the manufacturer . student s t - test was used to compare differences in demographic variables and in qus and dxa parameters between the two groups . linear regression analysis was carried out to determine the correlation between qus and dxa parameters in ( i ) patients only ( n=13 ) , ( ii ) control group only ( n=19 ) ( iii ) all participating subjects ( n=32 ) . the following comparisons were made : spine bmd v ad - sos ; spine bmd v btt ; spine bmd v qus z - score ; spine z - score v ad - sos ; spine z - score v btt ; spine z - score v qus z - score . student s t - test was used to compare differences in demographic variables and in qus and dxa parameters between the two groups . linear regression analysis was carried out to determine the correlation between qus and dxa parameters in ( i ) patients only ( n=13 ) , ( ii ) control group only ( n=19 ) ( iii ) all participating subjects ( n=32 ) . the following comparisons were made : spine bmd v ad - sos ; spine bmd v btt ; spine bmd v qus z - score ; spine z - score v ad - sos ; spine z - score v btt ; spine z - score v qus z - score . table 1 gives a summary of the demographic data , bmd parameters and qus parameters for the patient group and the controls . student s t - test showed no significant differences in any of the demographic or measurement parameters between the two groups . the correlation between remaining qus parameters and spine bmd are shown in table 2 . a significant correlation ( r=0.66 ) this relationship remained significant ( r=0.48 ) when all 32 subjects were included in the analysis ( figure 1 ) . all qus variables ( ad - sos , btt and z - score ) showed significant correlations ( r=0.47 to 0.60 ) with spine z - scores ( table 2 , figures 24 ) . the t - score is a linear function of ad - sos ( for qus measurements ) and bmd for ( dxa measurements ) . qus correlations with dxa measurements at the hip ( total hip and femoral neck sites ) were not significant ( table 2 ) . normalising the qus parameters by age and bmi did not improve the strength of correlation between any of above comparisons . as well as providing the mean ad - sos values , the bone profiler displays data for the thickness and ad - sos for each of the four phalanges measured . correlations between the ad - sos values for individual fingers and the dxa parameters were again moderate ( r= 0.44 0.63 ) but only significant when compared to spine z - scores ( table 3 ) . our results show a reasonably strong inverse correlation between all 3 qus parameters ( ad - sos , ubpi and btt ) and bmi ( table 4 ) . the strongest correlation was with ubpi in the pof patient group ( r = 0.78 ) ( figure 5 ) . similar inverse correlations were observed ( r = 0.48 to 0.76 ) between qus parameters and subjects weight . similar values were also observed when comparing weight and bmi with ad - sos values from individual fingers . the correlation between height and bmi was also established for dxa measurements at the spine and hip . moderate but significant positive correlations were observed with spine and hip bmd ( r = 0.37 0.62 ) . finally , the correlation of ad - sos values between the digits was established and found to be highly significant ( p<0.0001 ) . the strongest correlation ( r=0.96 ) student s t - test demonstrated significant differences in ad - sos and thickness values between fingers . the ad - sos values were greatest in phalanx iii , followed by iv , ii and v ( p < 0.02 ) . because of the growing demand for bone densitometry services , there is a need for cheap , safe and portable devices that can be used in a primary care setting to assess bmd and fracture risk . when evaluating new bone density scanners it is of interest to examine the correlation with dxa measurements at the spine and hip the established gold standard bone density measurements . in this study , we investigated the correlation between qus parameters obtained using the dbm sonic bone profiler and spine and hip measurements acquired on a lunar prodigy system . the precision for the prodigy dxa device has been previously quoted as 1.0% for the spine ( l1l4 ) , 0.9% for hip ( total ) and 1.5% for femoral neck . linear regression analysis between dxa and qus parameters showed a significant correlation of 0.66 ( p=0.01 ) between the btt value and spine bmd in women with pof , although this was not the case for other qus parameters . btt is a measure of the time delay between the two signals arriving at the receiver probe , with one having passed through bone and the other through the soft tissue phase . the significant correlation observed in our study could indicate that an improved bone structure at the phalanx correlates with higher bone density at the spine , and thus allows a more rapid transmission of the ultrasound signal through bone compared to soft tissue . although much of the literature currently focuses on the ad - sos values provided by the qus device , montagnani et al have presented data highlighting the importance of btt as the parameter most comparable to dxa and with the greatest ability to predict osteoporotic fractures . significant correlations of r=0.5 to 0.6 were observed between spine z - scores and ad - sos , as well as with btt . qus and dxa z - scores showed a moderate correlation when the data from patients and controls was pooled ( r=0.51 ) . although z - scores are not used in the diagnosis of osteoporosis our results suggest that this relationship between z - scores merits further investigation . if qus z - scores can predict dxa z - scores , it may suggest a role for qus measurements as a screening tool . it has previously been suggested that phalangeal qus z - scores may be a useful tool in the screening of bone disturbances in young patients with type i diabetes mellitus . it is possible that phalangeal qus may also have a role in other disease states affecting bone . regression analysis demonstrated that ad - sos , ubpi and qus z - score parameters are all inversely related with both bmi and weight . although the bone profiler accounts for soft tissue content , weak but significant correlations ( r = 0.30 to 0.32 ) between ad - sos and bmi have been reported previously . interestingly , btt does not show a similar correlation but was the only qus parameter to show a positive correlation with height in the pof group . a similarly significant correlation was seen between spine bmd and height ( r = 0.56 ) , which strengthens the argument for further research into the potential value of btt as a predictor of spine bmd . currently however , the association between qus and bmi remains unclear with some showing evidence of a significant correlation , whilst other data remains inconclusive . postulate qus may depend on body weight and bmi as the soft tissue surrounding the phalanges influences both acoustical contact and velocity . however , the mean ad - sos values were significantly greater in the middle finger , followed by the ring , index and small fingers , implying that the qus ad - sos parameter is indeed not simply a function of the distance between the probes , but can identify quantitative differences between the structures of the digits . the potential of qus measurement to reflect bone structure , as well as bone density , has in fact been one of the driving forces for qus densitometry . analysis of the collected data has shown significant correlations between a number of qus and dxa device parameters , as well as upon comparison with demographics . perhaps the most promising of these is the correlation of spinal bmd with btt and , with an r2 value of 0.44 in the pof group , offers the best predictive ability of bmd compared to the other qus parameters . alexandersen et al demonstrated weak but significant correlations between ad - sos and bmd at the spine and hip ( femoral neck ) ( r = 0.21 for both sites ) , whereas wuster et al obtained higher correlations ( r = 0.46 and r=0.36 spine and hip bmd , respectively ) . furthermore , the bone profiler is a device with a higher sensitivity to skeletal changes occurring in the early postmenopausal period ( in the range 5065 ) , which encompasses the population used by wuster et al and thus providing the strongest correlations . one advantage of assessing the human phalanges is their sensitivity to early changes in bone mass , and work originally based on animal studies has supported the argument that phalangeal ultrasound can identify architectural characteristics of bone . furthermore , inaccurate positioning of the calliper on the phalanx can lead to errors , as the joint itself has greater trabecular bone content than the metaphysis and can therefore contribute to operator errors . qus values are also dependent on the temperature of the limb , which can be difficult to control in variable climatic conditions . the stronger correlation of our results occurring between qus and dxa at the spine , as opposed to at the hip , may partly be explained by the fact that trabecular rich vertebrae are more sensitive in identifying changes occurring with age or therapy compared to the hip . as well as the relatively small sample size , there were further limitations to our study . uygur et al have suggested that current hrt regimes , designed for women undergoing natural menopause , may be inappropriate for some women with pof and thus cause further variations in the effects of hrt in pof groups . the potential benefits for phalangeal qus to be adopted as a screening device could extend beyond its correlation with dxa . other studies have shown the ability of the phalangeal qus device to identify changes associated with age and with the menopause , but also to predict fracture risk [ 5659 ] . our study suggests that there may be a need to move away from focusing on the ad - sos parameter in place of the btt , and even a greater role for z - scores . btt has shown stronger correlation with dxa values and stronger predictive r2 ability compared to other qus parameters . in either case , the aim is to identify patients at risk of fracture before the fracture occurs . the strongest correlation was observed between btt and spine bmd , as well as between the z - scores from the two devices . such studies will help establish whether phalangeal qus can be used as a screening tool in the primary care setting . currently , the best measurement sites for qus and the ideal parameters with which to form guidelines still have to be established .

a little over a decade ago , it was generally accepted that the human heart is a terminally differentiated organ lacking any intrinsic regenerative capacity . after birth , the cardiomyocytes undergo a final round of cell division with a portion of the cardiomyocytes becoming binuclear and/or polypoid [ 14 , 82 , 83 ] . the observed post - natal growth is attributed to mainly to hypertrophic growth of the resident cardiomyocytes , with very no contribution of cell division . in line with these findings it was observed that amputation of the ventricular apex at 1 day after birth in mice tolerates full regeneration of this region , whereas at 7 days post natal this capacity is completely lost . the dogma that the adult heart is a post - mitotic organ , was challenged for the first time in 1998 by kajstura et al . , who showed that a small ( 0.001% ) portion of cardiomyocytes undergo mitosis in normal hearts . though numerous subsequent follow - up studies have reported slightly differing numbers , the issue remained controversial . bergmann et al . , finally added real weight to the idea that cardiomyocytes can and do undergo mitosis in adult hearts , using c14 radiocarbon dating of human genomic dna . this experiment resembles that of a pulse - chase experiment in which genomic dna was labelled as a result of trace amounts of radioactive nuclear fallout produced as a result of nuclear weapons testing during the cold war . according to their study , at the age of 25 cardiomyocytes have a mitotic turn - over of approximately 1% per year . although this value gradually decreases with age , the numbers suggest that about 50% of resident cardiomyocytes are renewed during life . however , the initial challengers of the dogma , revisited and expanded their initial study using state of the art tools reporting a similar decrease in myocyte turn - over rate with age . interestingly , based on their methods and estimations the cardiomyocytes of the heart would are renewed 10-times during a normal life span . although this long lasting discussion has regained a renewed interest , it is evident that the adult human heart has a limited endogenous regenerative capacity . this capacity is insufficient ( 1 ) to maintain the number of cardiomyocytes during aging and ( 2 ) to replace cardiomyocytes lost upon injury and/or disease , leading to decreased heart function and eventually heart failure . a second question directly relating to this endogenous regenerative capacity is the cellular origin of the newly formed cardiomyocytes . the quest for progenitors of cardiomyocytes within the heart has led to the identification of a multitude of cardiac stem cells or progenitors , each with a different molecular signature [ 41 , 53 ] . ckit ( cd117 ) and flk-1 are but two of the markers used to identify precursor cells contributing to the cardiomyocyte , smooth muscle and endocardial lineage . facs sorting of the ckit - positive population revealed that their contribution to the heart gradually decreases with age , from 0.65% , of which 10% is cd45-(hematopoietic)positive , in neonatal mouse heart to 0.5% , of which 15% is cd45-positive , in the adult heart . such cells can also be isolated from the human heart as well . though absolute numbers are not known , they are rare and their number also decreases with age [ 29 , 30 ] . co - culturing neonatal ckit - positive cells with foetal cardiomyocytes results in robust differentiation into cardiomyocytes , whereas adult ckit - positive cells appear to have lost this capacity . in vivo testing using mouse models revealed that injecting these cells into an infarct showed no significant differentiation into cardiomyocytes [ 10 , 29 , 30 , 88 ] . though it is currently accepted that cardiac stem cells or progenitors exist , it is as yet not known what factors are needed to expand this population and to induce their differentiation into functional cardiomyocytes . with the derivation of the first human embryonic stem cells ( hesc ) by thomson et al . , the field of stem cell research caught the scientific and public domain s attention , bringing an understandable high level of hope for new breakthroughs in regenerative medicine . despite the ensuing reactions regarding the ethical issues that surround the isolation and use of cells that are of a human embryonic origin , the prospect of being able to treat a multiplicity of diseases , particularly the appeal of finally being able to restore function of tissue lost during a myocardial infarct , stem cell research took off at an incredible rate ( fig . 1 ) however , apart from the ethical issues surrounding this research , hesc , as we shall discuss , have thrown up many obstacles on their route to the clinic resulting in a re - evaluation of strategies being taken to implement their use . although other stem cell sources have been applied more readily in the clinical setting , hescs have only just received the first green light for an fda approved human trial to investigate their applicability for the treatment of complete thoracic spinal severance .fig . 1annual publication frequency within the field of stem cells and cardiac regeneration annual publication frequency within the field of stem cells and cardiac regeneration embryonic stem cells are derived from the inner cell mass of the blastocyst ~5 days post fertilization . given the diverse and sometimes incorrect use of the word stem cell , esc were described using the physical characteristics ( i ) derived from the pre - implanted embryo , ( ii ) prolonged , undifferentiated proliferation and ( iii ) the potential to develop and give rise to all three germinal layers ( endoderm , mesoderm and ectoderm ) . the molecular definition of hesc remains a challenge , due in part to the heterogeneity that already exists even at the blastocyst stage . this is perhaps not surprising since the inner cell mass will rapidly give rise to the ordered three layered germinal disc from which the embryo will develop . cells may therefore even at this stage differ from their neighbouring cells and begin to display the molecular characteristics of one of the germinal layers , which in turn could be composed of cells that differ from each other . however , markers , have been identified which serve as tools for sorting and cellular selection . the two glycosphingolipid surface antigen markers ssea-3 and ssea-4 are , are identified on human embryonal carcinoma ( ec ) cell lines , on early cell - cleavage mouse embryos and later on cells of the inner cell mass of human blastocysts . in contrast to mice , which have lost expression of these antigens by the blastocyst stage , these markers along with two others also identified previously in human ec cells ( tra-1 - 60 and tra-1 - 81 ) appear to become down - regulated during differentiation , a feature which is also observed in hesc cultures exposed to retinoic acid [ 23 , 33 ] . although the exact molecular definition of these surface markers remains to be resolved , experiments suggest that they represent carbohydrate modifications , possibly linked to the glycoprotein podocalyxin [ 51 , 64 ] . the expression of these markers on hesc and their subsequent loss during differentiation suggests a critical functional role in cell pluripotency . experiments involving the depletion of ssea-3 and ssea-4 on hescs using inhibitors of sphingolipid synthesis appear to show that these markers do not play a critical role in maintenance of the undifferentiated state . although the function of most of the cell surface markers used for hesc selection remain unknown , the value of identifying unique markers is obvious and remains an area of keen research . has identified a novel surface molecule , e1b - ap5 which co - localizes together with ssea-3 , ssea-4 , tra-1 - 60 and tra-1 - 81 and becomes down - regulated during differentiation . however , unlike these markers , e1b - ap5 s molecular identity is better defined , representing an adenovirus nuclear rna - binding protein which appears to also be expressed at the plasma membrane as well as in the nucleus of hesc cells . a further , perhaps more comprehensive , approach to characterizing undifferentiated hescs has been brought about by the introduction of high - throughput molecular techniques such as microarrays , chip - seq and rna - seq ( reviewed in ) . this work has led to the identification of a core set of transcription factors consisting of oct3/4 ( also known as pou5f1 ) , the sex determining region y protein sox2 , the homeobox protein nanog and the pr - domain containing zinc finger protein prdm14 . these factors are key in the process of pluripotency maintenance , though their individual expression patterns are not all restricted to this earliest phase of development , many being required during later key differentiation and cell lineage decisions . not coincidently then , sox2 and oct3/4 constitute 2 of the factors used in the initial studies aimed at direct reprogramming of somatic cells to pluripotent stem - like cells ( ips ) . the issues and details surrounding ips cells will be discussed later . in line with this , work from early 2000 demonstrated that a reduction of oct3/4 led to a regression of established es cell lines to a more trophoblast like state and that an increase in expression of less than two - fold led to differentiation towards the mesodermal and endodermal states . sox2 has been shown to directly interact with oct3/4 , forming a synergistic regulatory complex regulating many pluripotent stem cell genes . have proposed that the major essential function of sox2 is to stabilize es pluripotency by maintaining the strict levels of oct3/4 that a requisite to this undifferentiated state . in an attempt to try and provide a more comprehensive and systematic view of markers present on hesc lines present in different laboratories around the world , one of the major conclusions of this study indeed confirmed that the lines were divergent in character ; similar expression patterns of all the above mentioned markers were found along with several other unique identifiers . research surrounding the potential of hescs for regeneration therapies , although real , has encountered several major obstacles . one of the major , aside from the ethical issues , has been the high risk of teratoma formation . teratomas are in general benign tumours made up of mixtures of different cell and tissue types caused by the presence of contaminating co - transplanted differentiated cells . although the answer to this potential danger appeared to be pre - differentiation of hesc before injection , following experiments showing the apparent tumour free application of pre - differentiated hesc in animal models , this could not exclude a potential risk if used in the human clinical setting . this fear may recently have been justified with the first description of a patient treated with multiple injection rounds of foetal neural stem cells for a neurodegenerative disorder developed a multifocal brain tumour . although the potential for replacement therapy has received much of its acclaim in field of the embryonic stem cell , perhaps more due to the open ethical discussions surrounding such research , initial studies that recognized the potential of stem cells were focused on haematopoietic and bone marrow derived cells . the relative success and apparent safety of bone marrow replacement therapy , perhaps then contributed to the rapid cardiac clinical setting of this cell source . to this day , bmc has been the most frequently tapped source of cells used in clinical studies and trials aimed at cardiac repair after myocardial infarction . typically , bmcs are isolated by aspiration from the iliac crest of the patient themselves , reducing the risks of immunological response in terms of cellular therapy . after an initial phase of processing , often sedimentation or gradient centrifugation , the bmc cell suspension consists of progenitors of endothelial origin ( ~4% ) , mesenchymal stem cells ( < 0.1% ) and a very small number of so - called side population cells including a recently identified very small embryonic - like stem cell ( vsel ) [ 70 , 84 ] . the rest of the cellular material , by far the largest portion , is of non - progenitor type . the heterogeneity of the cell population itself and the techniques used for isolation has made the characterization and comparison of sub - populations using cell surface markers difficult . groups have often selected their own set of markers to define their set of enriched bmc derived cells . the cluster of differentiation ( cd ) antigens cd14 , cd34 , cd45 and cd133 [ 45 , 65 ] have been used to identify and enrich hematopoietic progenitor cell populations ( reviewed in ) . recently , riekstina and co - workers examined and determined the presence or absence of a large range of known stem cell markers in an attempt to characterize the mesenchymal progenitor populations from a number of sources including human bone marrow . like the hesc , the presence of ssea-4 , oct3/4 and nanog could be detected in bone marrow derived mesenchymal stem cells ( mscs ) , though sox2 appeared to be specifically absent . further , all sources of mscs tested appeared positive for the cell surface markers cd73 , cd90 and cd105 and lacked expression cd34 . as mentioned earlier , one feature that sets bmc and the sub - derived cell populations has been their rapid implementation in patient clinical trials . since the 12 month and 18 month data from a large number of these trials has been discussed and ( meta-)analyzed elsewhere [ 1 , 36 , 44 , 47 ] , we shall only discuss three of the longer - term follow - up clinical trials , though data from such combined analyzes would seem to support the notion of modest improvement in cardiac function after acute myocardial infarction . most randomized trials have typically only covered a short - term follow - up of 412 months , making assessment of the true efficacy and sustained effect of such a therapy difficult . further , although apparently safe in the short - term application , long - term safety issues have been raised based on reports of associated risks of arrhythmias and calcification [ 81 , 87 ] . the astami ( autologous stem cell transplantation in acute myocardial infarction ) , repair - ami ( reinfusion of enriched progenitor cells and infarct remodelling in acute myocardial infarction ) [ 5 , 62 ] and boost ( bone marrow transfer to enhance st - elevation infarct regeneration trials ) all made use of a randomized controlled patient study in which patients had received , after percutaneous coronary intervention with stenting , an intracoronary injection of bmcs . all three trials found that the application of bmcs in the clinical setting is safe . however , only the repair - ami ( 2 years follow - up ) , but not astami ( 3 year follow - up ) and boost ( 5 year follow - up ) studies , showed a significant positive benefit in left ventricular ejection fraction . this finding can also be concluded from the boost 18 month follow - up results and most other clinical bmc trials , with patients typically displaying a 23% improvement in left ventricular function . however , after this period , as with the astami study , boost results suggest that this effect steadily declines to become non - significant when compared to control groups . one can perhaps then conclude from these studies that in the long - term there is no observable benefit from the administration of bmcs to patients that have suffered an ami . however , one should perhaps not be too quick to be dismissive of this cell source . therefore , the lack of information as to why these positive effects disappear during a longer - term follow - up , demonstrates that a more basic approach to understanding the actual mechanisms underlying the observed effects is essential for correct interpretation of the end results . understanding the underlying mechanism one might be able to focus research and boost the initial as well as sustain these beneficial effects . one interesting and plausible explanation for at least some of the short - term positive effects observed could be the release of paracrine factors by the injected cell source , which serve to assist recovery of the damaged tissues . paracrine factors , such as cytokines and growth factors , are often released by cells surrounding areas of tissue damage , functioning as , for instance , circulatory chemoattractants mediating homing of macrophages and stem cells to the site of injury . effects may be as diverse as neovascularization associated with release of vegf and igf , fibrosis and of particular interest in ami repair down - regulation thereof ( for example hgf ) and even cell survival . this may also go some way to explaining why multiple cell sources can have positive effects during the first few months of ami cellular treatment and recovery . to test this hypothesis , ziebert and co - workers made use of an inducible thymidine kinase suicide system to deplete transplanted cells in animal models . induction of this system at specific time points after cell transplantation was observed to reduce the normally observed effects of neovascularization and decrease the level of recovery of left ventricular function and capillary density . although the paracrine effects in human clinical trials can not easily be determined , bmc have been shown to release such factors when transplanted into models of ischemia [ 39 , 74 ] . not only is the isolation of bone marrow a rather painful procedure , but also the absolute number of stem cells within the bone marrow is low and their molecular signature is poorly defined . in this respect , adipose tissue could be an attractive alternative source , as it is most often abundantly present in people suffering from cardiac disease , easy to harvest using liposuction , and relatively rich in stem cells . adipose tissue has been shown to have more than 100-fold higher stem cell density than bone marrow . adipose tissue gained an interest as a potential cellular source for cardiac regeneration when in vitro experiments showed that dispersed adipose tissue can be induced to differentiate into cells of various lineages and even into cardiomyocytes . these experiments revealed that ~0.05% of the plated cells spontaneously differentiated into cardiomyocytes within 20 days of culture . infusion of non pre - selected or pre - selection of cd44 ( hyaluronic acid receptor ) , cd105 ( endoglin ) and cd34-negative adipose - derived cells in animal models of acute myocardial infarction , displayed an improved lv - ejection fraction and wall thickness compared to pbs injected controls [ 8 , 16 , 75 , 89 ] in contrast to several studies which apparently demonstrated a loss of infused cells after injection , recent studies using bioluminescent imaging of luciferase transfected adipose - derived cells could be identified in the recipient heart 4 weeks after administration . histological analysis revealed that the adipose - derived cells had differentiated into cardiomyocytes , smooth muscle cells and endothelial cells in the infracted region . moreover , these cells were found to secrete growth factors , like vegf or igf - i , possibly contributing to the observed enhanced cardiac function indirectly [ 7 , 8 ] . despite several unresolved issues , two double - blind , randomized , placebo - controlled phase i trials , apollo and precise , were initiated in 2007 ; apollo , adipose - derived stem cells in the treatment of patients with st - elevation myocardial infarction , and precise , a randomized clinical trial of adipose - derived stem and regenerative cells in the treatment of patients with non revascularizable ischemic myocardium ( http://clinicaltrials.gov ) . nevertheless , at the american heart conference in 2010 the 6 month follow - up of the first 14 patients , of which 10 were treated with adipose - derived stem cells and 4 with placebo , were presented . though the patients treated with adopose - derived stem cells showed a reduction in infarct size , increased myocardial perfusion and ejection fraction , the results did not reach significance most probably due the small sample groups . perhaps one of the most obvious sources of cells for cardiac repair are skeletal muscle precursors . these skeletal myoblasts are progenitor cells of adult myofibers with several features which make them an attractive alternative to embryonic and somatic stem cells as replacement cell source ; autologous , easy to culture and high scalability in vitro , and because they are natural precursors of muscle cells there is a low risk of tumorigenesis . typically , these cells are isolated from the thigh muscle and then expanded in vitro for 34 weeks prior to reinjection into the patient . although early trials ( for short review see ) demonstrate that transplantation of skeletal myoblasts is feasible and leads to an improvement in heart function in very short - term follow - ups , longer term measurable effects in left ventricular eject fraction were lost . clinical trials ( marvel and seismic ) , however , are ongoing though results of these trials are scanty , often being announced at meetings or via company press releases ( see http://www.bioheartinc.com ) . seemingly a perfect source of new muscle , skeletal myoblasts possess one natural inherent barrier that seriously limits their use in directed cardiac cell therapies , namely , unsuitable electrophyiological coupling . although skeletal myoblasts can be grafted into the heart and can even be sustained for a considerable time , coupling between grafts and resident cardiomyocytes is poor to non - existent leading to the real risk of conduction slowing and re - entrant arrhythmias . one approach to try and overcome this problem is to prevent cx43 down - regulation , a key gap junction in cardiac conductance , during skeletal myoblast differentiation [ 20 , 58 ] . sustained expression of cx43 in differentiating skeletal myoblasts appeared to offer improve coupling in model systems , but may not have solved the problem of arrhythmogenicity [ 27 , 60 ] . the discovery that co - culturing hescs with endodermal cells led to higher numbers of cardiomyocytes within the differentiating pluripotent population , proved a key initiative in furthering research on cellular replacement strategies reviewed in . this fact , coupled with the finding that transplanted escs display an inability to self differentiate into cardiomyocytes in the natural cardiac milieu demonstrated a need for a more basic understanding of signals necessary to induce cardiomyogenesis . the discovery that a combination of at least two tgfb - superfamily members could induce cardiac differentiation in avians and amphibians [ 78 , 79 ] , prompted laflamme et al . to explore if the same was possible with cultured hescs . hescs pre - treated with activin a followed by bmp4 showed yields of > 30% beating cardiomyocytes . later yang and co - workers adapted and improved this protocol by including a later phase of wnt inhibition via dkk ( dikkopf homologue 1 ) and vegf ( vascular endothelial growth factor ) treatment . the advent of techniques to molecularly characterize cells provided a list of potential factors that could drive cardiomyogenesis , but at the same time opened a new door in terms of cellular programming . the possibility of driving a differentiated cell back towards a more pre - differential state began as a list of esc associated genes up - regulated in the pluripotent state and lost during / after differentiation . the systematic transfection of combinations of these factors finally gave rise to a seemingly magical cocktail of four transcription factors : oct3/4 , sox2 , klf4 and myc , which when transfected to adult fibroblasts could derive a stem cell - like state . this technique which has since been shown to induce pluripotent stem cells ( ips ) from many different types of fibroblast cells , including human fibroblasts ( technique reviewed in ) . although the ability to derive cardiomyocytes from this potential new cell source still needs to be fully explored , initial experiments using animal models appear to demonstrate that human fibroblast derived ips cells can potentially restore function to the damaged myocardium . though 90% of the cardiac volume comprises cardiomyocytes , they only constitute 30% of the cells . the vast majority of the cells in the heart are non - myocytes of which approximately half is cardiac fibroblast . upon a myocardial infarction the ventricular wall is rescued from rupture by the rapid infiltration of cardiac fibroblasts , healing the wound quickly with simple scar tissue . although in this respect the cardiac fibroblast is not regenerating the lost myocardium , but replacing it , the prospect of harnessing this naturally abundant cell source and altering its function has been a driving source for recent research into epicardial development and function [ 77 , 79 ] . a challenging though possible option would be to first induce pluripotency [ 32 , 68 ] and subsequently differentiate them into cardiomyocytes . taking fibroblasts as an undifferentiated mesenchymal precursor cell , one could potentially reprogramme this abundant cell source into cardiomyocytes directly , circumventing the necessity to induce a pluripotent state . zeisberg and co - workers demonstrated that this is a realistic possibility , that transduction of the transcription factor myod could induce skeletal muscle differentiation in cardiac fibroblasts . . . identified key regulators that upon transduction were able to induce myocardial differentiation of cardiac fibroblasts . making use of isolated gfp - negative cardiac cells from transgenic mice harbouring a gfp driven by alpha - myosin heavy chain promoter transduction of 14 key developmental cardiac regulators induced gfp expression . sequential removal of single factors revealed that the combination of the transcription factors gata4 , mef2c and tbx5 were necessary and sufficient to induce this effect . further analysis revealed not only the induction of gfp expression , but also the expression of an array of cardiomyocyte specific genes , a genomic methylation status equivalent to cardiomyocytes and spontaneous contractions . injection of these cells into the left ventricular wall of immunosuppressed mice , revealed gfp - expressing cells at the site of injection . nevertheless , the used isolation procedures do not exclude that cardiac stem cells are co - isolated . to get a handle on this , the transcription factor cocktail was tested in ckit , isl1 , or mesp1 depleted cardiac fibroblasts , and remained functional pointing to a direct reprogramming of cardiac fibroblasts rather than inducing cardiac differentiation of co - isolated stem cells . the next challenge will be to test this in human cardiac fibroblast and translate it into an approach that is applicable in vivo in humans . the clinical application of progenitor cell sources for cardiac repair rapidly took off with patient trials across europe and the united states . the promise of an improved cardiac function seemed to be a fact and not just fiction in the initial phases of almost all studies . although even the more sceptical accepted that initial results looked promising , the call for a better understanding of the mechanisms and molecular basis seemed at first to go unheeded as new , better controlled trials , using larger patient groups , were set - up . questions as to how the infused cell source was inducing positive effects went and remained for a large part , unanswered . even basic questions surrounding the actual number and type of cells surviving in the infarct after their injection caused a great deal of controversy . as clinical trials progressed and follow - up periods became extended , results began to show a trend change , tending more towards resembling the placebo controls with many of the initial positive benefits of cell therapy being lost over time . here , we have tried to present and discuss some of the basic molecular knowledge and clinical trails which attempted to apply the various cell sources in compromised hearts . although much has been learnt over the past 10 years , many basic scientific questions remain . understanding how cells develop and differentiate during normal embryogenesis can provide us with many answers to the fundamental questions surrounding this promising area of translational research . one can only hope that the hype within the scientific and medical community , surrounding the first clinical trials and the perspectives of this wonder cure for a growing problem in an aging society , has not destroyed public confidence in this novel area of research . taken at face value , it would now be easy to dismiss this prospective therapy , just as we are beginning to understand how it could be converted from hope into fact by implementing the mechanistic knowledge that has and is being gained . a fundamental question that could be posed is why the regenerative capacity of the human heart to repair itself up on damage is virtually non - existing and even insufficient to maintain the number of cardiomyocytes during aging , while lower vertebrates possess the capacity of heart regeneration ? infiltration of fibroblasts into an ischemic area , giving rise to scar tissue , could be considered a kind of quick and dirty repair mechanism to prevent heart s of animals with a high pressure circulatory blood system , ( simply ) from rupturing after the infarct . if , then , it were possible to assist the damaged heart not only by providing it with a suitable cell source for repair , but also provide a better environment for repair by , for instance , reducing critical hemodynamics and pressures during convalescence , nature may be encouraged to take a more favourable direction in terms of cardiac muscle regeneration . if it would prove possible to regenerate muscle mass sufficiently , than we should also take into account that heart failure is not simply due to muscle loss . it should be kept in mind that about half of the patients suffering heart failure do no die due of hemodynamic dysfunction , but rather due to electrical failure and re - infarction . it would , therefore , be prudent to not only focus on the basic mechanisms underlying heart muscle cell regeneration but also to stimulate research into other aspects of heart disease / failure .

cardiovascular diseases are the leading cause of mortality , morbidity , hospitalization and impaired quality of life . in most , if not all , pathologic cardiac ischemia ensues triggering a succession of events leading to massive death of cardiomyocytes , fibroblast and extracellular matrix accumulation , cardiomyocyte hypertrophy which culminates in heart failure and eventually death . though current pharmacological treatment is able to delay the succession of events and as a consequence the development of heart failure , the only currently available and effective treatment of end - stage heart failure is heart transplantation . however , donor heart availability and immunorejection upon transplantation seriously limit the applicability . cardiac regeneration could provide a solution , making real a dream of both scientist and clinician in the previous century and ending an ongoing challenge for this century . in this review , we present a basic overview of the various cell types that have been used in both the clinical and research setting with respect to myocardial differentiation .

Myocardial regeneration (Human) Embryonic stem cells derivation and potential Bone marrow mononuclear cells (BMCs) Adipose tissue-derived stem cells (ASC) Skeletal myoblast: a direct source of muscle? Deriving cardiomyocytes by programming Reprogramming cardiac fibroblasts Reflection and future perspectives

after birth , the cardiomyocytes undergo a final round of cell division with a portion of the cardiomyocytes becoming binuclear and/or polypoid [ 14 , 82 , 83 ] . the observed post - natal growth is attributed to mainly to hypertrophic growth of the resident cardiomyocytes , with very no contribution of cell division . in line with these findings it was observed that amputation of the ventricular apex at 1 day after birth in mice tolerates full regeneration of this region , whereas at 7 days post natal this capacity is completely lost . , who showed that a small ( 0.001% ) portion of cardiomyocytes undergo mitosis in normal hearts . though numerous subsequent follow - up studies have reported slightly differing numbers , the issue remained controversial . this experiment resembles that of a pulse - chase experiment in which genomic dna was labelled as a result of trace amounts of radioactive nuclear fallout produced as a result of nuclear weapons testing during the cold war . although this value gradually decreases with age , the numbers suggest that about 50% of resident cardiomyocytes are renewed during life . however , the initial challengers of the dogma , revisited and expanded their initial study using state of the art tools reporting a similar decrease in myocyte turn - over rate with age . interestingly , based on their methods and estimations the cardiomyocytes of the heart would are renewed 10-times during a normal life span . this capacity is insufficient ( 1 ) to maintain the number of cardiomyocytes during aging and ( 2 ) to replace cardiomyocytes lost upon injury and/or disease , leading to decreased heart function and eventually heart failure . a second question directly relating to this endogenous regenerative capacity is the cellular origin of the newly formed cardiomyocytes . the quest for progenitors of cardiomyocytes within the heart has led to the identification of a multitude of cardiac stem cells or progenitors , each with a different molecular signature [ 41 , 53 ] . ckit ( cd117 ) and flk-1 are but two of the markers used to identify precursor cells contributing to the cardiomyocyte , smooth muscle and endocardial lineage . facs sorting of the ckit - positive population revealed that their contribution to the heart gradually decreases with age , from 0.65% , of which 10% is cd45-(hematopoietic)positive , in neonatal mouse heart to 0.5% , of which 15% is cd45-positive , in the adult heart . co - culturing neonatal ckit - positive cells with foetal cardiomyocytes results in robust differentiation into cardiomyocytes , whereas adult ckit - positive cells appear to have lost this capacity . with the derivation of the first human embryonic stem cells ( hesc ) by thomson et al . , the field of stem cell research caught the scientific and public domain s attention , bringing an understandable high level of hope for new breakthroughs in regenerative medicine . despite the ensuing reactions regarding the ethical issues that surround the isolation and use of cells that are of a human embryonic origin , the prospect of being able to treat a multiplicity of diseases , particularly the appeal of finally being able to restore function of tissue lost during a myocardial infarct , stem cell research took off at an incredible rate ( fig . 1 ) however , apart from the ethical issues surrounding this research , hesc , as we shall discuss , have thrown up many obstacles on their route to the clinic resulting in a re - evaluation of strategies being taken to implement their use . although other stem cell sources have been applied more readily in the clinical setting , hescs have only just received the first green light for an fda approved human trial to investigate their applicability for the treatment of complete thoracic spinal severance .fig . 1annual publication frequency within the field of stem cells and cardiac regeneration annual publication frequency within the field of stem cells and cardiac regeneration embryonic stem cells are derived from the inner cell mass of the blastocyst ~5 days post fertilization . given the diverse and sometimes incorrect use of the word stem cell , esc were described using the physical characteristics ( i ) derived from the pre - implanted embryo , ( ii ) prolonged , undifferentiated proliferation and ( iii ) the potential to develop and give rise to all three germinal layers ( endoderm , mesoderm and ectoderm ) . cells may therefore even at this stage differ from their neighbouring cells and begin to display the molecular characteristics of one of the germinal layers , which in turn could be composed of cells that differ from each other . however , markers , have been identified which serve as tools for sorting and cellular selection . the two glycosphingolipid surface antigen markers ssea-3 and ssea-4 are , are identified on human embryonal carcinoma ( ec ) cell lines , on early cell - cleavage mouse embryos and later on cells of the inner cell mass of human blastocysts . experiments involving the depletion of ssea-3 and ssea-4 on hescs using inhibitors of sphingolipid synthesis appear to show that these markers do not play a critical role in maintenance of the undifferentiated state . although the function of most of the cell surface markers used for hesc selection remain unknown , the value of identifying unique markers is obvious and remains an area of keen research . however , unlike these markers , e1b - ap5 s molecular identity is better defined , representing an adenovirus nuclear rna - binding protein which appears to also be expressed at the plasma membrane as well as in the nucleus of hesc cells . this work has led to the identification of a core set of transcription factors consisting of oct3/4 ( also known as pou5f1 ) , the sex determining region y protein sox2 , the homeobox protein nanog and the pr - domain containing zinc finger protein prdm14 . these factors are key in the process of pluripotency maintenance , though their individual expression patterns are not all restricted to this earliest phase of development , many being required during later key differentiation and cell lineage decisions . not coincidently then , sox2 and oct3/4 constitute 2 of the factors used in the initial studies aimed at direct reprogramming of somatic cells to pluripotent stem - like cells ( ips ) . the issues and details surrounding ips cells will be discussed later . in an attempt to try and provide a more comprehensive and systematic view of markers present on hesc lines present in different laboratories around the world , one of the major conclusions of this study indeed confirmed that the lines were divergent in character ; similar expression patterns of all the above mentioned markers were found along with several other unique identifiers . one of the major , aside from the ethical issues , has been the high risk of teratoma formation . although the answer to this potential danger appeared to be pre - differentiation of hesc before injection , following experiments showing the apparent tumour free application of pre - differentiated hesc in animal models , this could not exclude a potential risk if used in the human clinical setting . this fear may recently have been justified with the first description of a patient treated with multiple injection rounds of foetal neural stem cells for a neurodegenerative disorder developed a multifocal brain tumour . although the potential for replacement therapy has received much of its acclaim in field of the embryonic stem cell , perhaps more due to the open ethical discussions surrounding such research , initial studies that recognized the potential of stem cells were focused on haematopoietic and bone marrow derived cells . to this day , bmc has been the most frequently tapped source of cells used in clinical studies and trials aimed at cardiac repair after myocardial infarction . typically , bmcs are isolated by aspiration from the iliac crest of the patient themselves , reducing the risks of immunological response in terms of cellular therapy . after an initial phase of processing , often sedimentation or gradient centrifugation , the bmc cell suspension consists of progenitors of endothelial origin ( ~4% ) , mesenchymal stem cells ( < 0.1% ) and a very small number of so - called side population cells including a recently identified very small embryonic - like stem cell ( vsel ) [ 70 , 84 ] . the rest of the cellular material , by far the largest portion , is of non - progenitor type . the heterogeneity of the cell population itself and the techniques used for isolation has made the characterization and comparison of sub - populations using cell surface markers difficult . the cluster of differentiation ( cd ) antigens cd14 , cd34 , cd45 and cd133 [ 45 , 65 ] have been used to identify and enrich hematopoietic progenitor cell populations ( reviewed in ) . like the hesc , the presence of ssea-4 , oct3/4 and nanog could be detected in bone marrow derived mesenchymal stem cells ( mscs ) , though sox2 appeared to be specifically absent . since the 12 month and 18 month data from a large number of these trials has been discussed and ( meta-)analyzed elsewhere [ 1 , 36 , 44 , 47 ] , we shall only discuss three of the longer - term follow - up clinical trials , though data from such combined analyzes would seem to support the notion of modest improvement in cardiac function after acute myocardial infarction . most randomized trials have typically only covered a short - term follow - up of 412 months , making assessment of the true efficacy and sustained effect of such a therapy difficult . further , although apparently safe in the short - term application , long - term safety issues have been raised based on reports of associated risks of arrhythmias and calcification [ 81 , 87 ] . all three trials found that the application of bmcs in the clinical setting is safe . however , only the repair - ami ( 2 years follow - up ) , but not astami ( 3 year follow - up ) and boost ( 5 year follow - up ) studies , showed a significant positive benefit in left ventricular ejection fraction . this finding can also be concluded from the boost 18 month follow - up results and most other clinical bmc trials , with patients typically displaying a 23% improvement in left ventricular function . however , after this period , as with the astami study , boost results suggest that this effect steadily declines to become non - significant when compared to control groups . one can perhaps then conclude from these studies that in the long - term there is no observable benefit from the administration of bmcs to patients that have suffered an ami . however , one should perhaps not be too quick to be dismissive of this cell source . therefore , the lack of information as to why these positive effects disappear during a longer - term follow - up , demonstrates that a more basic approach to understanding the actual mechanisms underlying the observed effects is essential for correct interpretation of the end results . understanding the underlying mechanism one might be able to focus research and boost the initial as well as sustain these beneficial effects . one interesting and plausible explanation for at least some of the short - term positive effects observed could be the release of paracrine factors by the injected cell source , which serve to assist recovery of the damaged tissues . induction of this system at specific time points after cell transplantation was observed to reduce the normally observed effects of neovascularization and decrease the level of recovery of left ventricular function and capillary density . although the paracrine effects in human clinical trials can not easily be determined , bmc have been shown to release such factors when transplanted into models of ischemia [ 39 , 74 ] . in this respect , adipose tissue could be an attractive alternative source , as it is most often abundantly present in people suffering from cardiac disease , easy to harvest using liposuction , and relatively rich in stem cells . adipose tissue gained an interest as a potential cellular source for cardiac regeneration when in vitro experiments showed that dispersed adipose tissue can be induced to differentiate into cells of various lineages and even into cardiomyocytes . these experiments revealed that ~0.05% of the plated cells spontaneously differentiated into cardiomyocytes within 20 days of culture . infusion of non pre - selected or pre - selection of cd44 ( hyaluronic acid receptor ) , cd105 ( endoglin ) and cd34-negative adipose - derived cells in animal models of acute myocardial infarction , displayed an improved lv - ejection fraction and wall thickness compared to pbs injected controls [ 8 , 16 , 75 , 89 ] in contrast to several studies which apparently demonstrated a loss of infused cells after injection , recent studies using bioluminescent imaging of luciferase transfected adipose - derived cells could be identified in the recipient heart 4 weeks after administration . histological analysis revealed that the adipose - derived cells had differentiated into cardiomyocytes , smooth muscle cells and endothelial cells in the infracted region . despite several unresolved issues , two double - blind , randomized , placebo - controlled phase i trials , apollo and precise , were initiated in 2007 ; apollo , adipose - derived stem cells in the treatment of patients with st - elevation myocardial infarction , and precise , a randomized clinical trial of adipose - derived stem and regenerative cells in the treatment of patients with non revascularizable ischemic myocardium ( http://clinicaltrials.gov ) . nevertheless , at the american heart conference in 2010 the 6 month follow - up of the first 14 patients , of which 10 were treated with adipose - derived stem cells and 4 with placebo , were presented . though the patients treated with adopose - derived stem cells showed a reduction in infarct size , increased myocardial perfusion and ejection fraction , the results did not reach significance most probably due the small sample groups . perhaps one of the most obvious sources of cells for cardiac repair are skeletal muscle precursors . although early trials ( for short review see ) demonstrate that transplantation of skeletal myoblasts is feasible and leads to an improvement in heart function in very short - term follow - ups , longer term measurable effects in left ventricular eject fraction were lost . clinical trials ( marvel and seismic ) , however , are ongoing though results of these trials are scanty , often being announced at meetings or via company press releases ( see http://www.bioheartinc.com ) . although skeletal myoblasts can be grafted into the heart and can even be sustained for a considerable time , coupling between grafts and resident cardiomyocytes is poor to non - existent leading to the real risk of conduction slowing and re - entrant arrhythmias . sustained expression of cx43 in differentiating skeletal myoblasts appeared to offer improve coupling in model systems , but may not have solved the problem of arrhythmogenicity [ 27 , 60 ] . the discovery that co - culturing hescs with endodermal cells led to higher numbers of cardiomyocytes within the differentiating pluripotent population , proved a key initiative in furthering research on cellular replacement strategies reviewed in . this fact , coupled with the finding that transplanted escs display an inability to self differentiate into cardiomyocytes in the natural cardiac milieu demonstrated a need for a more basic understanding of signals necessary to induce cardiomyogenesis . hescs pre - treated with activin a followed by bmp4 showed yields of > 30% beating cardiomyocytes . the possibility of driving a differentiated cell back towards a more pre - differential state began as a list of esc associated genes up - regulated in the pluripotent state and lost during / after differentiation . though 90% of the cardiac volume comprises cardiomyocytes , they only constitute 30% of the cells . the vast majority of the cells in the heart are non - myocytes of which approximately half is cardiac fibroblast . although in this respect the cardiac fibroblast is not regenerating the lost myocardium , but replacing it , the prospect of harnessing this naturally abundant cell source and altering its function has been a driving source for recent research into epicardial development and function [ 77 , 79 ] . taking fibroblasts as an undifferentiated mesenchymal precursor cell , one could potentially reprogramme this abundant cell source into cardiomyocytes directly , circumventing the necessity to induce a pluripotent state . zeisberg and co - workers demonstrated that this is a realistic possibility , that transduction of the transcription factor myod could induce skeletal muscle differentiation in cardiac fibroblasts . identified key regulators that upon transduction were able to induce myocardial differentiation of cardiac fibroblasts . making use of isolated gfp - negative cardiac cells from transgenic mice harbouring a gfp driven by alpha - myosin heavy chain promoter transduction of 14 key developmental cardiac regulators induced gfp expression . sequential removal of single factors revealed that the combination of the transcription factors gata4 , mef2c and tbx5 were necessary and sufficient to induce this effect . nevertheless , the used isolation procedures do not exclude that cardiac stem cells are co - isolated . to get a handle on this , the transcription factor cocktail was tested in ckit , isl1 , or mesp1 depleted cardiac fibroblasts , and remained functional pointing to a direct reprogramming of cardiac fibroblasts rather than inducing cardiac differentiation of co - isolated stem cells . the next challenge will be to test this in human cardiac fibroblast and translate it into an approach that is applicable in vivo in humans . the clinical application of progenitor cell sources for cardiac repair rapidly took off with patient trials across europe and the united states . the promise of an improved cardiac function seemed to be a fact and not just fiction in the initial phases of almost all studies . although even the more sceptical accepted that initial results looked promising , the call for a better understanding of the mechanisms and molecular basis seemed at first to go unheeded as new , better controlled trials , using larger patient groups , were set - up . even basic questions surrounding the actual number and type of cells surviving in the infarct after their injection caused a great deal of controversy . as clinical trials progressed and follow - up periods became extended , results began to show a trend change , tending more towards resembling the placebo controls with many of the initial positive benefits of cell therapy being lost over time . here , we have tried to present and discuss some of the basic molecular knowledge and clinical trails which attempted to apply the various cell sources in compromised hearts . one can only hope that the hype within the scientific and medical community , surrounding the first clinical trials and the perspectives of this wonder cure for a growing problem in an aging society , has not destroyed public confidence in this novel area of research . a fundamental question that could be posed is why the regenerative capacity of the human heart to repair itself up on damage is virtually non - existing and even insufficient to maintain the number of cardiomyocytes during aging , while lower vertebrates possess the capacity of heart regeneration ? if , then , it were possible to assist the damaged heart not only by providing it with a suitable cell source for repair , but also provide a better environment for repair by , for instance , reducing critical hemodynamics and pressures during convalescence , nature may be encouraged to take a more favourable direction in terms of cardiac muscle regeneration . if it would prove possible to regenerate muscle mass sufficiently , than we should also take into account that heart failure is not simply due to muscle loss . it should be kept in mind that about half of the patients suffering heart failure do no die due of hemodynamic dysfunction , but rather due to electrical failure and re - infarction . it would , therefore , be prudent to not only focus on the basic mechanisms underlying heart muscle cell regeneration but also to stimulate research into other aspects of heart disease / failure .

ethical approval was obtained from the ethics committee of the university of ibadan / university college hospital review board ( ui / ec/13/0153 ) . the patients were recruited from the neurology outpatient clinic at the university college hospital ( tertiary public health institution ) , adeoyo state hospital ( secondary public health facility ) and oluyoro catholic hospital ( secondary private health facility ) , ibadan . all consenting patients with the diagnosis of pd that could stand and walk with or without a history of falls and fulfilled the inclusion criteria were recruited into the study . the diagnosis of pd was made using the united kingdom parkinson s disease society ( ukpds ) criteria . we excluded patients who did not give consent , those with atypical parkinsonian syndrome and those who were totally bed ridden . a detailed questionnaire ( interviewer administered ) was used to collect socio - demographic variables ( age , sex , occupation , and marital status ) and relevant clinical information ( date of first pd diagnosis , medication history , fall history in the last one , six , and 12 months , history of associated injury during falls and circumstances surrounding falls , static and dynamic balance , cadence and stability , and past history of hypertension and/or diabetes ) . a fall was defined as an event that resulted in a person coming to rest inadvertently on the ground or floor . a patient was subjectively asked about fear of falling if the fall events and circumstances surrounding the fall were recorded . cognition was assessed by using the community screening interview for dementia ( csi d ) . pd severity was assessed using unified parkinson s disease rating scale ( updrs ) criteria . autonomic function was assessed using standard bedside tests ( heart rate and blood pressure in response to standing ) . postural hypotension was assessed using a difference of > 20 mm hg in systolic blood pressure or > 10 mm hg in diastolic blood pressure between standing and supine positions , respectively . the tinetti balance and gait test scale has a 71% sensitivity and 79% specificity to predict falls in both the elderly and pd patients . the balance scale contains nine items with a total summation of 16 points while the gait scale contains eight items with a total summation of 12 points for a total of both 28 points for both scales . a score < 18 implies high risk , while scores between 19 and 23 imply moderate risk , and a score > 24 implies low fall risk . hypertension was diagnosed using sphygmomanometer measurements of blood pressure > 140/90 mm hg measured at two different times or a history of antihypertensive therapy . diabetes mellitus was diagnosed based on either a fasting plasma glucose of > 126 mg / dl or random plasma glucose of > 200 mg / dl measured on two different occasions at least 24 h apart . normally distributed continuous variables were assessed using independent samples t - test , where data were not normally distributed , non - parametric methods were used . the mann - whitney u test for variables that were not normally distributed and kruskal - wallis test were used to compare ranked variables . categorical variables were tabulated and statistically assessed for association using the test or fisher s exact test ( where the expected counts were < five per cell ) to compare falling with non - falling patients . all variables that showed association with falls in univariate analysis were included in the regression model . a detailed questionnaire ( interviewer administered ) was used to collect socio - demographic variables ( age , sex , occupation , and marital status ) and relevant clinical information ( date of first pd diagnosis , medication history , fall history in the last one , six , and 12 months , history of associated injury during falls and circumstances surrounding falls , static and dynamic balance , cadence and stability , and past history of hypertension and/or diabetes ) . a fall was defined as an event that resulted in a person coming to rest inadvertently on the ground or floor . a patient was subjectively asked about fear of falling if the fall events and circumstances surrounding the fall were recorded . cognition was assessed by using the community screening interview for dementia ( csi d ) . pd severity was assessed using unified parkinson s disease rating scale ( updrs ) criteria . autonomic function was assessed using standard bedside tests ( heart rate and blood pressure in response to standing ) . postural hypotension was assessed using a difference of > 20 mm hg in systolic blood pressure or > 10 mm hg in diastolic blood pressure between standing and supine positions , respectively . the tinetti balance and gait test scale has a 71% sensitivity and 79% specificity to predict falls in both the elderly and pd patients . the balance scale contains nine items with a total summation of 16 points while the gait scale contains eight items with a total summation of 12 points for a total of both 28 points for both scales . a score < 18 implies high risk , while scores between 19 and 23 imply moderate risk , and a score > 24 implies low fall risk . hypertension was diagnosed using sphygmomanometer measurements of blood pressure > 140/90 mm hg measured at two different times or a history of antihypertensive therapy . diabetes mellitus was diagnosed based on either a fasting plasma glucose of > 126 mg / dl or random plasma glucose of > 200 mg / dl measured on two different occasions at least 24 h apart . normally distributed continuous variables were assessed using independent samples t - test , where data were not normally distributed , non - parametric methods were used . the mann - whitney u test for variables that were not normally distributed and kruskal - wallis test categorical variables were tabulated and statistically assessed for association using the test or fisher s exact test ( where the expected counts were < five per cell ) to compare falling with non - falling patients . all variables that showed association with falls in univariate analysis were included in the regression model . the mean age of the recruited participants was 65.8 9.0 years , and the male to female ratio was 2:1 . the median disease duration was 36 months [ interquartile range ( iqr ) : 2060 months ] . seventy - seven ( 95.1% ) were on medications , with 76 ( 93.8% ) on carbidopa - levodopa , 57 ( 70.4% ) on trihexyphenidyl and 6 ( 7.4% ) on other anti - pd medications . median occurrence of fall in our respondents was 30 months ( iqr : 760 months ) after diagnosis . thirty - four ( 42% ) respondents had history of falls in the last 12 months with 11 ( 32.3% ) having single falls and 23 ( 67.7% ) recurrent falls . there were no statistically significant differences in age and gender between the falling and non - falling patients . falling patients reported having longer disease durations with a median duration of 60 months as opposed to 24 months among non - falling patients ( p < 0.001 ) . more of the falling patients ( 22 ; 64.7% ) had a fear of falling when compared to non - falling patients ( 9 ; 25.7% ) . d score of the falling patients was significantly lower than the non - falling patients ( p = 0.034 ) . patients with previous falls , when compared to non - falling patients , had worse pd severity as measured using updrs ( p < 0.001 ) ( table 2 ) . static and dynamic balance was also worse in falling than in non - falling patients . there was no difference in cadence and stability ( measured using the timed up and go test ) between the falling and non - falling patients ( p = 0.117 ) ( table 2 ) . total updrs and tinetti score ( not their sub - scores ) were used in the model . when all predictor variables were considered together , they significantly predicted fall occurrence ( = 34.4 , p = 0.000 , r = 0.336 ) . the results showed that pd severity [ odds ratio ( or ) : 1.09 , 95% confidence interval ( ci ) : 1.021.16 ] , disease duration measured in months ( or : 1.01 , 95% ci : 1.001.03 ) , and fear of falling ( or : 3.86 , 95% ci : 1.1712.7 ) were significant fall predictors in this study ( table 3 ) . to the best of our knowledge , this is the first documentation of falls among pd patients in sub - saharan africa . the frequency of falls was 42% compared to 14.8% of age - matched healthy adults , and the median disease duration in the pd patients was 36 months . most of the pd patients ( 71.6% ) were on two or more anti - pd drugs . factors responsible for falls in pd patients are multiple and most likely include similar factors associated with falls in healthy elderly patients . the frequency of falls in our study was similar to those described in previous studies in pd patients . in a study to assess the risk of falls in 160 pd patients , contreras and grandas documented a 38.8% prevalence , a figure slightly lower than the finding in this study . however , a meta - analysis of six fall studies in pd patients reported fall frequencies ranging between 38% and 50% . the frequency in our study lies within this range , and variations in study methodology and clinical characteristics could account for the varied frequencies . it is difficult to speculate on cultural factors playing a very significant role in falls in pd patients because the frequency was similar to what was obtained in other environments . factors such as special flooring ( marble , tiles , and polished surfaces ) that could predispose a patient to fall are absent in most traditional nigerian homes but did not seem to have any significant effects on falling frequencies when compared to other types of homes . additionally , there were no increases in the incidence of neuropathy , visual impairment , or medications that predispose pd patients to fall . falling patients had significantly worse disease and longer durations when compared to non - falling patients . these findings are similar to findings by bloem et al . and wood et al . , who also reported higher updrs motor scores in falling versus non - falling patients ; therefore , this is not a predictor of falls . postural instability was prominent in the falling patients more than the non - falling patients due to poor preparatory adjustments , abnormal postural balance , and delayed reaction time . a study by contreras and grandas reported predictability of tinetti balance test to predict falls with 71% sensitivity and 79% specificity . he confirmed the tinetti test tool as an independent fall predictor after using a multivariate logistic regression model . this is consistent with studies of gait and balance abnormalities that were carried out in other environments where gait abnormality was considered the most consistent predictor of falls . however , in this study the tinetti balance and gait tool did not independently predict the risk of falls . the frequency of falls has also been reported to have a direct correlation with age , disease severity , medication , cognitive dysfunction , orthostatic hypotension , and visual impairment . a previous study of falls both in pd patients and healthy adults had shown an association between adults > 65 years and increased falls of 30 to 40% . this was most likely because most of the pd patients in that study were younger compared to other studies conducted in different environments . advancing age ( > 70 years ) has been associated with increased risk of falls due to advanced pd progression . furthermore , older age is associated with frailty and reduced muscle mass and strength . in this study , this is different from previous epidemiological studies of pd in nigeria where relatively low proportions of young onset pd patients were evaluated . however , femi et al . reported an increasing older age of pd onset in the northwestern nigeria . these differences might be due to an inappropriate record of birth dates due to low level of literacy , shorter average life expectancy of those living in the underdeveloped and developing countries , poverty and/or poor access to health care services . furthermore , different genetic makeups and environmental exposures of the study participants might be responsible for the observed differences . a previous fall has been shown to be a significant risk factor for more falls . reported in a meta - analysis that > 2 falls was the best predictor of more falls in pd . this might be the result of lower recovery performances in forward falls and lower muscle strength in pd patients who fall . in this study , 67.7% of our pd patients had recurrent falls , out of which 44% had a fear of falling . the reported frequency of recurrent falls was higher than what was obtained by landers et al . . however , the findings in this study are similar to a prospective study of falls by cole et al . . these differences may be due to differences in methodology and characteristics of disease severity . . demonstrated low balance confidence scores , which is an indicator for more falls , in pd patients compared to controls . patients who fall more frequently often have a self - induced restriction of activities due to fear of more falls , thus , leading to reduced muscle strength and increased risk of more falls , resulting in poor health - related quality of life . studies have demonstrated increased incidence of trochanteric fractures in pd patients due to adduction of arms against the trunk when falling , while older adults without pd stretched out their arms in the direction of impending fall and , thus , sustained minor injuries such as abrasion . worthy of note in this study is the predictability of fear of falling as a strong modifiable risk factor with an adjusted or of 3.86 . thus , clinical strategies to reduce fear of falling in pd patients will reduce the frequency of falls and their health - related and economic burden . in this study , it was observed that falling pd patients sustained other injuries such as fractures and lacerations ; however , bloem et al . recorded no fractures in their study but reported a high proportion of soft tissue injuries ( 60% ) , a figure slightly lower than the finding in our study . these injuries may be a result of the inter - segmental stiffness and postural inflexibility observed in pd patients . being a retrospective study , there was the possibility of recall bias , and subjects with cognitive impairment may not have been accurate about fall frequency . it has been said that older people tend to forget occurrences of previous falls ; however , this can be improved upon by getting a fall diary to be used in a prospective study . secondly , this was a hospital - based study , and thus , the proportion of falling patients in our study may not represent community - dwelling pd patients who are at greater risk of falls . although the results show that fear of falling could be an independent risk factor for falls in pd patients , additional prospective studies are required to confirm if this is a cause or consequence of falling or both meanwhile , our findings suggest that addressing the fear of falling may help to reduce falling events in pd patients . in conclusion , our study confirmed an increased frequency of falls among pd patients , and this frequency was shown to be multifactorial . the predictive risk factors were disease severity and duration and fear of falling . early detection of risk factors can prevent the untoward effects of falls in pd patients . this will facilitate tailored management to suit such patients and ultimately reduce the economic health - related burden of falls in these patients . to the best of our knowledge , this is the first documentation of falls among pd patients in sub - saharan africa . the frequency of falls was 42% compared to 14.8% of age - matched healthy adults , and the median disease duration in the pd patients was 36 months . most of the pd patients ( 71.6% ) were on two or more anti - pd drugs . factors responsible for falls in pd patients are multiple and most likely include similar factors associated with falls in healthy elderly patients . the frequency of falls in our study was similar to those described in previous studies in pd patients . in a study to assess the risk of falls in 160 pd patients , contreras and grandas documented a 38.8% prevalence , a figure slightly lower than the finding in this study . however , a meta - analysis of six fall studies in pd patients reported fall frequencies ranging between 38% and 50% . the frequency in our study lies within this range , and variations in study methodology and clinical characteristics could account for the varied frequencies . it is difficult to speculate on cultural factors playing a very significant role in falls in pd patients because the frequency was similar to what was obtained in other environments . factors such as special flooring ( marble , tiles , and polished surfaces ) that could predispose a patient to fall are absent in most traditional nigerian homes but did not seem to have any significant effects on falling frequencies when compared to other types of homes . additionally , there were no increases in the incidence of neuropathy , visual impairment , or medications that predispose pd patients to fall . falling patients had significantly worse disease and longer durations when compared to non - falling patients . these findings are similar to findings by bloem et al . and wood et al . , who also reported higher updrs motor scores in falling versus non - falling patients ; therefore , this is not a predictor of falls . postural instability was prominent in the falling patients more than the non - falling patients due to poor preparatory adjustments , abnormal postural balance , and delayed reaction time . a study by contreras and grandas reported predictability of tinetti balance test to predict falls with 71% sensitivity and 79% specificity . he confirmed the tinetti test tool as an independent fall predictor after using a multivariate logistic regression model . this is consistent with studies of gait and balance abnormalities that were carried out in other environments where gait abnormality was considered the most consistent predictor of falls . however , in this study the tinetti balance and gait tool did not independently predict the risk of falls . the frequency of falls has also been reported to have a direct correlation with age , disease severity , medication , cognitive dysfunction , orthostatic hypotension , and visual impairment . a previous study of falls both in pd patients and healthy adults had shown an association between adults > 65 years and increased falls of 30 to 40% . this was most likely because most of the pd patients in that study were younger compared to other studies conducted in different environments . advancing age ( > 70 years ) has been associated with increased risk of falls due to advanced pd progression . furthermore , older age is associated with frailty and reduced muscle mass and strength . in this study , this is different from previous epidemiological studies of pd in nigeria where relatively low proportions of young onset pd patients were evaluated . however , femi et al . reported an increasing older age of pd onset in the northwestern nigeria . these differences might be due to an inappropriate record of birth dates due to low level of literacy , shorter average life expectancy of those living in the underdeveloped and developing countries , poverty and/or poor access to health care services . furthermore , different genetic makeups and environmental exposures of the study participants might be responsible for the observed differences . a previous fall has been shown to be a significant risk factor for more falls . reported in a meta - analysis that > 2 falls was the best predictor of more falls in pd . this might be the result of lower recovery performances in forward falls and lower muscle strength in pd patients who fall . in this study , 67.7% of our pd patients had recurrent falls , out of which 44% had a fear of falling . the reported frequency of recurrent falls was higher than what was obtained by landers et al . ( 51% ) and dibble and lange ( 55% ) . however , the findings in this study are similar to a prospective study of falls by cole et al . . demonstrated low balance confidence scores , which is an indicator for more falls , in pd patients compared to controls . patients who fall more frequently often have a self - induced restriction of activities due to fear of more falls , thus , leading to reduced muscle strength and increased risk of more falls , resulting in poor health - related quality of life . studies have demonstrated increased incidence of trochanteric fractures in pd patients due to adduction of arms against the trunk when falling , while older adults without pd stretched out their arms in the direction of impending fall and , thus , sustained minor injuries such as abrasion . worthy of note in this study is the predictability of fear of falling as a strong modifiable risk factor with an adjusted or of 3.86 . thus , clinical strategies to reduce fear of falling in pd patients will reduce the frequency of falls and their health - related and economic burden . in this study , it was observed that falling pd patients sustained other injuries such as fractures and lacerations ; however , bloem et al . recorded no fractures in their study but reported a high proportion of soft tissue injuries ( 60% ) , a figure slightly lower than the finding in our study . these injuries may be a result of the inter - segmental stiffness and postural inflexibility observed in pd patients . being a retrospective study , there was the possibility of recall bias , and subjects with cognitive impairment may not have been accurate about fall frequency . it has been said that older people tend to forget occurrences of previous falls ; however , this can be improved upon by getting a fall diary to be used in a prospective study . secondly , this was a hospital - based study , and thus , the proportion of falling patients in our study may not represent community - dwelling pd patients who are at greater risk of falls . although the results show that fear of falling could be an independent risk factor for falls in pd patients , additional prospective studies are required to confirm if this is a cause or consequence of falling or both . meanwhile , our findings suggest that addressing the fear of falling may help to reduce falling events in pd patients . in conclusion , our study confirmed an increased frequency of falls among pd patients , and this frequency was shown to be multifactorial . the predictive risk factors were disease severity and duration and fear of falling . early detection of risk factors can prevent the untoward effects of falls in pd patients . this will facilitate tailored management to suit such patients and ultimately reduce the economic health - related burden of falls in these patients .

objectivefalls are a devastating consequence of parkinson s disease ( pd ) and are due to motor imbalance . however , the frequency of falls and their risk factors among nigerians with pd is not known despite the significant increase in pd cases in the country . to assess fall risk factors and frequency in nigerian pd patients.methodsusing an analytical design to compare falling versus non - falling patients , 81 pd patients were assessed for clinical factors , frequency of falls , and candidate risk factors for falls according to the tinetti balance and gait , unified parkinson s disease rating scale subsection 1 , and timed up and go tests . descriptive , bivariate , and multivariate analyses were performed at the 95% confidence level.resultsthe mean age of participants was 65.6 9.7 years . falls were about three times ( p < 0.001 ) more common in pd patients . of the falling patients , 67.7% sustained injuries , 67.7% had recurrent falls and 44.9% admitted to having a fear of falling . the independent statistical predictors of fall were fear of falling [ odds ratio ( or ) : 3.86 ] , disease severity ( or : 1.09 ) and disease duration ( or : 1.01).conclusionthe frequency of falls in pd patients was significantly higher when compared with the healthy adult population , and the modifiable predictor was fear of falling with a potential to significantly reduce falls when strategically addressed .

MATERIALS & METHODS Clinical assessments Statistical analysis RESULTS DISCUSSION Fall frequency Factors associated with falls

ethical approval was obtained from the ethics committee of the university of ibadan / university college hospital review board ( ui / ec/13/0153 ) . the patients were recruited from the neurology outpatient clinic at the university college hospital ( tertiary public health institution ) , adeoyo state hospital ( secondary public health facility ) and oluyoro catholic hospital ( secondary private health facility ) , ibadan . all consenting patients with the diagnosis of pd that could stand and walk with or without a history of falls and fulfilled the inclusion criteria were recruited into the study . the diagnosis of pd was made using the united kingdom parkinson s disease society ( ukpds ) criteria . a detailed questionnaire ( interviewer administered ) was used to collect socio - demographic variables ( age , sex , occupation , and marital status ) and relevant clinical information ( date of first pd diagnosis , medication history , fall history in the last one , six , and 12 months , history of associated injury during falls and circumstances surrounding falls , static and dynamic balance , cadence and stability , and past history of hypertension and/or diabetes ) . a patient was subjectively asked about fear of falling if the fall events and circumstances surrounding the fall were recorded . cognition was assessed by using the community screening interview for dementia ( csi d ) . pd severity was assessed using unified parkinson s disease rating scale ( updrs ) criteria . the tinetti balance and gait test scale has a 71% sensitivity and 79% specificity to predict falls in both the elderly and pd patients . the balance scale contains nine items with a total summation of 16 points while the gait scale contains eight items with a total summation of 12 points for a total of both 28 points for both scales . a score < 18 implies high risk , while scores between 19 and 23 imply moderate risk , and a score > 24 implies low fall risk . diabetes mellitus was diagnosed based on either a fasting plasma glucose of > 126 mg / dl or random plasma glucose of > 200 mg / dl measured on two different occasions at least 24 h apart . normally distributed continuous variables were assessed using independent samples t - test , where data were not normally distributed , non - parametric methods were used . the mann - whitney u test for variables that were not normally distributed and kruskal - wallis test were used to compare ranked variables . categorical variables were tabulated and statistically assessed for association using the test or fisher s exact test ( where the expected counts were < five per cell ) to compare falling with non - falling patients . all variables that showed association with falls in univariate analysis were included in the regression model . a detailed questionnaire ( interviewer administered ) was used to collect socio - demographic variables ( age , sex , occupation , and marital status ) and relevant clinical information ( date of first pd diagnosis , medication history , fall history in the last one , six , and 12 months , history of associated injury during falls and circumstances surrounding falls , static and dynamic balance , cadence and stability , and past history of hypertension and/or diabetes ) . a patient was subjectively asked about fear of falling if the fall events and circumstances surrounding the fall were recorded . pd severity was assessed using unified parkinson s disease rating scale ( updrs ) criteria . autonomic function was assessed using standard bedside tests ( heart rate and blood pressure in response to standing ) . the tinetti balance and gait test scale has a 71% sensitivity and 79% specificity to predict falls in both the elderly and pd patients . the balance scale contains nine items with a total summation of 16 points while the gait scale contains eight items with a total summation of 12 points for a total of both 28 points for both scales . a score < 18 implies high risk , while scores between 19 and 23 imply moderate risk , and a score > 24 implies low fall risk . normally distributed continuous variables were assessed using independent samples t - test , where data were not normally distributed , non - parametric methods were used . the mann - whitney u test for variables that were not normally distributed and kruskal - wallis test categorical variables were tabulated and statistically assessed for association using the test or fisher s exact test ( where the expected counts were < five per cell ) to compare falling with non - falling patients . all variables that showed association with falls in univariate analysis were included in the regression model . the mean age of the recruited participants was 65.8 9.0 years , and the male to female ratio was 2:1 . the median disease duration was 36 months [ interquartile range ( iqr ) : 2060 months ] . median occurrence of fall in our respondents was 30 months ( iqr : 760 months ) after diagnosis . thirty - four ( 42% ) respondents had history of falls in the last 12 months with 11 ( 32.3% ) having single falls and 23 ( 67.7% ) recurrent falls . there were no statistically significant differences in age and gender between the falling and non - falling patients . falling patients reported having longer disease durations with a median duration of 60 months as opposed to 24 months among non - falling patients ( p < 0.001 ) . more of the falling patients ( 22 ; 64.7% ) had a fear of falling when compared to non - falling patients ( 9 ; 25.7% ) . d score of the falling patients was significantly lower than the non - falling patients ( p = 0.034 ) . patients with previous falls , when compared to non - falling patients , had worse pd severity as measured using updrs ( p < 0.001 ) ( table 2 ) . static and dynamic balance was also worse in falling than in non - falling patients . there was no difference in cadence and stability ( measured using the timed up and go test ) between the falling and non - falling patients ( p = 0.117 ) ( table 2 ) . total updrs and tinetti score ( not their sub - scores ) were used in the model . the results showed that pd severity [ odds ratio ( or ) : 1.09 , 95% confidence interval ( ci ) : 1.021.16 ] , disease duration measured in months ( or : 1.01 , 95% ci : 1.001.03 ) , and fear of falling ( or : 3.86 , 95% ci : 1.1712.7 ) were significant fall predictors in this study ( table 3 ) . to the best of our knowledge , this is the first documentation of falls among pd patients in sub - saharan africa . the frequency of falls was 42% compared to 14.8% of age - matched healthy adults , and the median disease duration in the pd patients was 36 months . most of the pd patients ( 71.6% ) were on two or more anti - pd drugs . factors responsible for falls in pd patients are multiple and most likely include similar factors associated with falls in healthy elderly patients . the frequency of falls in our study was similar to those described in previous studies in pd patients . in a study to assess the risk of falls in 160 pd patients , contreras and grandas documented a 38.8% prevalence , a figure slightly lower than the finding in this study . however , a meta - analysis of six fall studies in pd patients reported fall frequencies ranging between 38% and 50% . the frequency in our study lies within this range , and variations in study methodology and clinical characteristics could account for the varied frequencies . it is difficult to speculate on cultural factors playing a very significant role in falls in pd patients because the frequency was similar to what was obtained in other environments . factors such as special flooring ( marble , tiles , and polished surfaces ) that could predispose a patient to fall are absent in most traditional nigerian homes but did not seem to have any significant effects on falling frequencies when compared to other types of homes . additionally , there were no increases in the incidence of neuropathy , visual impairment , or medications that predispose pd patients to fall . falling patients had significantly worse disease and longer durations when compared to non - falling patients . and wood et al . , who also reported higher updrs motor scores in falling versus non - falling patients ; therefore , this is not a predictor of falls . postural instability was prominent in the falling patients more than the non - falling patients due to poor preparatory adjustments , abnormal postural balance , and delayed reaction time . a study by contreras and grandas reported predictability of tinetti balance test to predict falls with 71% sensitivity and 79% specificity . he confirmed the tinetti test tool as an independent fall predictor after using a multivariate logistic regression model . this is consistent with studies of gait and balance abnormalities that were carried out in other environments where gait abnormality was considered the most consistent predictor of falls . however , in this study the tinetti balance and gait tool did not independently predict the risk of falls . the frequency of falls has also been reported to have a direct correlation with age , disease severity , medication , cognitive dysfunction , orthostatic hypotension , and visual impairment . a previous study of falls both in pd patients and healthy adults had shown an association between adults > 65 years and increased falls of 30 to 40% . this was most likely because most of the pd patients in that study were younger compared to other studies conducted in different environments . advancing age ( > 70 years ) has been associated with increased risk of falls due to advanced pd progression . in this study , this is different from previous epidemiological studies of pd in nigeria where relatively low proportions of young onset pd patients were evaluated . however , femi et al . reported an increasing older age of pd onset in the northwestern nigeria . these differences might be due to an inappropriate record of birth dates due to low level of literacy , shorter average life expectancy of those living in the underdeveloped and developing countries , poverty and/or poor access to health care services . furthermore , different genetic makeups and environmental exposures of the study participants might be responsible for the observed differences . reported in a meta - analysis that > 2 falls was the best predictor of more falls in pd . this might be the result of lower recovery performances in forward falls and lower muscle strength in pd patients who fall . in this study , 67.7% of our pd patients had recurrent falls , out of which 44% had a fear of falling . the reported frequency of recurrent falls was higher than what was obtained by landers et al . however , the findings in this study are similar to a prospective study of falls by cole et al . these differences may be due to differences in methodology and characteristics of disease severity . demonstrated low balance confidence scores , which is an indicator for more falls , in pd patients compared to controls . patients who fall more frequently often have a self - induced restriction of activities due to fear of more falls , thus , leading to reduced muscle strength and increased risk of more falls , resulting in poor health - related quality of life . studies have demonstrated increased incidence of trochanteric fractures in pd patients due to adduction of arms against the trunk when falling , while older adults without pd stretched out their arms in the direction of impending fall and , thus , sustained minor injuries such as abrasion . worthy of note in this study is the predictability of fear of falling as a strong modifiable risk factor with an adjusted or of 3.86 . thus , clinical strategies to reduce fear of falling in pd patients will reduce the frequency of falls and their health - related and economic burden . in this study , it was observed that falling pd patients sustained other injuries such as fractures and lacerations ; however , bloem et al . these injuries may be a result of the inter - segmental stiffness and postural inflexibility observed in pd patients . being a retrospective study , there was the possibility of recall bias , and subjects with cognitive impairment may not have been accurate about fall frequency . it has been said that older people tend to forget occurrences of previous falls ; however , this can be improved upon by getting a fall diary to be used in a prospective study . secondly , this was a hospital - based study , and thus , the proportion of falling patients in our study may not represent community - dwelling pd patients who are at greater risk of falls . although the results show that fear of falling could be an independent risk factor for falls in pd patients , additional prospective studies are required to confirm if this is a cause or consequence of falling or both meanwhile , our findings suggest that addressing the fear of falling may help to reduce falling events in pd patients . in conclusion , our study confirmed an increased frequency of falls among pd patients , and this frequency was shown to be multifactorial . the predictive risk factors were disease severity and duration and fear of falling . early detection of risk factors can prevent the untoward effects of falls in pd patients . this will facilitate tailored management to suit such patients and ultimately reduce the economic health - related burden of falls in these patients . to the best of our knowledge , this is the first documentation of falls among pd patients in sub - saharan africa . the frequency of falls was 42% compared to 14.8% of age - matched healthy adults , and the median disease duration in the pd patients was 36 months . most of the pd patients ( 71.6% ) were on two or more anti - pd drugs . factors responsible for falls in pd patients are multiple and most likely include similar factors associated with falls in healthy elderly patients . the frequency of falls in our study was similar to those described in previous studies in pd patients . in a study to assess the risk of falls in 160 pd patients , contreras and grandas documented a 38.8% prevalence , a figure slightly lower than the finding in this study . however , a meta - analysis of six fall studies in pd patients reported fall frequencies ranging between 38% and 50% . the frequency in our study lies within this range , and variations in study methodology and clinical characteristics could account for the varied frequencies . it is difficult to speculate on cultural factors playing a very significant role in falls in pd patients because the frequency was similar to what was obtained in other environments . factors such as special flooring ( marble , tiles , and polished surfaces ) that could predispose a patient to fall are absent in most traditional nigerian homes but did not seem to have any significant effects on falling frequencies when compared to other types of homes . additionally , there were no increases in the incidence of neuropathy , visual impairment , or medications that predispose pd patients to fall . falling patients had significantly worse disease and longer durations when compared to non - falling patients . and wood et al . , who also reported higher updrs motor scores in falling versus non - falling patients ; therefore , this is not a predictor of falls . postural instability was prominent in the falling patients more than the non - falling patients due to poor preparatory adjustments , abnormal postural balance , and delayed reaction time . a study by contreras and grandas reported predictability of tinetti balance test to predict falls with 71% sensitivity and 79% specificity . he confirmed the tinetti test tool as an independent fall predictor after using a multivariate logistic regression model . this is consistent with studies of gait and balance abnormalities that were carried out in other environments where gait abnormality was considered the most consistent predictor of falls . however , in this study the tinetti balance and gait tool did not independently predict the risk of falls . the frequency of falls has also been reported to have a direct correlation with age , disease severity , medication , cognitive dysfunction , orthostatic hypotension , and visual impairment . a previous study of falls both in pd patients and healthy adults had shown an association between adults > 65 years and increased falls of 30 to 40% . this was most likely because most of the pd patients in that study were younger compared to other studies conducted in different environments . advancing age ( > 70 years ) has been associated with increased risk of falls due to advanced pd progression . in this study , this is different from previous epidemiological studies of pd in nigeria where relatively low proportions of young onset pd patients were evaluated . however , femi et al . reported an increasing older age of pd onset in the northwestern nigeria . these differences might be due to an inappropriate record of birth dates due to low level of literacy , shorter average life expectancy of those living in the underdeveloped and developing countries , poverty and/or poor access to health care services . furthermore , different genetic makeups and environmental exposures of the study participants might be responsible for the observed differences . a previous fall has been shown to be a significant risk factor for more falls . reported in a meta - analysis that > 2 falls was the best predictor of more falls in pd . this might be the result of lower recovery performances in forward falls and lower muscle strength in pd patients who fall . in this study , 67.7% of our pd patients had recurrent falls , out of which 44% had a fear of falling . the reported frequency of recurrent falls was higher than what was obtained by landers et al . ( 51% ) and dibble and lange ( 55% ) . however , the findings in this study are similar to a prospective study of falls by cole et al . . demonstrated low balance confidence scores , which is an indicator for more falls , in pd patients compared to controls . patients who fall more frequently often have a self - induced restriction of activities due to fear of more falls , thus , leading to reduced muscle strength and increased risk of more falls , resulting in poor health - related quality of life . studies have demonstrated increased incidence of trochanteric fractures in pd patients due to adduction of arms against the trunk when falling , while older adults without pd stretched out their arms in the direction of impending fall and , thus , sustained minor injuries such as abrasion . worthy of note in this study is the predictability of fear of falling as a strong modifiable risk factor with an adjusted or of 3.86 . thus , clinical strategies to reduce fear of falling in pd patients will reduce the frequency of falls and their health - related and economic burden . in this study , it was observed that falling pd patients sustained other injuries such as fractures and lacerations ; however , bloem et al . these injuries may be a result of the inter - segmental stiffness and postural inflexibility observed in pd patients . being a retrospective study , there was the possibility of recall bias , and subjects with cognitive impairment may not have been accurate about fall frequency . it has been said that older people tend to forget occurrences of previous falls ; however , this can be improved upon by getting a fall diary to be used in a prospective study . secondly , this was a hospital - based study , and thus , the proportion of falling patients in our study may not represent community - dwelling pd patients who are at greater risk of falls . although the results show that fear of falling could be an independent risk factor for falls in pd patients , additional prospective studies are required to confirm if this is a cause or consequence of falling or both . meanwhile , our findings suggest that addressing the fear of falling may help to reduce falling events in pd patients . in conclusion , our study confirmed an increased frequency of falls among pd patients , and this frequency was shown to be multifactorial . the predictive risk factors were disease severity and duration and fear of falling . early detection of risk factors can prevent the untoward effects of falls in pd patients . this will facilitate tailored management to suit such patients and ultimately reduce the economic health - related burden of falls in these patients .

periodontitis has been described as a probable risk factor for evoking a procoagulant state mediated by periodontal pathogens and its bioactive products , which contributes to an induction of a low - grade systemic inflammation that complies with a transient repeated bacteremia . on the other hand , endothelial dysfunction and hypercoagulability are thought to be a result of the concomitant involvement of hypertension and the physical friction of blood on the vascular wall . apparently , the activation of platelets and the endothelial cells is reflected by an increase in the plasmatic vascular and thrombotic markers , for example , platelets count ( plt ) , fibrinogen ( fib ) , von willebrand factor antigen activity ( vwf : ag ) and d - dimers ( dd ) . periodontal debridement itself has been assumed to induce additional transient bacteraemia through the concomittant traumatic procedures . the aim of this study was to assess changes in blood levels of several vascular thrombotic markers ; platelets count , fib , vwf : ag and the dd , accompanying surgical and non - surgical periodontal debridement in hypertensive patients with periodontitis . a total of 40 consecutive patients , 27 males and 13 females , aged 37 - 68 years old , ( mean 51.2 years ) , of whom recalled the department of periodontology , faculty of dental surgery at damascus university from march 2010 to april 2010 , met the inclusion criteria and agreed to participate signing an informed written consent . they were non - smokers , had more than 20 teeth , did not have any coagulopathies or metabolic disorders ( e.g. , diabetes , hypercholesterolemia , hypertriglyceridemia ) or any periodontal treatment or extractions during the last 6 months or any antibiotic therapy for 3 months or corticosteroids for the last year . pregnant and breast feeding women , uncontrolled hypertensive ( systolic blood pressure [ sbp ] > 160 mm / hg and/or diastolic blood pressure > 110 mm / hg ) and body mass index > 31 were excluded . personal information , medical history and blood pressure mean value before treatment were also registered . hypertension was diagnosed depending on the medical history and checking the current blood pressure as patients were on different antihypertensive agents ( losartan * , deltiazim * , metoprolol * , amlodepin * ) . periodontal disease was diagnosed depending on the existence of 4 teeth or more with one site or more in which pocket probing depth ( ppd ) 4 mm and/or clinical attachment loss 3 mm . accordingly , the study population was distributed into two groups : non - surgical group with pockets 4 - 6 mm , 12 males and 8 females and surgical group , pockets > 6 mm , 15 males and 5 females . the scientific research board and ethical committee of damascus university approval was obtained with the primary project plan . periodontal indices included plaque index ( pi ) , gingival inflammation ( gi ) , bleeding on probing ( bop ) and ppd , were measured by a trained examiner taking four - site registration ( midfacial , mesial , distal and lingual / palatal ) by means of university of north carolina-15 periodontal probe , ( medesy - italy ) . non - surgical mechanical debridement consisted of one session full mouth scaling and root planning under local anesthesia as required using ultrasonic instruments , ( satelec - switzerland ) and was accomplished by gracey 's manual instrumentation , ( lascad / zeffiro - italy ) . surgical group received full mouth supragingival scaling with widman flap reflected at one quadrant , mechanical debridement without any chemical conditioning , flap reduction and 4/0 silk interrupted suturing . actual working time was registered for each patient at the end of the treatment session . blood samples , 20 ml , were collected from the antecubital veins of all patients after 16 h of fasting at the first visit for baseline , by the same patient - status - blinded technician . blood samples were distributed into two types of tubes ; the first contained ethylenediaminetetraacetic acid for general blood chemistry tests ; ( cholesterol , triglycerides , blood glucose fasting , ( chemistry analyzer au 400-life and material science - germany ) , and complete blood count and platelets ( ref . interval 150 - 400 103/mm ) , ( abx-5 pentra-60 , horiba abx - france ) , while the second and the third tubes contained sodium citrate 3.8% for the coagulation tests ( clotting analyzer coadata 2001 , labitech - germany ) ; fib , ( ref . interval clauss 200 - 400 mg / dl ) , vwf : ag , ( ref . interval 100% ) , ( hitachi 911 , roche - diagnostics , u.s.a ) and d - dimers , ( ref . interval 0.05 g feu / ml ) , ( au400 olympus ) . all tests were performed at the police hospital central lab while vwf : ag test was performed at the laboratories department of al - assad hospital , damascus university . chlorhexidine 0.12% mouth rinses were prescribed and sutures removal after 7 days with a weekly recall . a second periodontal parameters determination was performed after 6 weeks for each patient while blood samples were re - collected after 48 h and after 6 weeks of treatment . independent t - test was used to study the differences in variables between the treatment groups and paired samples t - test to study differences in each group before and after periodontal treatment . general linear model anova analysis for repeated measures was applied to study the significance of the thrombotic variables changes through the three - time intervals , as well as the effect of interaction between time and group for each variable . all tests were performed at 5% of confidence level , spss 19 edition software ( chicago , il , usa ) . a total of 40 consecutive patients , 27 males and 13 females , aged 37 - 68 years old , ( mean 51.2 years ) , of whom recalled the department of periodontology , faculty of dental surgery at damascus university from march 2010 to april 2010 , met the inclusion criteria and agreed to participate signing an informed written consent . they were non - smokers , had more than 20 teeth , did not have any coagulopathies or metabolic disorders ( e.g. , diabetes , hypercholesterolemia , hypertriglyceridemia ) or any periodontal treatment or extractions during the last 6 months or any antibiotic therapy for 3 months or corticosteroids for the last year . pregnant and breast feeding women , uncontrolled hypertensive ( systolic blood pressure [ sbp ] > 160 mm / hg and/or diastolic blood pressure > 110 mm / hg ) and body mass index > 31 were excluded . personal information , medical history and blood pressure mean value before treatment were also registered . hypertension was diagnosed depending on the medical history and checking the current blood pressure as patients were on different antihypertensive agents ( losartan * , deltiazim * , metoprolol * , amlodepin * ) . periodontal disease was diagnosed depending on the existence of 4 teeth or more with one site or more in which pocket probing depth ( ppd ) 4 mm and/or clinical attachment loss 3 mm . accordingly , the study population was distributed into two groups : non - surgical group with pockets 4 - 6 mm , 12 males and 8 females and surgical group , pockets > 6 mm , 15 males and 5 females . the scientific research board and ethical committee of damascus university approval was obtained with the primary project plan . periodontal indices included plaque index ( pi ) , gingival inflammation ( gi ) , bleeding on probing ( bop ) and ppd , were measured by a trained examiner taking four - site registration ( midfacial , mesial , distal and lingual / palatal ) by means of university of north carolina-15 periodontal probe , ( medesy - italy ) . non - surgical mechanical debridement consisted of one session full mouth scaling and root planning under local anesthesia as required using ultrasonic instruments , ( satelec - switzerland ) and was accomplished by gracey 's manual instrumentation , ( lascad / zeffiro - italy ) . surgical group received full mouth supragingival scaling with widman flap reflected at one quadrant , mechanical debridement without any chemical conditioning , flap reduction and 4/0 silk interrupted suturing . actual working time was registered for each patient at the end of the treatment session . blood samples , 20 ml , were collected from the antecubital veins of all patients after 16 h of fasting at the first visit for baseline , by the same patient - status - blinded technician . blood samples were distributed into two types of tubes ; the first contained ethylenediaminetetraacetic acid for general blood chemistry tests ; ( cholesterol , triglycerides , blood glucose fasting , ( chemistry analyzer au 400-life and material science - germany ) , and complete blood count and platelets ( ref . interval 150 - 400 103/mm ) , ( abx-5 pentra-60 , horiba abx - france ) , while the second and the third tubes contained sodium citrate 3.8% for the coagulation tests ( clotting analyzer coadata 2001 , labitech - germany ) ; fib , ( ref . interval clauss 200 - 400 mg / dl ) , vwf : ag , ( ref . interval 100% ) , ( hitachi 911 , roche - diagnostics , u.s.a ) and d - dimers , ( ref . all tests were performed at the police hospital central lab while vwf : ag test was performed at the laboratories department of al - assad hospital , damascus university . chlorhexidine 0.12% mouth rinses were prescribed and sutures removal after 7 days with a weekly recall . a second periodontal parameters determination was performed after 6 weeks for each patient while blood samples were re - collected after 48 h and after 6 weeks of treatment . independent t - test was used to study the differences in variables between the treatment groups and paired samples t - test to study differences in each group before and after periodontal treatment . general linear model anova analysis for repeated measures was applied to study the significance of the thrombotic variables changes through the three - time intervals , as well as the effect of interaction between time and group for each variable . all tests were performed at 5% of confidence level , spss 19 edition software ( chicago , il , usa ) . statistical study showed higher sbp mean value at baseline in the non - surgical group compared with the surgical one ( p = 0.047 ) [ table 1 ] . there was no statistically significant differences in age , body mass index , cholesterol , triglycerides , blood glucose fasting ( p > 0.05 ) [ table 1 ] . means of variables in the non - surgical and before periodontal debridement ( baseline ) surgical groups ( n=40 ) ppd was higher at baseline in the surgical group compared with the non - surgical one , ( p = 0,001 ) , [ table 1 ] . the periodontal indices decreased significantly after 6 weeks in both groups ( p = 0.001 ) , [ table 2 ] despite of the fact that bop and ppd values remained higher in the surgical group compared with the non - surgical one ( p = 0.041 - 0.001 respectively ) , [ table 2 ] . working time was significantly higher in the surgical group compared with the non - surgical ( 40.2 5.1 vs. 74.3 10.4 , respectively p = 0.001 ) . means of periodontal indices in the non - surgical and surgical groups no significant differences appeared between groups regarding vascular thrombotic variables at baseline , ( p > 0.05 ) , [ table 1 ] . nonetheless , all vascular thrombotic markers levels increased significantly after 48 h in both groups ( p < 0.05 ) , [ table 3 ] . after 6 weeks , plt and fib decreased significantly while vwf : ag sustained higher than baseline , ( p < 0.05 ) and dd restored approximately baseline , ( p > 0.05 ) , [ table 3 , figure 1 ] . no significant differences were noticed between the two groups after 48 h and 6 weeks , ( p > 0.05 ) , [ table 4 ] . however , anova general linear model test revealed significant changes through the three - time interval within each group , ( p = 0.001 ) and a significant effect of interaction between time and group for vwf : ag , ( p = 0.005 ) , [ table 4 ] . means of vascular thrombotic markers in the non - surgical and surgical groups changes in vascular thrombotic markers across the three - time intervals anova test for vascular thrombotic markers changes across time intervals within the two groups and the t test results for acute and healing phases statistical study showed higher sbp mean value at baseline in the non - surgical group compared with the surgical one ( p = 0.047 ) [ table 1 ] . there was no statistically significant differences in age , body mass index , cholesterol , triglycerides , blood glucose fasting ( p > 0.05 ) [ table 1 ] . means of variables in the non - surgical and before periodontal debridement ( baseline ) surgical groups ( n=40 ) ppd was higher at baseline in the surgical group compared with the non - surgical one , ( p = 0,001 ) , [ table 1 ] . the periodontal indices decreased significantly after 6 weeks in both groups ( p = 0.001 ) , [ table 2 ] despite of the fact that bop and ppd values remained higher in the surgical group compared with the non - surgical one ( p = 0.041 - 0.001 respectively ) , [ table 2 ] . working time was significantly higher in the surgical group compared with the non - surgical ( 40.2 5.1 vs. 74.3 10.4 , respectively p = 0.001 ) . no significant differences appeared between groups regarding vascular thrombotic variables at baseline , ( p > 0.05 ) , [ table 1 ] . nonetheless , all vascular thrombotic markers levels increased significantly after 48 h in both groups ( p < 0.05 ) , [ table 3 ] . after 6 weeks , plt and fib decreased significantly while vwf : ag sustained higher than baseline , ( p < 0.05 ) and dd restored approximately baseline , ( p > 0.05 ) , [ table 3 , figure 1 ] . no significant differences were noticed between the two groups after 48 h and 6 weeks , ( p > 0.05 ) , [ table 4 ] . however , anova general linear model test revealed significant changes through the three - time interval within each group , ( p = 0.001 ) and a significant effect of interaction between time and group for vwf : ag , ( p = 0.005 ) , [ table 4 ] . means of vascular thrombotic markers in the non - surgical and surgical groups changes in vascular thrombotic markers across the three - time intervals anova test for vascular thrombotic markers changes across time intervals within the two groups and the t test results for acute and healing phases statistical study revealed groups homogeneity regarding age , gender and body mass index ( p > 0.05 ) while higher blood pressure in the non - surgical group compared with surgical one ( 148.9 9.8 vs. 142.4 10.1 mm / hg , respectively , p = 0.047 ) , which might be contributed to coincidence or the small size of population and groups . ageing was described as a common risk factor for chronic periodontitis and hypertension as both diseases have a middle age predilection , bearing in mind that age and gender are determinant factors that can not be modified with the fact of lower healing responses are assumed in males and females older than 40 years . angeli et al . , suggested a direct association between periodontitis and the left ventricular volume in hypertensives . also reported that blood pressure was higher in individuals who had 0 to 6 teeth in their mouth compared with those who had 27 - 28 teeth . recent reports have pointed out that ageing is accompanied by changes at the hemostatic level presented with hypercoagulability . cholesterol , triglycerides and blood glucose fasting levels in both groups were comparable ( p > 0.05 ) and within the reference interval , table 1 , which negates the metabolic syndrome existence in our study population . however , benguigui et al . revealed an association between the metabolic syndrome , especially increased insulin resistance and severe periodontitis . moreover , oz et al . reported that the systemic inflammation in periodontitis is accompanied with dyslipidemia , which improved after periodontal treatment . ppd was higher in the surgical group at baseline ( 4.4 0.6 vs. 3.7 0.3 mm , respectively , p = 0.001 ) . nevertheless , after 6 weeks of periodontal treatment all periodontal parameters improved in each group ( p < 0.05 ) . however , both of bop and ppd remained higher in the surgical group compared with the non - surgical ( 39.1 13.1 vs. 31.3 1% , p = 0.041 ) , ( 3.6 0.4 vs. 3 0.3 mm , p = 0.001 ) respectively . oral hygiene instructions and periodontal treatment , after different follow - up periods extended from 6 weeks to 6 years , lead to an improvement in periodontal indices such as plaque index , gingival inflammation , pocket probing depth and clinical attachment loss , regardless of the treatment modality . the differences in the healing degree reflect a difference in the healing volume depending on the initial pocket depth when it is 7 mm . considering that our study population individuals were administering a wide - spectrum of antihypertensive agents , which may influence the periodontal disease process , since the results of our previous study comparing hypertensives on antihypertensive agents with normotensives as higher values in ppd ( 4.1 0.6 vs. 3.75 0.55 respectively , p = 0.008 ) and clinical attachment loss ( 3.3 0.93 vs. 2.31 1.43 , respectively , p = 0.001 ) . despite of the difference in pockets depth and the periodontal disease intensity , there were no statistically significant differences in vascular thrombotic markers between the two groups at baseline , ( p > 0.05 ) . platelets count increased after 48 h of treatment in both groups and decreased after 6 weeks ( p < 0.05 ) , with no significant differences between the two groups at both time intervals , ( p > 0.05 ) . these findings comply with the study of balwant et al . on 26 healthy patients aged 30 - 65 years with severe periodontitis , ppd > 6 mm , reporting that plt count decreased significantly after 10 weeks of scaling and root planning ( 245 vs. 223 10/mm ) as it has been assumed that platelets have a role in the inflammatory process through adhesion and aggregation . platelets are activated in hypertension and in our previous study on 80 patients with periodontitis , plt count was higher in the hypertensive group compared with those with periodontitis alone ( 266 43.5 vs. 289.3 43.6 respectively , p = 0.022 ) . on the other hand , other studies on the effect of periodontal treatment on vascular thrombotic markers in healthy individuals with periodontitis , reported a significant increase in plt count after 48 h in the non - surgical group compared with the surgical one in addition to a significant time group interaction ( p = 0.001 ) , which might be attributed to the extension of working surface . fib levels increased after 48 h in both groups and decreased after 6 weeks ( p < 0.05 ) , with no significant differences between groups at both time intervals , ( p > 0.05 ) . reported that intensive periodontal treatment in severe periodontitis was accompanied with an increase in fib levels , which peaked after 24 h and decreased significantly after 30 weeks while balwant et al . suggested that non - surgical periodontal treatment was effective in improving bop and ppd indices and in reducing inflammatory markers interleukin-6 , c - reactive protein ( crp ) , including fib in severe periodontally diseased hypertensives . in contrast , radafshar et al . did not find a significant decrease in fib levels after 1 week and after 3 months following mechanical periodontal treatment in severe periodontitis , even though there was a significant decrease in crp and white blood cell count . vwf : ag activity increased significantly in both groups after 48 h ( p < 0.05 ) and it decreased after 6 weeks retaining higher values in comparison to baseline with no significant differences between groups at both time intervals , ( p > 0.05 ) . nonetheless , despite there was no statistical significant difference between the two groups at baseline , anova test showed a significant effect of interaction of time group , ( p < 0.05 ) . this outcome would be attributed to the intensity of periodontal disease in the surgical group compared to the non - surgical one represented by the deeper ppd ( 4.4 0.6 vs. 3.7 0.3 respectively , p = 0.001 ) . these results may enhance the suggestion that poor oral hygiene is accompanied with increased levels of von willebrand factor . reported that vwf : ag activity was higher in the intensive group after 24 h of treatment compared to multi - sessions . mentioned that intensive treatment was accompanied with a mild inflammatory response , 30% higher than baseline , which persisted for 1 week . it was also associated with clear endothelial cell activating during 30 day , adding that population contained smokers as smoking had influenced the acute release of the bioactive markers . likewise , dd levels raised after48 h in both groups ( p < 0.05 ) and decreased after 6 weeks , with no significant differences between groups at both time intervals , ( p > 0.05 ) . endothelial surface coursing as noticed in atherosclerosis , trauma and infection , would initiate thrombosis . postulated that intensive periodontal treatment evoked changes at the hemostatic system plane manifested by an increase in dd level , which peaked after 24 h and resettled within 1 month . mentioned that non - surgical periodontal treatment in systemic healthy individuals lead to a significant increase in dd levels after 24 h , which was more evident in the surgical group compared with the non - surgical . did not find any significant differences before and after periodontal treatment regarding dd in patients exhibiting periodontitis with coronary heart disease . small size of population formed a limitation of this study , but the overall data might infer that both modalities of periodontal treatment had induced , after 48 h , a mild activation of several thrombotic system mediators presenting a procoagulatory state , even though not exceeding the normal range of values , which recessed within 6 weeks . despite of the longer operating time in the surgical group , the degree of changes was close in both groups . according to the chronic nature of both periodontitis and hypertension , the periodontal intervention , when one of the two diseases exists or both do , may not decrease the symptoms or lower the risk of future thrombotic events , but it may augment the overall prophylactic circumstances to decrease this risk by introducing better oral hygiene and the suitable intervention . this might be supported by systemic antibiotics and anti- inflammatory agents that lower the microbial and inflammatory burden , that 's why development of new anticoagulants or antithrombotics that reduce the contribution of the inflammatory reaction to the vascular thrombotic disease is being warranted . data presented in this study may subject using the adjunctive therapies such as systemic antibiotics and justifies not interrupting platelets antagonists prescribed for hypertensive . periodontal debridement , surgical and non - surgical , has improved the periodontal status in hypertensives . periodontal treatment activated the coagulation system in hypertensives and recessed later while the treatment modality did not affect the degree of activation . ultimately , more studies are suggested to investigate the effect of periodontal disease and its treatment , in healthy and systemically diseased individuals , on the overall coagulation activation process output which is presented by thrombin activation using advanced thrombin generation assays such as the calibrated automated thrombogram that would help in assessing additional meticulous markers such as the microparticles levels that play , like thrombin , a role in both of the inflammatory and coagulation process , which would enhance more strict prophylactic procedures when treating health - compromised individuals .

background : periodontal debridement has an impact on the vascular thrombotic markers in healthy individuals . this study aimed to investigate changes in several vascular thrombotic markers after surgical and non - surgical periodontal debridement in hypertensives with periodontitis.materials and methods:40 hypertensives , 27 males and 13 females , 37 - 68 year old , mean 51.2 years , with moderate to severe periodontitis , were divided into two groups , ( n = 20 for each ) ; the first received comprehensive one session non - surgical periodontal debridement , ( pockets 4 - 6 mm ) , while the second received comprehensive supragingival scaling with surgical debridement at one quadrant , ( pockets > 6 mm ) . periodontal parameters included ; plaque index ( pi ) , gingival inflammation ( gi ) , bleeding on probing ( bop ) , pocket probing depth ( ppd ) . vascular thrombotic tests included ; platelets count ( plt ) , fibrinogen ( fib ) , von willebrand factor antigen activity ( vwf : ag ) , and d - dimers ( dd).results : pi , gi , bop , ppd , decreased significantly ( p = 0.001 ) after 6 weeks of periodontal debridement in both groups , while bop and ppd remained higher in the surgical one ( p < 0.05 ) . thrombotic vascular markers changes through the three - time intervals were significant in each group ( p = 0.001 ) , and time - group interception effect was significant for vwf : ag ( p = 0.005 ) , while no significant differences between groups after treatment ( p > 0.05).conclusion : periodontal debridement , surgical and non - surgical , improved the periodontal status in hypertensives . periodontal treatment activated the coagulation system in hypertensives and recessed later while the treatment modality did not affect the degree of activation .

INTRODUCTION MATERIALS AND METHODS Study population and general examination Periodontal examination Periodontal debridement procedures Laboratory biochemical tests Follow-up Statistical study RESULTS General examination Periodontal parameters Vascular thrombotic markers DISCUSSION CONCLUSIONS

apparently , the activation of platelets and the endothelial cells is reflected by an increase in the plasmatic vascular and thrombotic markers , for example , platelets count ( plt ) , fibrinogen ( fib ) , von willebrand factor antigen activity ( vwf : ag ) and d - dimers ( dd ) . the aim of this study was to assess changes in blood levels of several vascular thrombotic markers ; platelets count , fib , vwf : ag and the dd , accompanying surgical and non - surgical periodontal debridement in hypertensive patients with periodontitis . a total of 40 consecutive patients , 27 males and 13 females , aged 37 - 68 years old , ( mean 51.2 years ) , of whom recalled the department of periodontology , faculty of dental surgery at damascus university from march 2010 to april 2010 , met the inclusion criteria and agreed to participate signing an informed written consent . they were non - smokers , had more than 20 teeth , did not have any coagulopathies or metabolic disorders ( e.g. periodontal disease was diagnosed depending on the existence of 4 teeth or more with one site or more in which pocket probing depth ( ppd ) 4 mm and/or clinical attachment loss 3 mm . accordingly , the study population was distributed into two groups : non - surgical group with pockets 4 - 6 mm , 12 males and 8 females and surgical group , pockets > 6 mm , 15 males and 5 females . periodontal indices included plaque index ( pi ) , gingival inflammation ( gi ) , bleeding on probing ( bop ) and ppd , were measured by a trained examiner taking four - site registration ( midfacial , mesial , distal and lingual / palatal ) by means of university of north carolina-15 periodontal probe , ( medesy - italy ) . non - surgical mechanical debridement consisted of one session full mouth scaling and root planning under local anesthesia as required using ultrasonic instruments , ( satelec - switzerland ) and was accomplished by gracey 's manual instrumentation , ( lascad / zeffiro - italy ) . surgical group received full mouth supragingival scaling with widman flap reflected at one quadrant , mechanical debridement without any chemical conditioning , flap reduction and 4/0 silk interrupted suturing . actual working time was registered for each patient at the end of the treatment session . blood samples were distributed into two types of tubes ; the first contained ethylenediaminetetraacetic acid for general blood chemistry tests ; ( cholesterol , triglycerides , blood glucose fasting , ( chemistry analyzer au 400-life and material science - germany ) , and complete blood count and platelets ( ref . interval 150 - 400 103/mm ) , ( abx-5 pentra-60 , horiba abx - france ) , while the second and the third tubes contained sodium citrate 3.8% for the coagulation tests ( clotting analyzer coadata 2001 , labitech - germany ) ; fib , ( ref . interval clauss 200 - 400 mg / dl ) , vwf : ag , ( ref . interval 100% ) , ( hitachi 911 , roche - diagnostics , u.s.a ) and d - dimers , ( ref . interval 0.05 g feu / ml ) , ( au400 olympus ) . all tests were performed at the police hospital central lab while vwf : ag test was performed at the laboratories department of al - assad hospital , damascus university . a second periodontal parameters determination was performed after 6 weeks for each patient while blood samples were re - collected after 48 h and after 6 weeks of treatment . independent t - test was used to study the differences in variables between the treatment groups and paired samples t - test to study differences in each group before and after periodontal treatment . general linear model anova analysis for repeated measures was applied to study the significance of the thrombotic variables changes through the three - time intervals , as well as the effect of interaction between time and group for each variable . a total of 40 consecutive patients , 27 males and 13 females , aged 37 - 68 years old , ( mean 51.2 years ) , of whom recalled the department of periodontology , faculty of dental surgery at damascus university from march 2010 to april 2010 , met the inclusion criteria and agreed to participate signing an informed written consent . periodontal disease was diagnosed depending on the existence of 4 teeth or more with one site or more in which pocket probing depth ( ppd ) 4 mm and/or clinical attachment loss 3 mm . accordingly , the study population was distributed into two groups : non - surgical group with pockets 4 - 6 mm , 12 males and 8 females and surgical group , pockets > 6 mm , 15 males and 5 females . periodontal indices included plaque index ( pi ) , gingival inflammation ( gi ) , bleeding on probing ( bop ) and ppd , were measured by a trained examiner taking four - site registration ( midfacial , mesial , distal and lingual / palatal ) by means of university of north carolina-15 periodontal probe , ( medesy - italy ) . non - surgical mechanical debridement consisted of one session full mouth scaling and root planning under local anesthesia as required using ultrasonic instruments , ( satelec - switzerland ) and was accomplished by gracey 's manual instrumentation , ( lascad / zeffiro - italy ) . surgical group received full mouth supragingival scaling with widman flap reflected at one quadrant , mechanical debridement without any chemical conditioning , flap reduction and 4/0 silk interrupted suturing . blood samples were distributed into two types of tubes ; the first contained ethylenediaminetetraacetic acid for general blood chemistry tests ; ( cholesterol , triglycerides , blood glucose fasting , ( chemistry analyzer au 400-life and material science - germany ) , and complete blood count and platelets ( ref . interval 150 - 400 103/mm ) , ( abx-5 pentra-60 , horiba abx - france ) , while the second and the third tubes contained sodium citrate 3.8% for the coagulation tests ( clotting analyzer coadata 2001 , labitech - germany ) ; fib , ( ref . interval clauss 200 - 400 mg / dl ) , vwf : ag , ( ref . interval 100% ) , ( hitachi 911 , roche - diagnostics , u.s.a ) and d - dimers , ( ref . all tests were performed at the police hospital central lab while vwf : ag test was performed at the laboratories department of al - assad hospital , damascus university . a second periodontal parameters determination was performed after 6 weeks for each patient while blood samples were re - collected after 48 h and after 6 weeks of treatment . independent t - test was used to study the differences in variables between the treatment groups and paired samples t - test to study differences in each group before and after periodontal treatment . general linear model anova analysis for repeated measures was applied to study the significance of the thrombotic variables changes through the three - time intervals , as well as the effect of interaction between time and group for each variable . statistical study showed higher sbp mean value at baseline in the non - surgical group compared with the surgical one ( p = 0.047 ) [ table 1 ] . there was no statistically significant differences in age , body mass index , cholesterol , triglycerides , blood glucose fasting ( p > 0.05 ) [ table 1 ] . means of variables in the non - surgical and before periodontal debridement ( baseline ) surgical groups ( n=40 ) ppd was higher at baseline in the surgical group compared with the non - surgical one , ( p = 0,001 ) , [ table 1 ] . the periodontal indices decreased significantly after 6 weeks in both groups ( p = 0.001 ) , [ table 2 ] despite of the fact that bop and ppd values remained higher in the surgical group compared with the non - surgical one ( p = 0.041 - 0.001 respectively ) , [ table 2 ] . working time was significantly higher in the surgical group compared with the non - surgical ( 40.2 5.1 vs. 74.3 10.4 , respectively p = 0.001 ) . means of periodontal indices in the non - surgical and surgical groups no significant differences appeared between groups regarding vascular thrombotic variables at baseline , ( p > 0.05 ) , [ table 1 ] . nonetheless , all vascular thrombotic markers levels increased significantly after 48 h in both groups ( p < 0.05 ) , [ table 3 ] . after 6 weeks , plt and fib decreased significantly while vwf : ag sustained higher than baseline , ( p < 0.05 ) and dd restored approximately baseline , ( p > 0.05 ) , [ table 3 , figure 1 ] . no significant differences were noticed between the two groups after 48 h and 6 weeks , ( p > 0.05 ) , [ table 4 ] . however , anova general linear model test revealed significant changes through the three - time interval within each group , ( p = 0.001 ) and a significant effect of interaction between time and group for vwf : ag , ( p = 0.005 ) , [ table 4 ] . means of vascular thrombotic markers in the non - surgical and surgical groups changes in vascular thrombotic markers across the three - time intervals anova test for vascular thrombotic markers changes across time intervals within the two groups and the t test results for acute and healing phases statistical study showed higher sbp mean value at baseline in the non - surgical group compared with the surgical one ( p = 0.047 ) [ table 1 ] . there was no statistically significant differences in age , body mass index , cholesterol , triglycerides , blood glucose fasting ( p > 0.05 ) [ table 1 ] . means of variables in the non - surgical and before periodontal debridement ( baseline ) surgical groups ( n=40 ) ppd was higher at baseline in the surgical group compared with the non - surgical one , ( p = 0,001 ) , [ table 1 ] . the periodontal indices decreased significantly after 6 weeks in both groups ( p = 0.001 ) , [ table 2 ] despite of the fact that bop and ppd values remained higher in the surgical group compared with the non - surgical one ( p = 0.041 - 0.001 respectively ) , [ table 2 ] . working time was significantly higher in the surgical group compared with the non - surgical ( 40.2 5.1 vs. 74.3 10.4 , respectively p = 0.001 ) . no significant differences appeared between groups regarding vascular thrombotic variables at baseline , ( p > 0.05 ) , [ table 1 ] . nonetheless , all vascular thrombotic markers levels increased significantly after 48 h in both groups ( p < 0.05 ) , [ table 3 ] . after 6 weeks , plt and fib decreased significantly while vwf : ag sustained higher than baseline , ( p < 0.05 ) and dd restored approximately baseline , ( p > 0.05 ) , [ table 3 , figure 1 ] . no significant differences were noticed between the two groups after 48 h and 6 weeks , ( p > 0.05 ) , [ table 4 ] . however , anova general linear model test revealed significant changes through the three - time interval within each group , ( p = 0.001 ) and a significant effect of interaction between time and group for vwf : ag , ( p = 0.005 ) , [ table 4 ] . means of vascular thrombotic markers in the non - surgical and surgical groups changes in vascular thrombotic markers across the three - time intervals anova test for vascular thrombotic markers changes across time intervals within the two groups and the t test results for acute and healing phases statistical study revealed groups homogeneity regarding age , gender and body mass index ( p > 0.05 ) while higher blood pressure in the non - surgical group compared with surgical one ( 148.9 9.8 vs. 142.4 10.1 mm / hg , respectively , p = 0.047 ) , which might be contributed to coincidence or the small size of population and groups . cholesterol , triglycerides and blood glucose fasting levels in both groups were comparable ( p > 0.05 ) and within the reference interval , table 1 , which negates the metabolic syndrome existence in our study population . ppd was higher in the surgical group at baseline ( 4.4 0.6 vs. 3.7 0.3 mm , respectively , p = 0.001 ) . nevertheless , after 6 weeks of periodontal treatment all periodontal parameters improved in each group ( p < 0.05 ) . however , both of bop and ppd remained higher in the surgical group compared with the non - surgical ( 39.1 13.1 vs. 31.3 1% , p = 0.041 ) , ( 3.6 0.4 vs. 3 0.3 mm , p = 0.001 ) respectively . oral hygiene instructions and periodontal treatment , after different follow - up periods extended from 6 weeks to 6 years , lead to an improvement in periodontal indices such as plaque index , gingival inflammation , pocket probing depth and clinical attachment loss , regardless of the treatment modality . the differences in the healing degree reflect a difference in the healing volume depending on the initial pocket depth when it is 7 mm . considering that our study population individuals were administering a wide - spectrum of antihypertensive agents , which may influence the periodontal disease process , since the results of our previous study comparing hypertensives on antihypertensive agents with normotensives as higher values in ppd ( 4.1 0.6 vs. 3.75 0.55 respectively , p = 0.008 ) and clinical attachment loss ( 3.3 0.93 vs. 2.31 1.43 , respectively , p = 0.001 ) . despite of the difference in pockets depth and the periodontal disease intensity , there were no statistically significant differences in vascular thrombotic markers between the two groups at baseline , ( p > 0.05 ) . platelets count increased after 48 h of treatment in both groups and decreased after 6 weeks ( p < 0.05 ) , with no significant differences between the two groups at both time intervals , ( p > 0.05 ) . on 26 healthy patients aged 30 - 65 years with severe periodontitis , ppd > 6 mm , reporting that plt count decreased significantly after 10 weeks of scaling and root planning ( 245 vs. 223 10/mm ) as it has been assumed that platelets have a role in the inflammatory process through adhesion and aggregation . platelets are activated in hypertension and in our previous study on 80 patients with periodontitis , plt count was higher in the hypertensive group compared with those with periodontitis alone ( 266 43.5 vs. 289.3 43.6 respectively , p = 0.022 ) . on the other hand , other studies on the effect of periodontal treatment on vascular thrombotic markers in healthy individuals with periodontitis , reported a significant increase in plt count after 48 h in the non - surgical group compared with the surgical one in addition to a significant time group interaction ( p = 0.001 ) , which might be attributed to the extension of working surface . fib levels increased after 48 h in both groups and decreased after 6 weeks ( p < 0.05 ) , with no significant differences between groups at both time intervals , ( p > 0.05 ) . reported that intensive periodontal treatment in severe periodontitis was accompanied with an increase in fib levels , which peaked after 24 h and decreased significantly after 30 weeks while balwant et al . suggested that non - surgical periodontal treatment was effective in improving bop and ppd indices and in reducing inflammatory markers interleukin-6 , c - reactive protein ( crp ) , including fib in severe periodontally diseased hypertensives . did not find a significant decrease in fib levels after 1 week and after 3 months following mechanical periodontal treatment in severe periodontitis , even though there was a significant decrease in crp and white blood cell count . vwf : ag activity increased significantly in both groups after 48 h ( p < 0.05 ) and it decreased after 6 weeks retaining higher values in comparison to baseline with no significant differences between groups at both time intervals , ( p > 0.05 ) . nonetheless , despite there was no statistical significant difference between the two groups at baseline , anova test showed a significant effect of interaction of time group , ( p < 0.05 ) . this outcome would be attributed to the intensity of periodontal disease in the surgical group compared to the non - surgical one represented by the deeper ppd ( 4.4 0.6 vs. 3.7 0.3 respectively , p = 0.001 ) . these results may enhance the suggestion that poor oral hygiene is accompanied with increased levels of von willebrand factor . reported that vwf : ag activity was higher in the intensive group after 24 h of treatment compared to multi - sessions . likewise , dd levels raised after48 h in both groups ( p < 0.05 ) and decreased after 6 weeks , with no significant differences between groups at both time intervals , ( p > 0.05 ) . mentioned that non - surgical periodontal treatment in systemic healthy individuals lead to a significant increase in dd levels after 24 h , which was more evident in the surgical group compared with the non - surgical . did not find any significant differences before and after periodontal treatment regarding dd in patients exhibiting periodontitis with coronary heart disease . small size of population formed a limitation of this study , but the overall data might infer that both modalities of periodontal treatment had induced , after 48 h , a mild activation of several thrombotic system mediators presenting a procoagulatory state , even though not exceeding the normal range of values , which recessed within 6 weeks . despite of the longer operating time in the surgical group , the degree of changes was close in both groups . this might be supported by systemic antibiotics and anti- inflammatory agents that lower the microbial and inflammatory burden , that 's why development of new anticoagulants or antithrombotics that reduce the contribution of the inflammatory reaction to the vascular thrombotic disease is being warranted . periodontal debridement , surgical and non - surgical , has improved the periodontal status in hypertensives . periodontal treatment activated the coagulation system in hypertensives and recessed later while the treatment modality did not affect the degree of activation . ultimately , more studies are suggested to investigate the effect of periodontal disease and its treatment , in healthy and systemically diseased individuals , on the overall coagulation activation process output which is presented by thrombin activation using advanced thrombin generation assays such as the calibrated automated thrombogram that would help in assessing additional meticulous markers such as the microparticles levels that play , like thrombin , a role in both of the inflammatory and coagulation process , which would enhance more strict prophylactic procedures when treating health - compromised individuals .

type 2 diabetes is a common metabolic disorder defined by the presence of markedly elevated levels of plasma glucose , which arise from dysregulations in the complex interplay between pancreatic -cell function and insulin sensitivity in hepatic and skeletal muscle cells . genetic association studies have recently revealed nearly 40 robustly replicated loci for type 2 diabetes . parallel , high - powered genome - wide analyses of quantitative glycemic traits [ 36 , 7 ] have provided alternative insights into the function of these loci , which are informing our understanding of disease pathophysiology . glycated hemoglobin a1c ( hba1c ) , or the percentage of total hemoglobin that is bound by glucose , provides a better estimate of average glycemia than routine determinations of blood glucose concentration , and is the most widely used index of chronic glycemia [ 8 , 9 ] . it results from the nonenzymatic chemical modification of hemoglobin molecules carried in erythrocytes by glucose . the glycation process involves the nonenzymatic attachment of glucose to the nh - terminal n - terminal valine and internal lysine amino groups of hemoglobin . the glycation reaction is mostly irreversible , so that the concentration of hba1c is a function of the concentration of glucose to which the erythrocytes are exposed over their lifespan ( 120 days on average ) . hba1c therefore represents a surrogate marker of average blood glucose concentration over the previous 8 to 12 weeks , thus representing a longer - term indicator of glycemic status compared with fasting glucose . hba1c levels are better predictors than fasting glucose of the development of long - term complications in type 1 and type 2 diabetes , and higher levels in the subdiabetic range have been shown to predict type 2 diabetes risk and cardiovascular disease [ 13 , 14 ] . for these reasons an international expert committee has recently proposed a revision of the diagnostic criteria for diabetes , recommending that hba1c may be a better means of diagnosing diabetes than measures of glucose ( fasting and/or postchallenge ) and that it be adopted as a diagnostic criterion for diabetes . the heritability of hba1c levels is relatively high ( 47% to 59% ) when compared with fasting glucose ( 34% to 36% ) or glucose level 2-hour post - oral glucose tolerance test ( ogtt ) ( 33% ) [ 16 , 17 ] , and thus amenable to genetic analysis . here i review current research aimed at discovering genetic determinants of hba1c levels , discussing insights into biologic factors influencing variability in the general and diabetic population , and across different ethnicities . there is a relative paucity of published reports on linkage and candidate gene association studies for hba1c levels . the framingham heart study conducted a genome - wide search for diabetes - related genes using measures of glycemia as quantitative traits ( 20-year mean fasting glucose , current fasting glucose , and hba1c ) . a total of 771 men and women from 330 pedigrees from the 5th offspring study exam cycle ( 19911995 ) had information on hba1c levels and were typed at 401 microsatellite markers ( at an average spacing of 10 cm ) . peak evidence for linkage to hba1c levels was on the long arm of chromosome 1 at 187 cm ( multipoint logarithm of the odds score , 2.81 ) , in a model accounting for age , cigarette smoking , alcohol and estrogen use , physical activity , and body mass index . the same broad chromosomal region had been reported as having evidence for linkage for type 2 diabetes and quantitative fasting traits . among the first candidate gene studies , shima et al . tested the association of variants in the calpain-10 gene ( capn10 ) gene a candidate gene for type 2 diabetes originally reported in mexican americans with several metabolic traits in 286 unselected japanese subjects . they found one single nucleotide polymorphism ( snp-19 ) associated with higher body mass index and hba1c levels at the nominal level under the dominant model ( p = 0.003 and p = 0.024 , respectively ) , indicating a contribution of capn10 variants to mild obesity and glucose intolerance in japanese . more recently , krzov et al . tested associations of variants at two candidate genes of insulin resistance and type 2 diabetes mellitus , adiponectin ( adipoq ) and resistin ( retn ) , in individuals of european ancestry . g and 276g > t in adipoq and 62g > a and 180c > g in retn in patients with obesity , anorexia nervosa , and in control healthy normal - weight women , and tested associations with serum concentrations of these hormones and measures of insulin sensitivity and metabolic traits , including tumor necrosis factor- , insulin , cholesterol , hba1c , and blood glucose levels . they found significant associations of snp adp+276g > t allele with higher cholesterol levels in patients with anorexia , higher adiponectin concentrations in obese patients , and lower hba1c levels in normal women . snp of the resistin gene 62g > a was associated with lower hba1c in normal women and higher cholesterol concentrations in the obese group . however , neither of these two associations at capn10 and retn was replicated in further , high - powered genome - wide association studies ( gwas ) . the advent of genome - wide snp arrays and imputation - based meta - analysis has provided a robust statistical framework for discovery and replication , and boosted locus discovery revealing a wealth of genetic loci associated with disease and quantitative end points . gwas of fasting glucose levels led to the identification of associations with hba1c at three loci ( g6pc2 , mtnr1b , and gck ) [ 4 , 5 , 2025 ] . the association of mtnr1b with hba1c was furthermore replicated in 3210 unrelated chinese hans from beijing , whereas to date no gwas have been reported in non - european samples . in the first gwas of hba1c levels , par et al . [ 27 ] investigated 337,343 snps in 14,618 healthy women of european ancestry from the women s genome health study , and validated their findings in 455 nondiabetic caucasian participants recruited from the boston metropolitan area . they detected four loci with significant association at the genome - wide level of 10 . of these , three were previously associated with type 2 diabetes or glycemic end points ( gck , slc30a8 , and g6pc2 ) [ 20 , 21 , 23 , 25 , 28 ] and in people free of diabetes . investigators of the magic recently completed the largest to date meta - analysis of hba1c levels [ 7 ] . the genome - wide discovery set used in this study included approximately 2.5 million genotyped and imputed autosomal snps genotyped in 35,920 participants , a sample size that has 80% power to detect snps explaining 0.12% of the trait variance at the genome - wide significant threshold of p = 5 10 . the analysis confirmed a strong statistical support at gck , g6pc2/abcb11 , mtnr1b , and hk1 , confirming previous reports [ 5 , 2125 , 27 ] . furthermore , novel evidence for association was detected at six loci ( in or near fn3k , hfe , tmprss6 , atp11a / tubgcp3 , ank1 , and spta1 ) . in parallel , franklin et al . completed a gwas meta - analysis of glycated hemoglobin levels in 1782 healthy individuals from three genetically isolated populations : the orkney isles in the north of scotland , the dalmatian islands of vis , and korula in croatia . they reported a genome - wide significant association at an intronic variant in the tcf7l2 gene , the strongest common genetic risk factor for type 2 diabetes . an association at the same locus was also reported by karns et al . in a small sample of 843 individuals . in addition to ambient glycemia , it is known that medical conditions that change erythrocyte turnover , hereditary anemias , and iron storage disorders can influence the variability of hba1c in populations . the former include hemolytic anemias , chronic malaria , major blood loss , or blood transfusion ; the latter are caused by rare causative mutations in genes involved in erythrocyte membrane stability , hemoglobin function , and glucose sensing and membrane transport in erythrocytes . it is thus of interest to assess if genetic variation segregating at high frequencies in the population , and causing subtler variation in these parameters , might also affect hba1c . furthermore , it is important to establish whether variation due to nonglycemic factors affects the utility of hba1c in clinical practice . evidence from gwas ( table 1 ) supports the notion that common variants might affect hba1c levels through their effects on glucose levels and also through erythrocyte biology . [ 27 ] , who identified associations at four loci , including gck , slc30a8 , g6pc2 , and hk1 . hk1 encodes the enzyme hexokinase , which catalyzed the first step in glycolysis and thus represents a likely candidate for the control of glucose metabolism . hk1 is the only isoform that is essential for red blood cell glucose metabolism , and is the predominant form among the four isozymes of the hexokinase family ( hk1 , hk2 , hk3 , and glucokinase ) . it is expressed in the vast majority of cells and tissues , including cells that are strictly dependent on glucose uptake for their metabolic needs . in humans , rare nonsynonymous substitutions in the active site of hk1 and intragenic deletions have been shown to cause hk1 enzymatic deficiency associated with autosomal - recessive severe nonspherocytic hemolytic anemia [ 3336 ] . the downeast anemia mice display hk1 deficiency and a similar anemic phenotype .table 1summary of genetic variants robustly associated with hba1c and correlated hematologic and metabolic traits in gwasregionlocussnpspubmedraf ( 95% ci)pcorrelated trait association1q23.1spta1rs2779116208586830.270.02 ( 0.010.03)% increase3 102q31.1g6pc2/abcb11rs1402837190965180.230.02 ( nr)% increase5 10rs560887-fpg ( 18451265)rs560887-fpg ( 19060907)rs552976208586830.640.05 ( 0.040.06)% increase8 10rs563694-fpg ( 18521185)rs560887-homa - b ( 20081858)6p22.2hfers1800562208586830.940.06 ( 0.050.07)% increase3 10rs1408272-mch ( 19862010)rs198846-hgb ( 19820698)7p13gckrs730497190965180.170.03 ( nr)% increase6 10rs4607517-fpg ( 19060907)rs4607517-homa - b ( 20081858)rs1799884208586830.180.04 ( 0.030.05)% increase1 108p11.21ank1rs6474359208586830.970.06 ( 0.040.08)% increase1 10rs4737009208586830.240.03 ( 0.020.04)% increase6 108q24.11slc30a8rs13266634190965180.30.02 ( nr)% decrease5 10rs13266634-t2d ( 17293876 , 17460697 , 17463246 , 17463248 , 17463249 , 19056611 , 19401414 ) , fpg ( 19734900)rs3802177-t2d ( 20581827)rs11558471-fpg ( 20081858)10q22.1hk1rs16926246208586830.90.09 ( 0.080.10)% increase3 10rs7072268190965180.50.05 ( nr)% increase2 1010q25.2tcf7l2rs7903146208494300.720.05 ( 0.020.08)% hba1c decrease1 10rs7901695-t2d ( 17463249)rs4506565-fpg ( 20081858)rs7903146-t2d ( 17293876 , 17460697 , 17463246 , 17463248,17668382 , 18372903 , 19056611 , 19401414 , 19734900 , 20581827)rs12243326-ogtt ( 20081857)11q14.3mtnr1brs1387153208586830.280.03 ( 0.020.04)% increase4 10rs1387153-t2d ( 20581827)rs1387153-fpg ( 19060909)rs2166706-fpg ( 19651812)rs10830963-homa - b ( 20081858 ) , -fpg ( 19060907)13q34atp11a / tubgcp3rs7998202208586830.140.03 ( 0.020.04)% increase5 1017q25.3fn3krs1046896208586830.310.04 ( 0.030.05)% increase2 1022q12.3tmprss6rs855791208586830.420.03 ( 0.020.04)% increase3 10rs855791-hgb ( 19820698)rs2413450-mch ( 19862010)note that this table includes only results from gwas studies , and intentionally omits candidate - snp and candidate - gene studiesnot reportedrsid , associated trait , pubmed i d ; values are given for associations from genome - wide scansfpg fasting plasma glucose ; gwas genome - wide association studies ; hba1c hemoglobin a1c ; hgb hemoglobin ; homa homeostatic model assessment ; mch mean corpuscular hemoglobin ; ogtt oral glucose tolerance test ; raf risk allele frequency ; snp single nucleotide polymorphism ; t2d type 2 diabetes summary of genetic variants robustly associated with hba1c and correlated hematologic and metabolic traits in gwas note that this table includes only results from gwas studies , and intentionally omits candidate - snp and candidate - gene studies rsid , associated trait , pubmed i d ; values are given for associations from genome - wide scans fpg fasting plasma glucose ; gwas genome - wide association studies ; hba1c hemoglobin a1c ; hgb hemoglobin ; homa homeostatic model assessment ; mch mean corpuscular hemoglobin ; ogtt oral glucose tolerance test ; raf risk allele frequency ; snp single nucleotide polymorphism ; t2d type 2 diabetes these observations led bonnefond et al . [ 37 ] to postulate that hk1 genetic variation may indirectly alter hba1c measurements by generating a proanemic state , and independently of ambient blood glucose levels . they assessed the impact of the sentinel snp at hk1 on hba1c , glucose control - related traits ( fasting- and 2-hour post - ogtt related parameters ) , type 2 diabetes risk , and red blood cell related parameters in europeans . surprisingly , the most associated snp at this locus showed no association with any other markers of glucose control , whereas it was significantly associated with hemoglobin levels , hematocrit , and anemia . the subsequent study by the magic investigators [ 7 ] provided additional evidence of hba1c - associated loci with no evidence for association with glucose - control traits . of the 10 loci associated with hba1c at the genome - wide level , three had significant evidence for association of the hba1c - raising allele with fasting glucose and -cell function ( gck , mtnr1b , and g6pc2 ) in this and previous studies , and gck also with 2-hour glucose [ 36 , 24 , 25 , 27 , 38 , 39 ] . two of them ( gck and mtnr1b ) were also associated with type 2 diabetes . for the remaining seven we found no evidence for association with glycemic traits and diabetes , nor with insulin levels , despite adequate power . of these , associations at the hfe and tmprss6 loci mapped to known functional variants in two complementary and directionally consistent pathways and were associated with quantitative hematologic traits [ 7 , 41 , 42 ] . at hfe the a allele at rs1800562 ( cys262tyr ) , which is responsible for hereditary hemochromatosis ( mim 235200 ) , was associated with lower levels of hba1c , rather than the higher levels one would predict from epidemiologic observations of the increased hfe mutation prevalence in patients with type 2 diabetes [ 43 , 44 ] . the reciprocal observation was seen for tmprss6 , where the a allele at snp rs855791 ( val736ala ) was associated with lower hemoglobin levels and higher hba1c levels . three additional loci ( spta1 , ank1 , and including hk1 discussed earlier ) showed suggestive associations with erythrocyte indexes , and rare variants at these loci cause hereditary anemias [ 45 , 46 ] . we postulated that functional variants at hk1 may affect a potential dissociation between ambient plasma glucose and intracellular cytoplasmic glucose , and that the hemoglobin - lowering variant may affect the overall percent of hba1c through an increased glucose / hemoglobin molar ratio , which in turn could increase the rate of hemoglobin that is glycated at a given glucose level . we further postulated a possible role of erythrocyte membrane stability and altered erythrocyte lifespan ( ank1 , spta1 ) and hemoglobin deglycation ( fn3k ) based on the known function of the respective gene products mapping to the vicinity of the association signals . these patterns were confirmed in analyses conditioned on fasting glucose or hematologic traits , which provided statistical support for an effect on hba1c via regulation of systemic glucose concentrations for gck , g6pc2 , and mtnr1b , and via hematologic parameters for hfe , tmprss6 , and hk1 . taken together , these results suggest that these common variants influence hba1c levels via glycemic levels as well as erythrocyte physiology . specific mechanisms are suggested by existing knowledge on the function of leading candidate genes in each region . these hypotheses will need to be tested to understand mechanistically and physiologically the effects of these genetic variants . the gwas described before were all carried out in healthy individuals , using standardized analysis protocols and trait definition . the strength of this approach is that association results are not influenced by strong environmental confounders , principally disease status and antidiabetic medication . the observation that genetic associations underlying hba1c levels at some loci are independent from fasting glucose reflects earlier observations in a classical discordant monozygotic ( mz ) twin design by snieder et al . . such analysis revealed a significant correlation in hba1c levels in 45 mz twins discordant for the disease ( r = 0.52 , p < 0.001 ; as opposed to r = 0.68 , p < 0.001 in 33 mz twins concordant for diabetes ) , suggesting that a substantial proportion of heritability was due to diabetes - independent familial effects . although robust estimates of the proportion of phenotypic variance attributable to diabetes - independent genetic effects are still lacking , these results showed for the first time that familial factors might explain variation in hba1c levels that is not dependent on glycemic control . recently , [ 48 ] carried out a genome - wide analysis on repeated measures of hba1c from the dcct with the aim to identify genetic loci underlying glycemic control in individuals with type 1 diabetes . the study sample consisted of two sets of individuals sampled from the conventional ( n = 667 ) and intensive ( n = 637 ) treatment groups of the dcct . genome - wide snps were tested for association with mean hba1c , stratified by intervention arm as well as in the combined cohort . this analysis yielded a large number of snps ( 233 ) significant at the set threshold of p = 10 , indicating an excess of false - positive owing to relatively underpowered study samples . successive steps were used to prioritize loci for replication , assessing the association of these loci with capillary glucose and repeated measures of multiple complications of diabetes . for replication , associations were assessed through a series of ( non - independent ) analyses testing associations with quarterly hba1c using repeated measures , with mean daily glucose and baseline c - peptide ( to confirm a glycemic mechanism ) , with hba1c repeated measures in the combined cohort ( adjusting for treatment group and testing for interaction with treatment ) , and with glycemic complications including coronary calcium , neuropathy , hypoglycemia , and time to renal or retinal complications ( in each arm separately ) . loci having evidence for association after these steps , including snps near sorcs1 at 10q25.1 , gsc at 14q32.13 , bnc2 at 9p22 , and wdr72 at 15q21.3 , were carried forward for replication in two independent replication samples . these included the gokind study , a case control collection of patients with type 1 diabetes with and without diabetic nephropathy , and healthy subjects from the magic meta - analysis described earlier . these analyses revealed a locus ( rs1358030 near sorcs1 ) where association reached the widely accepted threshold for genome - wide significance of p < 5 10 in the conventional treatment group , which was also associated with mean glucose and showed suggestive evidence for replication in the intensive treatment group and in the control group in gokind . however , this association was not seen among gokind participants with nephropathy nor in the magic sample . another signal near bnc2 showed suggestive evidence in magic but did not replicate in gokind . snps in both regions were associated with diabetes complications in the expected direction : sorcs1 with hypoglycemia ( and less robustly with both retinopathy and nephropathy ) and bnc2 with microvascular end points . it remains to be established if these variable replication outcomes stem from limited study power , or reflect the variable effect of the environment most notably insulin treatment in the two study arms of the study . we direct the readers to an accompanying editorial to the study for a more detailed exploration of these effects . a recent study explored the same four loci bnc2 , sorcs1 , gsc , and wdr72 for their effect on glycemic control in type 2 diabetes . the authors typed 1486 subjects with type 2 diabetes from a norwegian population - based cohort ( hunt2 ) , and tested their effects on hba1c and non - fasting glucose levels individually and in a combined genetic score model . they detected no significant associations with hba1c or glucose and partially inconsistent direction of associations . further studies in other populations are needed to identify whether genetic variants affect glycemic control in type 1 and type 2 diabetes . recently , an international expert committee has proposed a revision of the diagnostic criteria for diabetes , recommending that hba1c may be a better means of diagnosing diabetes than measures of glucose ( fasting and/or postchallenge ) and that it be adopted as a diagnostic criterion for diabetes . the 2010 american diabetes association standards of medical care in diabetes added the hba1c 48 mmol / l ( 6.5% ) as another criterion for the diagnosis of diabetes . the recommendation is based on the association of microvascular complications with hba1c being at least as strong as those with fasting or postchallenge glucose , that hba1c is subject to less day - to - day variability than fasting or postchallenge glucose , and that it can be measured at any time of the day without preparations such as fasting or a glucose challenge . it is likely that practical , medical , methodologic , and financial factors will prevent implementation of the recommendation in the majority of clinical settings . it is nevertheless important to understand how genetic factors underlying normal variation in hba1c through nonglycemic routes might influence diabetes diagnosis . in addition to severe pathologies characterized by altered erythrocyte physiology ( e.g. , inherited hemoglobinopathies ) that may influence the utility of hba1c in diabetes diagnosis [ 31 , 51 , 53 , 54 ] , we and others showed that genetically determined physiologic variation in the general population can also play a role , affecting hba1c levels through subtler but more widespread alterations of iron levels and/or hemoglobin concentration . we sought to quantify these genetic effects in population - level terms , and to evaluate the resulting risk of misclassifying individuals as diabetic or nondiabetic owing to genetic influences on hba1c . using net reclassification analysis , we estimated that the population - level impact of the seven nonglycemic loci when hba1c 6.5% is used as the reference cutoff for diabetes diagnosis was approximately 2% ( p = 0.002 ) . this estimate represents an upper boundary for the effect of these common variants , as most people ( the majority in the center of the distribution ) are expected to have a smaller individual genotype effect size . this suggests that variation in hba1c levels due to common , nonglycemic effect variants might influence only minimally diagnosis or misclassification of diabetes . furthermore , variants at loci controlling iron metabolism associated with hba1c levels are known to vary across ethnic groups . for instance , the a allele frequency at rs1800562 ( hfe ) is absent in populations of west african and east asian ancestry ( www.hapmap.org ) but is relatively common ( ~5% ) in europe . the t allele at rs855791 ( tmprss6 ) is at approximately 39% in europeans , but relatively rare in west african ( ~11% ) and east asian ( ~5% ) populations . these observations raise the question of how variation in frequency and effect size in diverse populations may affect reclassification of diabetes status by hba1c . although the effect of individual loci has not been explored , maruthur et al . [ 55 ] explored the contribution of inherited interethnic differences in hba1c levels in a cross - sectional analysis of 2294 individuals of african american ancestry from the community - based aric study . as rates of admixture with europeans vary among african americans , the percentage of european genetic ancestry for each individual , estimated from ancestry - informative markers , was compared with hba1c levels categorized using american diabetes association diagnostic cut points ( < 5.7 , 5.76.4 , and 6.5% ) . this analysis showed that hba1c levels were positively correlated to the fraction of the genome that was of european origin ( p < 0.001 ) , although this correlation accounted for a minimal fraction ( < 1% ) of the overall variability . compared with genetic ancestry , socioeconomic , demographic , and metabolic risk factors were estimated to play a considerably greater role in governing changes in hba1c . as previously discussed for europeans , these results suggest that the inherited variability among populations is likely to have a negligible impact on hba1c - based diabetes classification , and that the relative contribution of demographic and metabolic factors far outweighs the contribution of genetic ancestry to hba1c values in african americans . current evidence suggests that high - powered genetic analyses provide important new opportunities for dissecting genetic influences of hba1c levels . these initiatives will be important not only to better understand genetic and biologic determinants of hba1c variation in the general population , but also to inform recent initiatives to focus diabetes diagnosis and care more centrally on hba1c . it will be of considerable interest in the future to explore additional areas of study . first , as more variants are discovered through sequencing and fine - mapping efforts , it will be important to reassess genetic predisposition and reclassification rates in european and non - european populations . second , it will be important to extend the study of genetic influences to hba1c in prediabetic and diabetic populations , although the confounding effects of treatment might obscure any role of these polymorphisms in the diabetic population . finally , additional genetic associations may be revealed from studies of low - to - intermediate frequency variants through imputation from the 1000 genomes project , direct association using whole - genome sequencing data , and in - depth replication and locus fine - mapping through custom arrays . these hypothesis - generating genetic efforts will pave the way for further studies of the role of the new loci in hemoglobin glycation , glucose metabolism , and diabetes .

glycated hemoglobin a1c ( hba1c ) indicates the percentage of total hemoglobin that is bound by glucose , produced from the nonenzymatic chemical modification by glucose of hemoglobin molecules carried in erythrocytes . hba1c represents a surrogate marker of average blood glucose concentration over the previous 8 to 12 weeks , or the average lifespan of the erythrocyte , and thus represents a more stable indicator of glycemic status compared with fasting glucose . hba1c levels are genetically determined , with heritability of 47% to 59% . over the past few years , inroads into understanding genetic predisposition by glycemic and nonglycemic factors have been achieved through genome - wide analyses . here i review current research aimed at discovering genetic determinants of hba1c levels , discussing insights into biologic factors influencing variability in the general and diabetic population , and across different ethnicities . furthermore , i discuss briefly the relevance of findings for diabetes monitoring and diagnosis .

Introduction The First Generation of Genetic Studies: Linkage Scans and Candidate Gene Association Studies Locus Discovery through GWAS in Healthy Individuals Associations with Correlated Metabolic and Hematologic Traits and Disease Provide Insights into Biologic Determinants of HbA Discovery Studies in Individuals with Diabetes Impact of Genetic Discoveries on the Use of HbA Interethnic Differences in the Allelic Architecture of HbA Conclusions and Outlook

type 2 diabetes is a common metabolic disorder defined by the presence of markedly elevated levels of plasma glucose , which arise from dysregulations in the complex interplay between pancreatic -cell function and insulin sensitivity in hepatic and skeletal muscle cells . parallel , high - powered genome - wide analyses of quantitative glycemic traits [ 36 , 7 ] have provided alternative insights into the function of these loci , which are informing our understanding of disease pathophysiology . glycated hemoglobin a1c ( hba1c ) , or the percentage of total hemoglobin that is bound by glucose , provides a better estimate of average glycemia than routine determinations of blood glucose concentration , and is the most widely used index of chronic glycemia [ 8 , 9 ] . it results from the nonenzymatic chemical modification of hemoglobin molecules carried in erythrocytes by glucose . the glycation process involves the nonenzymatic attachment of glucose to the nh - terminal n - terminal valine and internal lysine amino groups of hemoglobin . hba1c therefore represents a surrogate marker of average blood glucose concentration over the previous 8 to 12 weeks , thus representing a longer - term indicator of glycemic status compared with fasting glucose . hba1c levels are better predictors than fasting glucose of the development of long - term complications in type 1 and type 2 diabetes , and higher levels in the subdiabetic range have been shown to predict type 2 diabetes risk and cardiovascular disease [ 13 , 14 ] . the heritability of hba1c levels is relatively high ( 47% to 59% ) when compared with fasting glucose ( 34% to 36% ) or glucose level 2-hour post - oral glucose tolerance test ( ogtt ) ( 33% ) [ 16 , 17 ] , and thus amenable to genetic analysis . here i review current research aimed at discovering genetic determinants of hba1c levels , discussing insights into biologic factors influencing variability in the general and diabetic population , and across different ethnicities . the framingham heart study conducted a genome - wide search for diabetes - related genes using measures of glycemia as quantitative traits ( 20-year mean fasting glucose , current fasting glucose , and hba1c ) . a total of 771 men and women from 330 pedigrees from the 5th offspring study exam cycle ( 19911995 ) had information on hba1c levels and were typed at 401 microsatellite markers ( at an average spacing of 10 cm ) . peak evidence for linkage to hba1c levels was on the long arm of chromosome 1 at 187 cm ( multipoint logarithm of the odds score , 2.81 ) , in a model accounting for age , cigarette smoking , alcohol and estrogen use , physical activity , and body mass index . they found significant associations of snp adp+276g > t allele with higher cholesterol levels in patients with anorexia , higher adiponectin concentrations in obese patients , and lower hba1c levels in normal women . snp of the resistin gene 62g > a was associated with lower hba1c in normal women and higher cholesterol concentrations in the obese group . however , neither of these two associations at capn10 and retn was replicated in further , high - powered genome - wide association studies ( gwas ) . the advent of genome - wide snp arrays and imputation - based meta - analysis has provided a robust statistical framework for discovery and replication , and boosted locus discovery revealing a wealth of genetic loci associated with disease and quantitative end points . in the first gwas of hba1c levels , par et al . they detected four loci with significant association at the genome - wide level of 10 . investigators of the magic recently completed the largest to date meta - analysis of hba1c levels [ 7 ] . the genome - wide discovery set used in this study included approximately 2.5 million genotyped and imputed autosomal snps genotyped in 35,920 participants , a sample size that has 80% power to detect snps explaining 0.12% of the trait variance at the genome - wide significant threshold of p = 5 10 . furthermore , novel evidence for association was detected at six loci ( in or near fn3k , hfe , tmprss6 , atp11a / tubgcp3 , ank1 , and spta1 ) . completed a gwas meta - analysis of glycated hemoglobin levels in 1782 healthy individuals from three genetically isolated populations : the orkney isles in the north of scotland , the dalmatian islands of vis , and korula in croatia . they reported a genome - wide significant association at an intronic variant in the tcf7l2 gene , the strongest common genetic risk factor for type 2 diabetes . in addition to ambient glycemia , it is known that medical conditions that change erythrocyte turnover , hereditary anemias , and iron storage disorders can influence the variability of hba1c in populations . the former include hemolytic anemias , chronic malaria , major blood loss , or blood transfusion ; the latter are caused by rare causative mutations in genes involved in erythrocyte membrane stability , hemoglobin function , and glucose sensing and membrane transport in erythrocytes . it is thus of interest to assess if genetic variation segregating at high frequencies in the population , and causing subtler variation in these parameters , might also affect hba1c . furthermore , it is important to establish whether variation due to nonglycemic factors affects the utility of hba1c in clinical practice . hk1 encodes the enzyme hexokinase , which catalyzed the first step in glycolysis and thus represents a likely candidate for the control of glucose metabolism . hk1 is the only isoform that is essential for red blood cell glucose metabolism , and is the predominant form among the four isozymes of the hexokinase family ( hk1 , hk2 , hk3 , and glucokinase ) . in humans , rare nonsynonymous substitutions in the active site of hk1 and intragenic deletions have been shown to cause hk1 enzymatic deficiency associated with autosomal - recessive severe nonspherocytic hemolytic anemia [ 3336 ] . the downeast anemia mice display hk1 deficiency and a similar anemic phenotype .table 1summary of genetic variants robustly associated with hba1c and correlated hematologic and metabolic traits in gwasregionlocussnpspubmedraf ( 95% ci)pcorrelated trait association1q23.1spta1rs2779116208586830.270.02 ( 0.010.03)% increase3 102q31.1g6pc2/abcb11rs1402837190965180.230.02 ( nr)% increase5 10rs560887-fpg ( 18451265)rs560887-fpg ( 19060907)rs552976208586830.640.05 ( 0.040.06)% increase8 10rs563694-fpg ( 18521185)rs560887-homa - b ( 20081858)6p22.2hfers1800562208586830.940.06 ( 0.050.07)% increase3 10rs1408272-mch ( 19862010)rs198846-hgb ( 19820698)7p13gckrs730497190965180.170.03 ( nr)% increase6 10rs4607517-fpg ( 19060907)rs4607517-homa - b ( 20081858)rs1799884208586830.180.04 ( 0.030.05)% increase1 108p11.21ank1rs6474359208586830.970.06 ( 0.040.08)% increase1 10rs4737009208586830.240.03 ( 0.020.04)% increase6 108q24.11slc30a8rs13266634190965180.30.02 ( nr)% decrease5 10rs13266634-t2d ( 17293876 , 17460697 , 17463246 , 17463248 , 17463249 , 19056611 , 19401414 ) , fpg ( 19734900)rs3802177-t2d ( 20581827)rs11558471-fpg ( 20081858)10q22.1hk1rs16926246208586830.90.09 ( 0.080.10)% increase3 10rs7072268190965180.50.05 ( nr)% increase2 1010q25.2tcf7l2rs7903146208494300.720.05 ( 0.020.08)% hba1c decrease1 10rs7901695-t2d ( 17463249)rs4506565-fpg ( 20081858)rs7903146-t2d ( 17293876 , 17460697 , 17463246 , 17463248,17668382 , 18372903 , 19056611 , 19401414 , 19734900 , 20581827)rs12243326-ogtt ( 20081857)11q14.3mtnr1brs1387153208586830.280.03 ( 0.020.04)% increase4 10rs1387153-t2d ( 20581827)rs1387153-fpg ( 19060909)rs2166706-fpg ( 19651812)rs10830963-homa - b ( 20081858 ) , -fpg ( 19060907)13q34atp11a / tubgcp3rs7998202208586830.140.03 ( 0.020.04)% increase5 1017q25.3fn3krs1046896208586830.310.04 ( 0.030.05)% increase2 1022q12.3tmprss6rs855791208586830.420.03 ( 0.020.04)% increase3 10rs855791-hgb ( 19820698)rs2413450-mch ( 19862010)note that this table includes only results from gwas studies , and intentionally omits candidate - snp and candidate - gene studiesnot reportedrsid , associated trait , pubmed i d ; values are given for associations from genome - wide scansfpg fasting plasma glucose ; gwas genome - wide association studies ; hba1c hemoglobin a1c ; hgb hemoglobin ; homa homeostatic model assessment ; mch mean corpuscular hemoglobin ; ogtt oral glucose tolerance test ; raf risk allele frequency ; snp single nucleotide polymorphism ; t2d type 2 diabetes summary of genetic variants robustly associated with hba1c and correlated hematologic and metabolic traits in gwas note that this table includes only results from gwas studies , and intentionally omits candidate - snp and candidate - gene studies rsid , associated trait , pubmed i d ; values are given for associations from genome - wide scans fpg fasting plasma glucose ; gwas genome - wide association studies ; hba1c hemoglobin a1c ; hgb hemoglobin ; homa homeostatic model assessment ; mch mean corpuscular hemoglobin ; ogtt oral glucose tolerance test ; raf risk allele frequency ; snp single nucleotide polymorphism ; t2d type 2 diabetes these observations led bonnefond et al . [ 37 ] to postulate that hk1 genetic variation may indirectly alter hba1c measurements by generating a proanemic state , and independently of ambient blood glucose levels . surprisingly , the most associated snp at this locus showed no association with any other markers of glucose control , whereas it was significantly associated with hemoglobin levels , hematocrit , and anemia . of the 10 loci associated with hba1c at the genome - wide level , three had significant evidence for association of the hba1c - raising allele with fasting glucose and -cell function ( gck , mtnr1b , and g6pc2 ) in this and previous studies , and gck also with 2-hour glucose [ 36 , 24 , 25 , 27 , 38 , 39 ] . at hfe the a allele at rs1800562 ( cys262tyr ) , which is responsible for hereditary hemochromatosis ( mim 235200 ) , was associated with lower levels of hba1c , rather than the higher levels one would predict from epidemiologic observations of the increased hfe mutation prevalence in patients with type 2 diabetes [ 43 , 44 ] . we postulated that functional variants at hk1 may affect a potential dissociation between ambient plasma glucose and intracellular cytoplasmic glucose , and that the hemoglobin - lowering variant may affect the overall percent of hba1c through an increased glucose / hemoglobin molar ratio , which in turn could increase the rate of hemoglobin that is glycated at a given glucose level . these patterns were confirmed in analyses conditioned on fasting glucose or hematologic traits , which provided statistical support for an effect on hba1c via regulation of systemic glucose concentrations for gck , g6pc2 , and mtnr1b , and via hematologic parameters for hfe , tmprss6 , and hk1 . the observation that genetic associations underlying hba1c levels at some loci are independent from fasting glucose reflects earlier observations in a classical discordant monozygotic ( mz ) twin design by snieder et al . such analysis revealed a significant correlation in hba1c levels in 45 mz twins discordant for the disease ( r = 0.52 , p < 0.001 ; as opposed to r = 0.68 , p < 0.001 in 33 mz twins concordant for diabetes ) , suggesting that a substantial proportion of heritability was due to diabetes - independent familial effects . although robust estimates of the proportion of phenotypic variance attributable to diabetes - independent genetic effects are still lacking , these results showed for the first time that familial factors might explain variation in hba1c levels that is not dependent on glycemic control . recently , [ 48 ] carried out a genome - wide analysis on repeated measures of hba1c from the dcct with the aim to identify genetic loci underlying glycemic control in individuals with type 1 diabetes . the study sample consisted of two sets of individuals sampled from the conventional ( n = 667 ) and intensive ( n = 637 ) treatment groups of the dcct . genome - wide snps were tested for association with mean hba1c , stratified by intervention arm as well as in the combined cohort . for replication , associations were assessed through a series of ( non - independent ) analyses testing associations with quarterly hba1c using repeated measures , with mean daily glucose and baseline c - peptide ( to confirm a glycemic mechanism ) , with hba1c repeated measures in the combined cohort ( adjusting for treatment group and testing for interaction with treatment ) , and with glycemic complications including coronary calcium , neuropathy , hypoglycemia , and time to renal or retinal complications ( in each arm separately ) . these included the gokind study , a case control collection of patients with type 1 diabetes with and without diabetic nephropathy , and healthy subjects from the magic meta - analysis described earlier . these analyses revealed a locus ( rs1358030 near sorcs1 ) where association reached the widely accepted threshold for genome - wide significance of p < 5 10 in the conventional treatment group , which was also associated with mean glucose and showed suggestive evidence for replication in the intensive treatment group and in the control group in gokind . it remains to be established if these variable replication outcomes stem from limited study power , or reflect the variable effect of the environment most notably insulin treatment in the two study arms of the study . the authors typed 1486 subjects with type 2 diabetes from a norwegian population - based cohort ( hunt2 ) , and tested their effects on hba1c and non - fasting glucose levels individually and in a combined genetic score model . recently , an international expert committee has proposed a revision of the diagnostic criteria for diabetes , recommending that hba1c may be a better means of diagnosing diabetes than measures of glucose ( fasting and/or postchallenge ) and that it be adopted as a diagnostic criterion for diabetes . the recommendation is based on the association of microvascular complications with hba1c being at least as strong as those with fasting or postchallenge glucose , that hba1c is subject to less day - to - day variability than fasting or postchallenge glucose , and that it can be measured at any time of the day without preparations such as fasting or a glucose challenge . it is likely that practical , medical , methodologic , and financial factors will prevent implementation of the recommendation in the majority of clinical settings . , inherited hemoglobinopathies ) that may influence the utility of hba1c in diabetes diagnosis [ 31 , 51 , 53 , 54 ] , we and others showed that genetically determined physiologic variation in the general population can also play a role , affecting hba1c levels through subtler but more widespread alterations of iron levels and/or hemoglobin concentration . this estimate represents an upper boundary for the effect of these common variants , as most people ( the majority in the center of the distribution ) are expected to have a smaller individual genotype effect size . furthermore , variants at loci controlling iron metabolism associated with hba1c levels are known to vary across ethnic groups . as rates of admixture with europeans vary among african americans , the percentage of european genetic ancestry for each individual , estimated from ancestry - informative markers , was compared with hba1c levels categorized using american diabetes association diagnostic cut points ( < 5.7 , 5.76.4 , and 6.5% ) . this analysis showed that hba1c levels were positively correlated to the fraction of the genome that was of european origin ( p < 0.001 ) , although this correlation accounted for a minimal fraction ( < 1% ) of the overall variability . these initiatives will be important not only to better understand genetic and biologic determinants of hba1c variation in the general population , but also to inform recent initiatives to focus diabetes diagnosis and care more centrally on hba1c . second , it will be important to extend the study of genetic influences to hba1c in prediabetic and diabetic populations , although the confounding effects of treatment might obscure any role of these polymorphisms in the diabetic population . finally , additional genetic associations may be revealed from studies of low - to - intermediate frequency variants through imputation from the 1000 genomes project , direct association using whole - genome sequencing data , and in - depth replication and locus fine - mapping through custom arrays . these hypothesis - generating genetic efforts will pave the way for further studies of the role of the new loci in hemoglobin glycation , glucose metabolism , and diabetes .

zoonotic tick - borne diseases in europe have become increasingly prominent since the emergence of lyme borreliosis ( lb ) in the early 1980s , and the incidence of this disease and that of tick - borne encephalitis ( tbe ) have risen dramatically over the last two decades . both diseases are transmitted by hard ticks of the ixodes ricinus species complex ( i. ricinus and i. persulcatus ) and since these ticks spend most of their time in the environment , climate change is likely to affect their distribution and abundance and , therefore , the incidence of disease . i. ricinus and i. persulcatus are particularly sensitive to environmental conditions since in their prolonged nonparasitic phases they require a microclimatic relative humidity of at least 80% to avoid fatal desiccation . they are , therefore , restricted to areas of moderate - to - high rainfall where there is a good cover of vegetation , so that the soil surface remains humid through the driest times of the year . the fourth assessment report of the intergovernmental panel on climate change reported that in northern temperate europe temperature increases of 1.52.5c may occur over the next few decades as a result of global warming . such climate change may extend or curtail host - seeking tick activity periods , potentially increasing or decreasing tick abundance and distribution , and effects on tick development rates can change seasonal activity patterns by altering the proportion of the tick population that are exposed to regulatory mechanisms such as diapause . in areas where lowered summer precipitation coincides with raised summer temperatures , the survival , activity , and distribution of i. ricinus and i. persulcatus are likely to be reduced because of their vulnerability to desiccation . these tick species acquire their hosts by ambushing them from the vegetation and a significant number of large animals , such as deer , must be present in the habitat in order to feed the adult females and thus maintain the tick populations . the more catholic host preferences of the immature tick stages ( larvae and nymphs ) , which parasitize reptiles , small and medium - sized mammals and birds , in addition to large mammals , contribute significantly to the circulation of diverse pathogens between the tick and host populations . climate change may , therefore , exert a major influence on both tick abundance and disease prevalence by affecting faunal diversity . other important european tick species such as rhipicephalus sanguineus and dermacentor reticulatus can be affected by climate change through similar mechanisms , but anthropogenic factors also have profound effects on disease incidence , and separating these from the influence of climate change represents a major challenge . in this review attention is mainly focused on evidence for the effects of climate change on the distribution and abundance of european ticks . the potential impacts of climate - change effects on the incidences of the diseases they transmit are discussed . the climate is regarded as the principal restricting factor at the northern limit of i. ricinus distribution . although i. ricinus is surprisingly cold - hardy and when winter - acclimatized can survive 24-hour exposure to temperatures ranging from 14.4c to 18.9c , the detrimental effects of cold are accumulative and exposure for 30 days to only 10c has been shown to be lethal for a high proportion of unfed nymphs and diapausing engorged larvae and nymphs . molting i. ricinus ticks are even more vulnerable so that if summer temperatures are not high enough to complete development before the onset of winter ( little or no development takes place between 7 and 10c [ 7 , 8 ] ) , they are unlikely to survive even moderate frosts . degree - day models have been developed which proved useful in elucidating the i. ricinus life cycle [ 9 , 10 ] , but more research on the winter biology of this species would help further understand its northern distribution limit . in sweden , the northern distribution limit of i. ricinus , together with that of several other animal and plant species , has shifted northwards since the climate started to noticeably change in the late 1980s . the geographical distribution range of i. ricinus used to be located below 61n , but ticks are now established along the whole baltic sea coastline ( up to 66n ) and along the river valleys and the larger lakes in the northern regions . this shift in latitude distribution has been shown to be related to changes over several seasons in the number of degree - days with temperatures vital for tick survival , activity and development . at the highest latitudes fewer days with cold winter temperatures ( well below 12c for longer periods ) had the clearest impact for new tick establishment ( figure 1 ) . in central sweden ( 59n to 61n ) in areas with medium - high densities of ticks , increases in tick abundance were correlated to a combination of mild winters ( fewer days with temperatures below 7c ) and extended spring and autumn seasons ( more days with minimum temperatures not lower than 5 to 8c ) . in south and central sweden , the current climate only allows a tick activity season of 68 months compared to as much as 11 months in some parts of the british isles , and further changes in seasonal climate in sweden are likely to continue to have a major impact on the prevalence of ticks . the combination of climate variables affecting ticks has also been shown to be significantly correlated with increases in the incidence of tbe in stockholm county ( 59.2n ) during the period 19601998 . the risks of lb are likely to increase as well because borrelia burgdorferi sensu lato ( s.l . ) spirochetes have been found in i. ricinus throughout its range in sweden . a comparable situation has been reported in changes to the altitude distribution of i. ricinus in the mountainous regions of the czech republic . field studies in 1957 and in 1979 - 80 showed that ticks were prevalent up to 700 meters above sea level . ticks were collected from dogs or by drag - sampling in the same locations in 2001 and 2002 and were then found as high as 1100 meters in areas where they had been absent from small mammal samples [ 1517 ] and where it has been shown that they could not complete their life cycle over the period 19571983 . furthermore , the prevalence of ticks carrying the tbe virus or b. burgdorferi s. l. spirochetes also seems to have increased at high altitude in the czech republic [ 16 , 19 ] . dermacentor reticulatus is a vector of canine babesiosis , tularemia , q - fever , and at least one zoonotic rickettsiosis and has vector competence for anaplasma marginale . unlike i. ricinus , only the adults quest for hosts on surface vegetation and they feed primarily on deer , frequently bite dogs , but only occasionally humans , whereas the larvae and nymphs parasitize rodents . the life cycle is much shorter than that of i. ricinus , with eggs deposited in the spring and developing to adults within the same year . the geographic range of the species extends from france and southwest england in the west to central asia in the east . in western and central europe its northern limit is northern germany , northern poland , and lithuania , and its southern limit is the mediterranean shore ( restricted to humid mountainous areas ) , whereas it has a more northern distribution in the east ( st . petersburg ) . within this area , its distribution is highly focal , and within germany in 1976 , it was only reported from four sites out of more than 3000 . in two recent studies , however , data were collected showing that this tick species has since colonized many more sites in germany . the first study ( 2003 ) consisted of the screening of 365 dogs from 171 sites . almost 10% of the ticks from 41 dogs were d. reticulatus and the infested dogs came from 26 sites , all previously unknown for the tick . seven of the sites were subsequently confirmed by flagging . in the second study ( 2004 ) , 721 deer were shot at 201 sites from 161 districts and their heads examined for ticks . a total of 23 ( 3.2% ) deer from 14 sites were infested and only two sites were already known for d. reticulatus . these results strongly suggest that d. reticulatus has expanded its range within the last three decades particularly in the eastern and southwestern parts of germany . further evidence for the changing distribution of d. reticulatus is provided by the occurrence of canine babesiosis in new areas of germany [ 24 , 25 ] , hungary , switzerland , and the netherlands . several factors , perhaps acting synergistically , could be responsible for this recent spread of d. reticulatus , including increased deer abundance and the availability of more fallow land as a result of eu agricultural policies . however , there are good reasons for implicating a warming climate as being at least partly involved . habitats with adult d. reticulatus are all characterized by more or less intense solar radiation and it is likely that the temperature sum ( cumulative day - degrees above the developmental zero within one vegetation period ) at the soil surface is a limiting factor for oviposition and embryonic development . the fact that d. reticulatus occurs further north in eastern europe supports this view , since the continental climate there is characterized by higher summer temperatures . whereas adults are cold - hardy and can survive continental winters , eggs and larvae that do not complete development would not have survived the cold season of a few decades ago in central europe . further research to clarify the situation could focus on suitable habitats in the north of germany . h. marginatum is well known as a vector of the dangerous viral zoonosis crimean - congo hemorrhagic fever ( cchf ) . its possible northern spread and establishment of permanent populations is thus of much significance , especially since immature stages are frequently found on migratory birds flying to temperate europe . the life cycle of this tick is faster in southern parts of its distribution range ( northern africa ) with larvae active as early as february , but clearly slower in northern , colder regions , with immatures active as late as june . analysis of the recorded distribution of the tick show that , according to climate requirements , there are two clear clusters of populations . one cluster extends from the northern geographical limit of the species in the balkans ( approx . the second one is restricted to africa north of the sahara and western parts of spain . analysis of the climate niche of the first cluster clearly points to a temperature - related limiting factor for these northern populations . temperatures between september and december are critical for the establishment of permanent populations . cumulative temperatures between september and december have an average of 800c in places where the tick has permanent populations , and below 400c in sites not colonized by h. marginatum . this finding seems to be related to the factors that affect molting of immature stages and are not connected to the extremely cold winter temperatures that prevent overwintering adults surviving into the next year , as suggested by hoogstraal et al . . if temperatures are high enough to allow molting before the cold winters , unfed adults can survive the next active season . field observations ( z. vatansever , personal comment ) recorded the feeding of nymphs in late summer in turkey , with the resulting unfed flat adults commonly overwintering in the first few centimeters below the soil surface . regulatory variables for these northern populations appear to act on thermodependent phases of the tick life cycle . on the other hand , climate niche analysis of the southern cluster of tick communities points to a strict dependence on rainfall and potential evaporation , but this may not be relevant if specimens from the southern range can adapt to the colder conditions of the northern cluster . although migratory birds are carriers of immature hyalomma ticks and could potentially introduce them into currently hyalomma - free areas in the spring , their climate requirements and current climate data do not suggest that they can become established . mid - march and early april are the main periods of mass arrival of birds in spain on their way to northern europe . data obtained from the climate research unit ( uk ) show that average temperature in that period is 16 - 17c in morocco and mauritania , 911c in southern spain , and 5 - 6c in southern germany . according to data on molting of engorged nymphs under laboratory conditions , about 300c cumulative degrees above the developmental zero ( 1416c ) are necessary to complete the molt . nymphs that engorge at the time of migratory bird arrival in early spring would need much longer to molt in southern germany , with a consequent increase in mortality , than in north - western africa , where only a few weeks are required . in the current climatic conditions , it is highly improbable that engorged nymphs can survive in sufficient numbers to be founders of new permanent populations in europe . immature h. marginatum are found on local ( nonmigratory ) birds in central spain around late may and early june , which is too late for northern african and southern european populations of h. marginatum to mix because of the current climate barriers imposed by their respective climate requirements at the moment of bird migration . if climate change includes the predicted temperature increases , h. marginatum ticks may become established in northern latitudes but it is debatable whether initial introduction will occur as a result of bird migration alone because very small numbers of ticks , all immature stages , would be involved . it is more likely that , as autumn and winter temperatures rise , establishment of h. marginatum will mainly result from the introduction of adult females feeding on wild and domestic ruminants via the middle east and the balkans , where there is much uncontrolled movement of livestock . a special case of distribution and association to environmental variables is that of the brown dog tick , rhipicephalus sanguineus . it has a worldwide distribution mainly because of introduction by dogs , but rarely occurs in temperate and cold regions . however , r. sanguineus is an endophilous tick ( associated with shelters like kennels , private gardens , or cracks in walls of human constructions ) , so may potentially cause temporary infestations in heated accommodation anywhere in the world . within its normal range , r. sanguineus can reach huge populations under adequate environmental conditions and continued presence of a blood source . currently , the brown dog tick is extremely common around the mediterranean region . in the coldest places of this region , the tick may undergo a winter dormancy within the cracks of the walls , while in localities with warmer winters continuing activity may take place . only sporadic cases of infestation by r. sanguineus have been described in central and northern europe . studies on infestations in some mediterranean cities showed that permanent populations of the tick are absent in apartments where dogs are present , even without any kind of ixodicide treatment . however , the tick is present and may occur in large numbers in small private gardens and kennels of houses ( or even within houses ) in the outskirts . hourly climate data recorded by probes installed indoor and outdoor in these different types of construction showed that adequate humidity is a critical factor for successful establishment of indoor populations . in central europe , there are no humidity restrictions for the development of the tick in the private gardens or kennels and spring and summer temperatures are the only limitation . recent studies on climate features have shown that particular events , such as the heat wave in europe in 2003 , can result in temporary conditions adequate for the development and molting of immature stages . it is clear that despite the endophilous nature of this tick species , climatic conditions in the outer environment are critical for its long - term establishment in an area . september could result in the establishment of permanent populations of the tick in regions of northern temperate europe where it is currently absent . it is common knowledge that the seasonal activity of i. ricinus nymphs and adults extends from march to october in most parts of central europe , whereas the larval stage begins questing only in may , at least in berlin forests ( kahl and dautel , unpublished ) . in contrast to parts of the british isles , any tick activity from mid - november to mid - february is unusual in that region . the strong influence that temperature can exert on tick activity patterns , even in the cold season , was demonstrated in eastern germany in the extraordinarily mild winter of 2006/7 . the continuously warm - to - mild autumn in 2006 ( 1st september to 30th november ) , which was 3.4c warmer than the long - term mean for 196190 , was followed by a winter ( 1st december to 28th february ) 4.6c milder than the long - term mean ( http://www.dwd.de , data from potsdam ) with only two days with a maximum temperature < 0c . on prepared field plots in a berlin forest , questing adult i. ricinus ( figure 2 ) were found on every observation date throughout the winter ( early november to early march ) and questing nymphal ticks were absent on only two out of 19 occasions . moreover , dautel and colleagues collected 88 nymphs and seven adult i. ricinus in two man - hours by flagging 1000 m in a nearby forest in mid - january 2007 . at the same locality on another occasion in mid - february , nymphal and adult questing tick abundance was still higher ( temperature maximum on both days approximately 7c ) . this is a good example of how flexible the seasonal questing activity of this widespread vector tick can be if the temperature conditions change from the local norm . the unusual registration of four cases of human tbe ( a notifiable disease in germany ) in early 2007 ( early january to late february ) shows that there was some tick questing activity in other parts of germany as well during that winter . if winter temperatures generally increase in the future , it can be assumed that seasonal periods with no questing i. ricinus will become shorter in central europe . it is evident that winter activity of vector ticks distinctly increases the risk for forest visitors of an infectious tick bite , especially if they do not expect ticks to be active at that time of the year . it is unclear , however , what the chances are for a winter - active tick to find a host at that time ( because of reduced host abundance in winter though this may also change with the advent of warmer winters ) . it is also unclear how winter activity may affect the remaining seasonal activity pattern ( winter - active ticks spend precious energy ) or whether any changes in the seasonal activity of i. ricinus nymphs and adults are beneficial or detrimental for the perpetuation of tick - borne pathogens . the infection of i. ricinus larvae during feeding is a crucial step in the circulation of many tick - borne pathogens and the effects of higher ambient temperatures on the seasonal activity of larvae and its chances to find a suitable host may be of great significance in determining the prevalence of some tick - borne diseases . in 1976 the early summer weather in county wicklow , ireland , included maximum air temperatures of 2931c recorded on sheep pastures where ixodes ricinus activity was being studied . such monthly maxima in early summer may be the rule rather than the exception in this region in the coming decades . retrospective use was made of 1976 summer temperature and tick data in an examination of the effects of high temperatures on tick development and activity in relation to the predicted global warming . in most parts of its range , i. ricinus shows some degree of bimodal seasonal activity and in 1975 , more ticks were collected in autumn than in spring / summer , which may be attributed to the presence of hosts on these particular sheep pastures in late summer and autumn but not in spring or early summer for several years . however , by 1977 , the pattern of tick activity had changed dramatically and more than 90% nymphal activity occurred from march to june . it was postulated that the elevated early and mid - summer temperatures of 1976 were the primary cause of the change from autumn to spring / summer - dominated nymphal activity . this possibility was investigated by studies on tick development under quasi - natural conditions . the threshold period for deposited engorged larvae to enter a developmental diapause was identified as the first two weeks of august , after which time larvae overwintered in an engorged state and did not reappear as nymphs until the following autumn . the tick abundance data suggested that the 1975 autumn - feeding adults gave rise to larvae that fed predominantly in the prediapause period , so that they had the opportunity to overwinter as unfed nymphs and thus join the spring - active ticks in 1977 . this interpretation was supported by a degree - day development model for i. ricinus originally described by gardiner et al . , that predicted the appearance of larvae from autumn - laid eggs a month earlier when exposed to 1976 temperatures than when exposed to more normal temperatures . the high summer temperatures of 1976 had apparently transferred ticks from an autumn - active cohort to a spring - active one . the process revealed by this study suggests that after hotter summers much of the host - seeking activity of i. ricinus will occur in late autumn , to a lesser extent in the winter months , and with strong activity again in early spring . larval activity is likely to be mainly restricted to mid - summer ( as long as humidity requirements are satisfied ) with the majority of larvae avoiding developmental diapause and becoming active as nymphs in late autumn or early spring of the following year . interestingly , this pattern of activity is very similar to that of the american i. scapularis in new jersey , usa [ 39 , 40 ] , where air temperatures exceed 26c for 5060 days of the year . suitable studies in southern europe have not been undertaken , but a similar phenology to the predicted scenario for i. ricinus in hot summers was described in a recent comprehensive study in central spain where maximum summer air temperatures generally reach 26c . it might be expected that with low precipitation in hot summers , survival and activity of ticks such as i. ricinus , which is very susceptible to desiccation , would be reduced . indeed , it has been reported that in switzerland saturation deficits depressed nymphal and adult i. ricinus activity . however , irish data showed that all active stages of i. ricinus will quest throughout hot dry weather as long as appropriate vegetation cover is present to provide opportunities for rehydration . the same situation appears to obtain for i. scapularis immature stages in the usa . it seems likely that with increased global warming , i. ricinus activity will occur more in the autumn and winter months in many areas and , furthermore , a greater proportion of the tick population may be active at this time than at present , with a consequent temporal change in the risk of tick - borne diseases . climate suitability for a tick population can be defined as the fitness of a set of climatic conditions for the existence of that population in a given region . however , many other factors operating at different levels restrict the effective dispersal and establishment of potential invaders . thus , while the climate in a particular location may be suitable for a given tick species , the potential for dispersal there and the ability to establish a new viable population may be very low . furthermore , microclimatic variables such as soil surface temperature and relative humidity ( which are affected by such things as slope and aspect , snow cover , vegetation , litter layer , humus and underlying soils ) may be crucial in determining the distribution pattern of specific niches for tick survival within an area . most data on climate preferences of ticks have been empirically derived from descriptions of the abiotic components of the environmental niche , as defined by the climate - supported native populations , and based on the assumption that they are homogeneously distributed in the native area . the basic concept underlying species occurrence modeling is the definition of the ecological niche : each species is found within specific ranges for environmental variables that support individual survival and reproduction . we refer here to climate instead of environmental or ecological space because these studies are aimed at understanding the relationship of ticks to climate , and ignore other basic aspects , such as vegetation patterns or host abundance , that are also involved in delineation of the ecological preferences of a tick species or population . species occurrence can be predicted by inclusion of appropriate climate variables in what are commonly referred to as climate suitability models ( csms ) : the relationships are generalized from a sample of correlations of species presence with specific values of environmental variables . while it is well recognized that the climate niche space occupied by a species across its geographic range may vary , this is rarely considered in current modeling approaches despite its obvious importance . when regional climate niche variations occur , a csm derived for a particular area may not apply to other areas , and a model derived from a large area may have comparatively weak local predictive power . a widely held assumption in traditional models of tick distribution is that responses of species to environmental gradients are unimodal and symmetrical . thus , climate suitability is predicted to decline from central ( and ecologically optimal ) areas of a species ' range towards the periphery . in suboptimal conditions , a species may compensate for physiological stress by a shift in niche position . for wide - ranging species , however , these csm are unsuitable if an adequate understanding of the factors operating over the transmission of a disease is necessary . the many variables involved in such processes , like hosts , densities of questing infected ticks , and a perception of the small scale of foci , are only adequately addressed with models designed to describe seasonal dynamics . while some models with biological content have been produced for tick species such as boophilus microplus , amblyomma americanum , and ixodes scapularis , none are currently available for european ticks . such models are a priority to adequately understand the impact of climate change on tick populations , provided that adequate data are used for important components such as host densities and microhabitat suitability . however , despite the absence of such data in climate suitability models , these models have proved useful in the elucidation of tick - borne disease foci for the recent outbreak of cchf in turkey . both regional climatic requirement variations and the existence of demes ( defined as populations of closely related interbreeding organisms of the same species with differing responses to the wide array of climate factors occurring across the geographical range of the species ) have been demonstrated for i. ricinus ( figure 3 ) . the study revealed at least 10 distinct i. ricinus groups with a pattern of distribution closely overlapping with the presence of previously reported phenotypic forms of the species , and is in general agreement with a 16s mitochondrial rdna study of genetic variation in i. ricinus . the importance of these findings is that the climate and vegetation features correlated with the genetic groups in the whole tick metapopulation , with different populations having specific climatic requirements and unique genetic fingerprints . a study on the changes in climate suitability for i. ricinus in the western palearctic has been carried out using deme - derived models based on the different populations recognized in the palaearctic . the distribution records available for the different demes were used to build partial models ( i.e. , applied to regions of the whole distribution range ) from which a complete map for the whole region was produced . the study used a long ( 19001999 ) series of climate data at coarse resolution ( 10 minutes of arc ) to examine the trends in climate and to estimate sustained variations in climate suitability for i. ricinus . while some areas showed a deterministic ( i.e. , continuous ) tendency towards increasing or decreasing suitability for the tick , others showed unambiguous cycles of climate suitability , termed areas of random walk . in these , populations of the tick may undergo periodical variations in their geographical range as a consequence of cyclic changes in climate . this analysis suggests that while climate suitability for i. ricinus did not change in a large area of europe during the 100-year study period , it increased in specific geographically limited locations and decreased in others ( figure 4 ) . these changes are not recent and are associated with yearly and summer rainfall patterns rather than with temperature . the reported increased abundance of i. ricinus in parts of europe ( e.g. , sweden ) coincides geographically with the regions where a recent increase in climate suitability has been detected , within zones having a marked random walk tendency . thus , the observation of higher tick abundance in recent years may not be due to a permanent shift in tick populations , but rather because the long - term climate cycle , which varies on a wide timescale , has been in a phase that is favorable to tick survival . no single variable was consistently associated in the study period with changes in climate suitability across sites where random walk was detected . the absence of a single regulatory variable seems to be connected with the different climate niche experienced by the tick populations in their distribution area . thus , rainfall and temperature have different regulatory abilities according to the portion of the tick 's climate envelope represented in a given area . the mediterranean region is expected to experience profound changes in climate , and furthermore , its close proximity to the african continent makes it a particularly sensitive area to invasion by ticks currently restricted to northern africa . it is , therefore , of interest to explore the predicted impact of climate changes on the suitability for ticks . the ticks involved ( genera boophilus , dermacentor , rhipicephalus , and hyalomma ) find their maximum suitability in mediterranean type vegetation , including areas with cold winters and dry summers , and are prevalent in wide areas of that region , extending well into the middle east . most of them are vectors of pathogens of animals and humans . predicted changes in climate are also expected to have a serious impact on the vegetation structure , driving the area toward a more open , brush - like vegetation , where these ticks find their ecological optima . an estimation of the climate niches for each of these ticks has already been published . global climate models remain relatively coarse in terms of spatial resolution ; this compromised the desired resolution of that analysis . to address this problem , new climate layers were created with monthly increases and decreases in temperature of 1 and 2c , and monthly variations in rainfall of 60 , 80 , 120 , and 140% of actual values . for each combination of temperature and rainfall , the climate suitability for each species in the region was evaluated . the baseline climate suitability was used as a framework to compute the impact of the different changes of climate over the expansion or retraction of the geographical range for each species . the expected impact is displayed in figure 5 . for b. annulatus and h. excavatum , an increase in temperature would lead to an increase in suitability within regions of northern africa and limited parts of southern europe . changes in rainfall are predicted to have little geographic impact on b. annulatus ( which in any case is a one - host - tick that is much less exposed to ambient conditions than three - host - ticks ) , but they show a clear effect on h. excavatum . in the case of d. marginatus , a decrease in temperature results in an increase in the extent of adequate suitability in northern africa , most of coastal southern europe , and wide areas of southern spain , whereas an increase in temperature would result in a northward expansion of the suitable habitat for this species . changes in rainfall result in similar effects to those described for the above - mentioned changes in temperature . large areas of europe would potentially be affected by an increase in the climate suitability for rhipicephalus spp . and h. marginatum after an increase in temperature and decrease in rainfall . decreasing temperatures in europe are predicted to result in habitat loss for b. annulatus , r. bursa , and h. marginatum . again , we must stress that this is an evaluation of the climate suitability for these tick species , since changes in vegetation , host availability , and animal movements were not included in the models . all the tick species considered in the earlier sections are important vectors of disease and an increasing incidence of these diseases is the most significant potential outcome of climate changes that affect ticks , directly or indirectly . as described above , there is little doubt that dermacentor reticulatus is currently extending its range and this is reflected in the occurrence of canine babesiosis ( caused by babesia canis canis ) in new areas ( 24 , 25 , 26 , 27 , 28 ) . hyalomma marginatum is one of the vectors of crimean - congo hemorrhagic fever ( cchf ) , which occurs in parts of africa , asia , the middle east , and south - east europe . the largest epidemic on - record occurred in turkey , which started in 2002 and is still ongoing , and further north , a recent outbreak was reported in the balkans , another known endemic area , but there is no evidence as yet that this disease is spreading further northwards . however , as discussed above , the potential for the introduction and establishment of vector populations in areas of predicted climate suitability is increasing . rhipicephalus sanguineus is the primary vector of mediterranean - spotted fever ( a rickettsial zoonosis caused by rickettsia conorii ) , and also ehrlichia canis and babesia canis vogeli ( causing , resp . , although r. sanguineus can establish in kennels in central and northern european latitudes , thus potentially causing short - lived localized disease outbreaks , sufficient survival in the external environment does not seem to occur for significant disease transmission in northern temperate europe . nevertheless , concerns about its possible introduction and establishment have prompted the authorities in some countries , such as the united kingdom and ireland , to make treatment against ticks a component of the pet - passport scheme . ixodes ricinus is the most abundant and widespread tick in europe and together with the eurasian species , i. persulcatus , transmits lyme borreliosis ( lb ) and tick - borne encephalitis ( tbe ) . lb occurs at a relatively high incidence for a zoonotic disease , ranging from 155 per 100,000 in slovenia to 0.6 per 100,000 in ireland . however , it is difficult to statistically relate lb incidence to climate change because the reliability of incidence data is uncertain due to diagnostic problems and limited or absent reporting in most countries . nevertheless , in some regions , it has proved possible to relate disease incidence to climate , and a positive association of incidence with mild winters and warm , humid summers was reported in southern sweden . the suggested effect of mild winters is possibly due to an extension of the tick activity season , resulting in increased numbers of infected ticks becoming available in the following year . warm humid summers might result in a more efficient transmission by the vector , but there is limited evidence for this and the authors suggest that the observed increased lb incidence may be due to increased human exposure in these conditions . since tbe is notifiable in most countries where it occurs , current incidence data are more reliable than those for lb , even though reporting standards differ between countries and there are increasing differences in tbe vaccination rates between countries . increases in swedish cases since the mid-80s were associated with two consecutive years with milder winters , earlier arrival of spring and prolonged autumn periods with temperatures above 58c . the possibility that this is caused by climate effects on ticks is suggested by the northward extension of i. ricinus distribution [ 11 , 12 ] . similarly , an upward movement of the tbe prevalence altitude ceiling correlating with increasing temperatures has been reported [ 15 , 56 ] , which accords with reports of increasing numbers of active ticks at higher altitudes . detecting climatic effects on the incidence of lb and tbe in areas close to the latitude and altitude distribution limits of the vector is less complex than in other parts of europe where the direct and indirect impacts of climate change on disease incidence are often confounded by other factors . such studies should either be based on reliable long - term historical datasets in an area ( e.g. , the 40 year surveillance program for tbe in stockholm county , sweden ) or based on data from different regions or countries , collected with similar methodologies ( e.g. , vector sampling and analysis ) and subject to similar variations ( e.g. , tbe vaccination rates and reporting criteria ) . there is a need for a pan - european surveillance network , for example based on the abundance of infected vectors , as suggested by who . randolph et al . correlated the occurrence of synchronous activity of i. ricinus larvae and nymphs ( resulting in cofeeding transmission ) with that of tbe cases , and also reported a relationship between these two variables and the rate of decline of autumn temperatures . it , therefore , seems likely that climate change will affect the dynamics of tbe transmission , thus altering the distribution of the disease , but the exact mechanisms of this interaction remain to be elucidated . despite the ready availability of data on tbe incidence , a firm causal relationship with climate change a detailed study of the records of tbe incidence over the last 2 - 3 decades in estonia , latvia , and lithuania showed that although tbe incidence rose dramatically in some areas , there was so much heterogeneity in the data that it was impossible to identify climate change as the main factor driving increased disease incidence . it was concluded that the many socioeconomic changes arising from the end of soviet rule probably acted synergistically with climate factors to increase tbe incidence . transmission of infection occurs when there is an overlap of activities between reservoir , vector , and humans , and differs according to the pathogens and the location . although changes in climate and in the length of the different seasons will directly affect tick survival , activity , and development , there is no good evidence that rising temperatures will result in a greater abundance of ticks by simply increasing rates of development ; rather changes in development rates will make tick cohorts available to different diapause windows ( largely determined by day length ) , thus changing patterns of seasonal activity . indirect effects of climate change will impact the number of infected ticks by affecting vegetation . for example , a warming climate in central europe is likely to result in a decrease of norway spruce ( picea abies ) and the areas involved will probably be colonized by beech ( fagus sylvatica ) , the fallen leaves of which provide a favorable microclimate for survival of the free - living tick stages . additionally , climate change will also have indirect effects on tick - borne pathogen transmission by affecting the survival and abundance of tick maintenance hosts , such as deer , and pathogen - reservoir hosts such as rodents and birds . climate change may also influence disease risk by affecting the long - term use of land ( e.g. , farming , tourism , etc . ) , and weather patterns have an effect by influencing short - term human behavior such as picnics and mushroom picking . climate effects are more easily noticeable close to the geographical distribution limits of both vector and disease . the magnitude of the effects of climate change in an endemic area depends on local conditions and vulnerability , and is determined not only by ecological conditions but may be influenced by socioeconomic factors , human migration and settlement , ecosystems and biodiversity , migrating patterns of birds , land - use and land cover changes , human cultural and behavioral patterns , and immunity in the population . since some of these conditions are in turn influenced by climate change , a complex chain of processes exists that makes the precise factors responsible for changes in disease incidence often difficult to determine . a further difficulty in determining future scenarios is presented by the fact that the predominant tick species in europe , ixodes ricinus is extremely flexible and adaptable and can exhibit rather different seasonal activity even in adjacent parts of its geographical range . much current research effort attempts to match datasets collected for different purposes and in order to reduce confounding variables , it is evident that data from long - term studies on disease incidence , tick biology , tick distribution and tick abundance , host abundance and distribution , and relevant vegetation biology , specifically in relation to climate change , are required . such data will permit the development of models to predict future tick - borne disease scenarios , which take account of dynamic biological processes instead of simply the likelihood of occurrence of climate suitability for particular tick species .

zoonotic tick - borne diseases are an increasing health burden in europe and there is speculation that this is partly due to climate change affecting vector biology and disease transmission . data on the vector tick ixodes ricinus suggest that an extension of its northern and altitude range has been accompanied by an increased prevalence of tick - borne encephalitis . climate change may also be partly responsible for the change in distribution of dermacentor reticulatus . increased winter activity of i. ricinus is probably due to warmer winters and a retrospective study suggests that hotter summers will change the dynamics and pattern of seasonal activity , resulting in the bulk of the tick population becoming active in the latter part of the year . climate suitability models predict that eight important tick species are likely to establish more northern permanent populations in a climate - warming scenario . however , the complex ecology and epidemiology of such tick - borne diseases as lyme borreliosis and tick - borne encephalitis make it difficult to implicate climate change as the main cause of their increasing prevalence . climate change models are required that take account of the dynamic biological processes involved in vector abundance and pathogen transmission in order to predict future tick - borne disease scenarios .

1. Introduction 2. Effects of Climate Change on Tick Distribution and Abundance 3. Effects of Climate Change on the Seasonal Aactivity of Ticks 4. The Role of Modeling in Analysis of Climate Change Effects on Ticks 5. Effects on Tick-Borne Diseases in Europe 6. Conclusions

zoonotic tick - borne diseases in europe have become increasingly prominent since the emergence of lyme borreliosis ( lb ) in the early 1980s , and the incidence of this disease and that of tick - borne encephalitis ( tbe ) have risen dramatically over the last two decades . both diseases are transmitted by hard ticks of the ixodes ricinus species complex ( i. ricinus and i. persulcatus ) and since these ticks spend most of their time in the environment , climate change is likely to affect their distribution and abundance and , therefore , the incidence of disease . such climate change may extend or curtail host - seeking tick activity periods , potentially increasing or decreasing tick abundance and distribution , and effects on tick development rates can change seasonal activity patterns by altering the proportion of the tick population that are exposed to regulatory mechanisms such as diapause . in areas where lowered summer precipitation coincides with raised summer temperatures , the survival , activity , and distribution of i. ricinus and i. persulcatus are likely to be reduced because of their vulnerability to desiccation . these tick species acquire their hosts by ambushing them from the vegetation and a significant number of large animals , such as deer , must be present in the habitat in order to feed the adult females and thus maintain the tick populations . climate change may , therefore , exert a major influence on both tick abundance and disease prevalence by affecting faunal diversity . other important european tick species such as rhipicephalus sanguineus and dermacentor reticulatus can be affected by climate change through similar mechanisms , but anthropogenic factors also have profound effects on disease incidence , and separating these from the influence of climate change represents a major challenge . in this review attention is mainly focused on evidence for the effects of climate change on the distribution and abundance of european ticks . the potential impacts of climate - change effects on the incidences of the diseases they transmit are discussed . the climate is regarded as the principal restricting factor at the northern limit of i. ricinus distribution . although i. ricinus is surprisingly cold - hardy and when winter - acclimatized can survive 24-hour exposure to temperatures ranging from 14.4c to 18.9c , the detrimental effects of cold are accumulative and exposure for 30 days to only 10c has been shown to be lethal for a high proportion of unfed nymphs and diapausing engorged larvae and nymphs . degree - day models have been developed which proved useful in elucidating the i. ricinus life cycle [ 9 , 10 ] , but more research on the winter biology of this species would help further understand its northern distribution limit . in sweden , the northern distribution limit of i. ricinus , together with that of several other animal and plant species , has shifted northwards since the climate started to noticeably change in the late 1980s . the geographical distribution range of i. ricinus used to be located below 61n , but ticks are now established along the whole baltic sea coastline ( up to 66n ) and along the river valleys and the larger lakes in the northern regions . in south and central sweden , the current climate only allows a tick activity season of 68 months compared to as much as 11 months in some parts of the british isles , and further changes in seasonal climate in sweden are likely to continue to have a major impact on the prevalence of ticks . a comparable situation has been reported in changes to the altitude distribution of i. ricinus in the mountainous regions of the czech republic . furthermore , the prevalence of ticks carrying the tbe virus or b. burgdorferi s. l. spirochetes also seems to have increased at high altitude in the czech republic [ 16 , 19 ] . the life cycle is much shorter than that of i. ricinus , with eggs deposited in the spring and developing to adults within the same year . in western and central europe its northern limit is northern germany , northern poland , and lithuania , and its southern limit is the mediterranean shore ( restricted to humid mountainous areas ) , whereas it has a more northern distribution in the east ( st . almost 10% of the ticks from 41 dogs were d. reticulatus and the infested dogs came from 26 sites , all previously unknown for the tick . analysis of the recorded distribution of the tick show that , according to climate requirements , there are two clear clusters of populations . on the other hand , climate niche analysis of the southern cluster of tick communities points to a strict dependence on rainfall and potential evaporation , but this may not be relevant if specimens from the southern range can adapt to the colder conditions of the northern cluster . in the current climatic conditions , it is highly improbable that engorged nymphs can survive in sufficient numbers to be founders of new permanent populations in europe . in the coldest places of this region , the tick may undergo a winter dormancy within the cracks of the walls , while in localities with warmer winters continuing activity may take place . studies on infestations in some mediterranean cities showed that permanent populations of the tick are absent in apartments where dogs are present , even without any kind of ixodicide treatment . however , the tick is present and may occur in large numbers in small private gardens and kennels of houses ( or even within houses ) in the outskirts . in central europe , there are no humidity restrictions for the development of the tick in the private gardens or kennels and spring and summer temperatures are the only limitation . recent studies on climate features have shown that particular events , such as the heat wave in europe in 2003 , can result in temporary conditions adequate for the development and molting of immature stages . september could result in the establishment of permanent populations of the tick in regions of northern temperate europe where it is currently absent . it is common knowledge that the seasonal activity of i. ricinus nymphs and adults extends from march to october in most parts of central europe , whereas the larval stage begins questing only in may , at least in berlin forests ( kahl and dautel , unpublished ) . this is a good example of how flexible the seasonal questing activity of this widespread vector tick can be if the temperature conditions change from the local norm . it is evident that winter activity of vector ticks distinctly increases the risk for forest visitors of an infectious tick bite , especially if they do not expect ticks to be active at that time of the year . it is unclear , however , what the chances are for a winter - active tick to find a host at that time ( because of reduced host abundance in winter though this may also change with the advent of warmer winters ) . it is also unclear how winter activity may affect the remaining seasonal activity pattern ( winter - active ticks spend precious energy ) or whether any changes in the seasonal activity of i. ricinus nymphs and adults are beneficial or detrimental for the perpetuation of tick - borne pathogens . the infection of i. ricinus larvae during feeding is a crucial step in the circulation of many tick - borne pathogens and the effects of higher ambient temperatures on the seasonal activity of larvae and its chances to find a suitable host may be of great significance in determining the prevalence of some tick - borne diseases . in most parts of its range , i. ricinus shows some degree of bimodal seasonal activity and in 1975 , more ticks were collected in autumn than in spring / summer , which may be attributed to the presence of hosts on these particular sheep pastures in late summer and autumn but not in spring or early summer for several years . however , by 1977 , the pattern of tick activity had changed dramatically and more than 90% nymphal activity occurred from march to june . it was postulated that the elevated early and mid - summer temperatures of 1976 were the primary cause of the change from autumn to spring / summer - dominated nymphal activity . the process revealed by this study suggests that after hotter summers much of the host - seeking activity of i. ricinus will occur in late autumn , to a lesser extent in the winter months , and with strong activity again in early spring . larval activity is likely to be mainly restricted to mid - summer ( as long as humidity requirements are satisfied ) with the majority of larvae avoiding developmental diapause and becoming active as nymphs in late autumn or early spring of the following year . interestingly , this pattern of activity is very similar to that of the american i. scapularis in new jersey , usa [ 39 , 40 ] , where air temperatures exceed 26c for 5060 days of the year . however , irish data showed that all active stages of i. ricinus will quest throughout hot dry weather as long as appropriate vegetation cover is present to provide opportunities for rehydration . it seems likely that with increased global warming , i. ricinus activity will occur more in the autumn and winter months in many areas and , furthermore , a greater proportion of the tick population may be active at this time than at present , with a consequent temporal change in the risk of tick - borne diseases . climate suitability for a tick population can be defined as the fitness of a set of climatic conditions for the existence of that population in a given region . thus , while the climate in a particular location may be suitable for a given tick species , the potential for dispersal there and the ability to establish a new viable population may be very low . most data on climate preferences of ticks have been empirically derived from descriptions of the abiotic components of the environmental niche , as defined by the climate - supported native populations , and based on the assumption that they are homogeneously distributed in the native area . we refer here to climate instead of environmental or ecological space because these studies are aimed at understanding the relationship of ticks to climate , and ignore other basic aspects , such as vegetation patterns or host abundance , that are also involved in delineation of the ecological preferences of a tick species or population . the many variables involved in such processes , like hosts , densities of questing infected ticks , and a perception of the small scale of foci , are only adequately addressed with models designed to describe seasonal dynamics . however , despite the absence of such data in climate suitability models , these models have proved useful in the elucidation of tick - borne disease foci for the recent outbreak of cchf in turkey . the study revealed at least 10 distinct i. ricinus groups with a pattern of distribution closely overlapping with the presence of previously reported phenotypic forms of the species , and is in general agreement with a 16s mitochondrial rdna study of genetic variation in i. ricinus . a study on the changes in climate suitability for i. ricinus in the western palearctic has been carried out using deme - derived models based on the different populations recognized in the palaearctic . , continuous ) tendency towards increasing or decreasing suitability for the tick , others showed unambiguous cycles of climate suitability , termed areas of random walk . this analysis suggests that while climate suitability for i. ricinus did not change in a large area of europe during the 100-year study period , it increased in specific geographically limited locations and decreased in others ( figure 4 ) . thus , the observation of higher tick abundance in recent years may not be due to a permanent shift in tick populations , but rather because the long - term climate cycle , which varies on a wide timescale , has been in a phase that is favorable to tick survival . thus , rainfall and temperature have different regulatory abilities according to the portion of the tick 's climate envelope represented in a given area . for each combination of temperature and rainfall , the climate suitability for each species in the region was evaluated . large areas of europe would potentially be affected by an increase in the climate suitability for rhipicephalus spp . again , we must stress that this is an evaluation of the climate suitability for these tick species , since changes in vegetation , host availability , and animal movements were not included in the models . all the tick species considered in the earlier sections are important vectors of disease and an increasing incidence of these diseases is the most significant potential outcome of climate changes that affect ticks , directly or indirectly . as described above , there is little doubt that dermacentor reticulatus is currently extending its range and this is reflected in the occurrence of canine babesiosis ( caused by babesia canis canis ) in new areas ( 24 , 25 , 26 , 27 , 28 ) . the largest epidemic on - record occurred in turkey , which started in 2002 and is still ongoing , and further north , a recent outbreak was reported in the balkans , another known endemic area , but there is no evidence as yet that this disease is spreading further northwards . however , as discussed above , the potential for the introduction and establishment of vector populations in areas of predicted climate suitability is increasing . , although r. sanguineus can establish in kennels in central and northern european latitudes , thus potentially causing short - lived localized disease outbreaks , sufficient survival in the external environment does not seem to occur for significant disease transmission in northern temperate europe . ixodes ricinus is the most abundant and widespread tick in europe and together with the eurasian species , i. persulcatus , transmits lyme borreliosis ( lb ) and tick - borne encephalitis ( tbe ) . however , it is difficult to statistically relate lb incidence to climate change because the reliability of incidence data is uncertain due to diagnostic problems and limited or absent reporting in most countries . nevertheless , in some regions , it has proved possible to relate disease incidence to climate , and a positive association of incidence with mild winters and warm , humid summers was reported in southern sweden . the suggested effect of mild winters is possibly due to an extension of the tick activity season , resulting in increased numbers of infected ticks becoming available in the following year . warm humid summers might result in a more efficient transmission by the vector , but there is limited evidence for this and the authors suggest that the observed increased lb incidence may be due to increased human exposure in these conditions . the possibility that this is caused by climate effects on ticks is suggested by the northward extension of i. ricinus distribution [ 11 , 12 ] . detecting climatic effects on the incidence of lb and tbe in areas close to the latitude and altitude distribution limits of the vector is less complex than in other parts of europe where the direct and indirect impacts of climate change on disease incidence are often confounded by other factors . correlated the occurrence of synchronous activity of i. ricinus larvae and nymphs ( resulting in cofeeding transmission ) with that of tbe cases , and also reported a relationship between these two variables and the rate of decline of autumn temperatures . it , therefore , seems likely that climate change will affect the dynamics of tbe transmission , thus altering the distribution of the disease , but the exact mechanisms of this interaction remain to be elucidated . despite the ready availability of data on tbe incidence , a firm causal relationship with climate change a detailed study of the records of tbe incidence over the last 2 - 3 decades in estonia , latvia , and lithuania showed that although tbe incidence rose dramatically in some areas , there was so much heterogeneity in the data that it was impossible to identify climate change as the main factor driving increased disease incidence . although changes in climate and in the length of the different seasons will directly affect tick survival , activity , and development , there is no good evidence that rising temperatures will result in a greater abundance of ticks by simply increasing rates of development ; rather changes in development rates will make tick cohorts available to different diapause windows ( largely determined by day length ) , thus changing patterns of seasonal activity . for example , a warming climate in central europe is likely to result in a decrease of norway spruce ( picea abies ) and the areas involved will probably be colonized by beech ( fagus sylvatica ) , the fallen leaves of which provide a favorable microclimate for survival of the free - living tick stages . additionally , climate change will also have indirect effects on tick - borne pathogen transmission by affecting the survival and abundance of tick maintenance hosts , such as deer , and pathogen - reservoir hosts such as rodents and birds . climate change may also influence disease risk by affecting the long - term use of land ( e.g. the magnitude of the effects of climate change in an endemic area depends on local conditions and vulnerability , and is determined not only by ecological conditions but may be influenced by socioeconomic factors , human migration and settlement , ecosystems and biodiversity , migrating patterns of birds , land - use and land cover changes , human cultural and behavioral patterns , and immunity in the population . a further difficulty in determining future scenarios is presented by the fact that the predominant tick species in europe , ixodes ricinus is extremely flexible and adaptable and can exhibit rather different seasonal activity even in adjacent parts of its geographical range . much current research effort attempts to match datasets collected for different purposes and in order to reduce confounding variables , it is evident that data from long - term studies on disease incidence , tick biology , tick distribution and tick abundance , host abundance and distribution , and relevant vegetation biology , specifically in relation to climate change , are required . such data will permit the development of models to predict future tick - borne disease scenarios , which take account of dynamic biological processes instead of simply the likelihood of occurrence of climate suitability for particular tick species .

cirrhosis of the liver the only cure for which is liver transplant is associated with several serious complications , including ascites , spontaneous bacterial peritonitis , variceal bleeding , and hepatic encephalopathy ( he ) . guidelines established by the american association for the study of liver diseases currently recommend referring patients with cirrhosis for liver transplant when their model for end - stage liver disease ( meld ) score is 10 and their child - turcotte - pugh ( ctp ) score is 7 or when they experience their first major complication ( e.g. , he , ascites , or variceal bleeding ) . however , the current united network for organ sharing allocation system only uses the meld score for prioritizing adults for liver transplant . the meld scoring system evaluates a patient 's short - term prognosis based on 3 common laboratory test results : serum bilirubin , international normalized ratio , and serum creatinine levels . however , this scoring system does not take into account several serious complications of cirrhosis , such as he , when prioritizing patients for liver transplant . this may have negative ramifications for patient care , as the development of he may be associated with substantial morbidity , mortality , and cost . he imposes a significant burden on patients , their families , and health care resources [ 5 , 6 ] . he is characterized by alterations in behavior , cognitive abilities , consciousness , and neuromuscular function . it negatively affects patient quality of life ( qol ) , and patients may be unable to drive , work , or adequately care for themselves because of its effects [ 811 ] . patients may be less compliant with all prescribed medications , and hospitalizations related to he may increase patient exposure to opportunistic infections and be associated with substantial costs . furthermore , he may be an independent predictor of mortality in patients with chronic liver disease . he occurring before liver transplant can also have a substantial negative impact on posttransplant outcomes [ 1322 ] . this paper will review the potential consequences of pretransplant he on posttransplant outcomes and therapeutic strategies for the pretransplant management of he . he may occur in patients with acute liver failure ( type a he ) , in patients with portosystemic shunting but no intrinsic hepatocellular disease ( type b he ) , or , as in the majority of he cases , in patients with cirrhosis and cirrhosis - related portosystemic shunting ( type c he ) . because he is a progressive neuropsychiatric condition , he may be graded or scored based on the severity of the clinical manifestations , which may range from subtle neurologic abnormalities in mild cases to coma in severe cases . minimal he ( sometimes referred to as covert he ) , which may occur in nearly 70% of patients with cirrhosis , is not associated with any clinical signs of brain dysfunction , but patients experience cognitive abnormalities that can lead to qol impairment [ 8 , 24 , 25 ] . unlike minimal he , overt he can manifest as a wide spectrum of symptoms that can be observed clinically , including those related to motor and neuropsychologic functions . overt he has been shown to occur in nearly half of patients with cirrhosis and , as with minimal he , also has a substantial negative impact on qol [ 710 ] . although research is ongoing , the pathogenesis of he is believed to primarily involve the exposure of the brain to elevated neurotoxin levels , particularly ammonia and other gut - derived toxins , leading to cellular morphologic changes ( e.g. , astrocyte swelling ) and the development of a variety of neurochemical , neurotransmitter , and neuroinflammatory changes [ 27 , 28 ] . many factors that can precipitate he ( e.g. , hypokalemia , infection , and gastrointestinal bleeding ) serve to increase the production of ammonia or other gut - derived toxins or to reduce toxin metabolism by the liver ( e.g. , dehydration , anemia ) . although the exact pathophysiology of he is unclear , data are accumulating to suggest that the cascade of neuropsychiatric and neuromuscular sequelae of he may have a longer term or more permanent negative impact on patients with chronic liver disease than originally suspected . neurologic complications are common following liver transplant and may include alterations in mental status , seizures , and focal motor deficits . the majority ( 75% ) of these complications are observed within the first month after liver transplant , suggesting a possible relationship between preoperative status and liver transplant rather than the effect of immunosuppression [ 13 , 19 , 31 ] . however , neurologic complications may be observed in the long term , even 1 year after transplant [ 19 , 32 ] . of the neurologic complications , encephalopathy is most commonly observed , although the reported incidence has varied widely from 12% to 84% of patients at some point postoperatively [ 19 , 20 , 31 , 3336 ] . a variety of factors may cause neurologic complications , including encephalopathy , infection ( e.g. , sepsis ) , perioperative complications , persistence of major portosystemic shunts , and immunosuppressant - associated toxicity . neurologic complications have also been associated with a greater risk of patient mortality [ 13 , 37 ] . thus , encephalopathy can be seen as a neurologic complication in itself and as a potential cause of neurologic complications . preoperative history of he is a significant predictor of posttransplant neurologic complications . in a prospective analysis of 84 patients with chronic liver disease who had undergone a liver transplant , the presence of an abnormal neurologic exam suggestive of he before transplant was an independent risk factor for developing in - hospital central nervous system complications after transplant ( p = 0.007 ) . in a retrospective study of 101 patients who had undergone a liver transplant , a history of he was strongly associated with neurologic complications after transplant ( univariate odds ratio ( or ) , 2.6 ; 95% confidence interval ( ci ) , 1.16.4 ; p = 0.03 ) . furthermore , using a multivariate analysis , he in the immediate preoperative period was associated with posttransplant neurologic complications ( adjusted or , 10.7 ; 95% ci , 3.829.9 ; p < 0.013 ) . data continue to accumulate suggesting that even with resolution of prior episodes of overt he , patients may continue to have cognitive deficits after transplant . patients who had undergone a liver transplant , on average 17 to 19 months previously , received a battery of cognitive tests ( e.g. , psychometric hepatic encephalopathy score ( phes ) and repeatable battery for the assessment of neuropsychological status ( rbans ; pearson education inc . , san antonio , tx ) ) to determine if the presence of he before liver transplant was associated with more substantial neurocognitive abnormalities within about 1.5 years after transplant . patients with a history of he before transplant ( n = 25 ) had significantly lower scores for 3 of 6 phes domains compared with healthy individuals ( n = 20 ) and for 2 of 6 phes domains ( attention domains ) compared with patients without he before transplant ( n = 14 ; figure 1 ) . the investigators of this study did not determine the total phes score because of a lack of available normative values . for rbans , patients with a history of he before transplant had significantly lower scores compared with healthy individuals in total rbans score and 4 of the 5 rbans subscores ( p < 0.05 ) . although the total rbans score , immediate memory , delayed memory , and attention subscores were lower for patients with he before transplant than for patients without he before transplant , no significant differences were observed . in cross - sectional ( n = 226 ) and prospective assessments ( n = 59 ) of patients with cirrhosis , patients who had experienced overt he had greater cognitive dysfunction compared with patients without overt he . patients who had an episode of overt he had persistent impairment in cognitive function despite normalization of mental status on lactulose therapy , and the severity of impairment increased with the number of overt he episodes . thus , patients who experience overt he may have persistent and cumulative deficits in working memory , response inhibitor , and learning that are chronic , cumulative , and not readily reversible ( i.e. , permanent ) . although not studied in patients who had eventually undergone a liver transplant , persistent cognitive impairment after overt he was also supported by a study in 106 patients with cirrhosis currently without overt he who were examined on 2 occasions within a 3-day period for the presence of mild cognitive impairment ( phes ) . among 45 patients ( 42% ) without a history of overt he and 34 patients ( 32% ) without a history of overt he but with a current diagnosis of minimal he , phes results improved significantly from the first to the second exam ( p = 0.04 and p = 0.016 , resp . ) , suggesting a learning capacity for taking the tests involved in phes . however , there was no significant improvement in phes results at the second exam for the 27 patients ( 25% ) who had experienced at least 1 prior episode of overt he , indicating a lack of learning capacity in patients with a history of overt he . therefore , patients with a history of overt he may have persistent cognitive impairment despite having a normal mental status and , in some cases , even in the presence of normal cognitive test results ( phes ) , which further supports the hypothesis that he is not a fully reversible condition . evidence also exists for the posttransplant persistence of cognitive dysfunction or radiologic abnormalities in patients exhibiting minimal he before transplant [ 1418 ] . in a small prospective study , 14 patients with minimal he underwent liver transplant and were assessed for visuomotor function ( average time of assessment , 21 months after transplant ) . improvement of visuomotor and visuoconstructive skills ( e.g. , trail making tests , reconstruction of drawing , or picture ) was observed in some patients after transplant , but worsening was observed in others . of note , no significant improvement in posttransplant visuomotor and visuoconstructive performance was noted compared with pretreatment performance , with 50% of patients showing deterioration in performance . in addition , mean posttransplant results for the 14 patients with minimal he were significantly lower than for 22 age - matched healthy individuals ( p = 0.04 ) . in another study of patients with minimal he before transplant ( n = 23 ) , most assessed cognitive functions improved at 6 months after transplant , with some cognitive functions improving only 18 months after transplant ( e.g. , verbal short - term memory ) . data support the hypothesis that patients with a history of he before transplant can have more pronounced cognitive dysfunction after transplant than patients without a history of he . however , results are confounded by some studies that suggest almost complete normalization of radiologic findings after transplant , including gradual normalization of glutamine / glutamate and choline signals in a majority of patients as measured by magnetic resonance spectroscopy [ 40 , 41 ] . in addition , the mechanisms by which more pronounced posttransplant cognitive impairment occurs in patients with pretransplant histories of he are unclear , especially the lack of clearly defined variables that may play a role in short - term or long - term cognitive dysfunction . in a prospective study of 52 patients with cirrhosis who had a liver transplant ( 54% with minimal he and 0% with overt he ) , cognitive function significantly improved from pretransplant values for memory , attention , executive function , motor function , and visuospatial domains of the battery of tests administered ( p < 0.05 ) . however , 13% of patients still had global cognitive impairment 6 to 12 months after transplant . in addition , after liver transplant , cognitive function in patients with cirrhosis of alcoholic etiology , diabetes mellitus , and prior he was more severely impaired compared with patients without these factors . posttransplant patients with alcohol - induced cirrhosis had memory decline , patients with diabetes mellitus exhibited attention impairment , and patients with histories of he had impaired motor function ( figure 2 ) . a multivariate analysis indicated that prior he , diabetes mellitus , and cirrhosis of alcoholic etiology were considered risk factors for poor cognitive function that persists after transplant . more research is necessary to gain a clearer understanding of the key demographics and disease characteristics that are involved to better identify patient subpopulations that are at the greatest risk for posttransplant neurologic complications . the development of minimal or overt he before liver transplant may affect posttransplant outcomes , and he is a probable risk factor for neuropsychiatric symptoms after transplantation . therefore , although the cause of neuropsychiatric symptoms following a liver transplant is likely multifactorial , improving patient he status before transplant may improve posttransplant outcomes . current pharmacologic therapies for he are primarily directed at reducing the systemic levels of ammonia and other toxins produced in the gastrointestinal tract , thereby reducing cerebral exposure . patients may be treated for minimal he , overt he , or the prevention of he relapse . however , most patients with minimal he do not currently receive treatment outside the context of clinical trials . in all of these cases , the mainstays of therapy are nonabsorbable disaccharides and antibiotics . nonabsorbable disaccharides , such as lactulose and lactitol ( not available in the united states ) , are metabolized in the colon by intestinal bacteria , resulting in a reduction in colonic ph . the acidic environment promotes uptake of ammonia by colonic bacteria , facilitates diffusion of ammonia from the blood into the intestine , and may reduce the survival of urease - producing bacteria . nonabsorbable disaccharides also increase the osmotic pressure of the intestinal lumen , which induces catharsis and elimination of potential sources of gut - derived toxins from the body . a 2004 meta - analysis of 22 randomized studies was conducted to evaluate the efficacy of nonabsorbable disaccharides compared with no treatment , placebo , or antibiotics in patients with acute , chronic , or minimal he . nonabsorbable disaccharides appeared to improve he ( i.e. , reduced the risk of no improvement ) when compared with no intervention or placebo ( p = 0.002 ) . however , when studies of poor methodologic quality were removed from the analysis , no significant effect was observed in the few high - quality trials that had been conducted . in addition , nonabsorbable disaccharides had no significant effect on mortality compared with no treatment or placebo intervention . the authors concluded that there was insufficient evidence to support or contest the use of lactulose or lactitol for the treatment of he . a 2011 meta - analysis of 5 studies specifically evaluated nonabsorbable disaccharides for the treatment of minimal he and concluded that compared with placebo these agents significantly improved minimal he ( i.e. , reduced the risk of no improvement ) ( p < 0.0001 ) . another 2011 meta - analysis of 9 studies evaluating lactulose for the treatment of minimal he confirmed that lactulose prevented the progression to overt he , compared with either placebo or no intervention . subsequent to this analysis , an open - label randomized study concluded lactulose to be effective for the primary prophylaxis of overt he in patients with cirrhosis . twenty ( 19% ) of 105 patients followed for 12 months developed an episode of overt he , six ( 11% ) in the lactulose group and 14 ( 28% ) in the nonlactulose treated group ( p = 0.02 ) . however , consistent with other studies , no significant difference in mortality was observed ( p = 0.16 ) . in the 2004 meta - analysis , nonabsorbable disaccharides were significantly less effective than antibiotics in improving he ( i.e. , they were associated with a higher risk of no he improvement ; p = 0.03 ) and did not have a significantly different impact on mortality . patients with he who received nonabsorbable disaccharides also had higher blood ammonia levels after treatment compared with patients who received antibiotics . however , a separate meta - analysis reported similar efficacy between antibiotics and nonabsorbable disaccharides in improving he . in addition , a few studies have evaluated nonabsorbable disaccharides for the prevention of he recurrence ( i.e. , secondary prophylaxis ) [ 4851 ] . in 1 randomized , and unblinded , placebo - controlled study , 140 patients with cirrhosis who had recovered from a previous he episode were randomly assigned within 1 week of recovery to receive either lactulose 30 to 60 ml / d ( n = 70 ) or placebo ( n = 70 ) . thirteen patients ( 9% ) were lost to follow - up ; 61 patients in the lactulose group and 64 patients in the placebo group were followed for a median of 14 months ( range , 120 months ) . lactulose significantly reduced the percentage of patients who experienced overt he recurrence compared with placebo ( 20% versus 47% , resp . ; however , no significant difference in the median time of he recurrence was observed ( 7.5 months ( range , 113 months ) versus 6.0 months ( range , 215 months ) , resp . ) . in addition , no significant differences between the 2 groups were reported in admissions to the liver intensive care unit for conditions other than he or deaths during the study . adverse events ( aes ) associated with nonabsorbable disaccharides are commonly gastrointestinal - related and include abdominal pain , diarrhea , flatulence , and nausea . diarrhea can also lead to secondary complications , including dehydration , hypokalemia , and hypernatremia . anorexia and vomiting have also been reported as aes occurring with use of nonabsorbable disaccharides ( rate of 2% for each ) . one additional concern with lactulose is that administration can cause abdominal distention , which may result in technical difficulties during liver transplant [ 54 , 55 ] . nutritional status before liver transplant has also been shown to correlate with posttransplant survival and is independently associated with the number of infection episodes after transplant . malnutrition , assessed by a subjective global nutritional assessment exam , was found to be an independent risk factor for the length of stay in the intensive care unit and the total number of days spent in the hospital after transplant . furthermore , alterations in specific laboratory measures ( e.g. , albumin , sodium , and potassium ) [ 5860 ] , which may be negatively impacted by gastrointestinal aes , have also been identified as risk factors for surgical complications in patients who received a liver transplant , and serum sodium levels are a prognostic factor for survival in patients awaiting liver transplant [ 61 , 62 ] . thus , gastrointestinal aes may increase patient risk , particularly for patients who are prone to malnutrition because of various comorbid variables or conditions ( e.g. , dietary restrictions or gastroparesis ) . it is possible that administration of nonabsorbable disaccharides such as lactulose may exacerbate pretransplant nutritional deficits , thereby contributing to poor posttransplant outcomes . antibiotics are administered to reduce systemic levels of ammonia and other gut - derived toxins by targeting gastrointestinal bacteria . because of the risk for systemic aes and bacterial antibiotic resistance with systemic antibiotics , nonsystemic antibiotics are preferred agents . rifaximin is a nonsystemic gut - selective antibiotic and more than 20 studies have evaluated rifaximin for the treatment of overt he ( see review by lawrence and klee ) or minimal he [ 6668 ] . in 2010 , rifaximin was approved by the us food and drug administration for the maintenance of overt he remission in adults . in a randomized , double - blind , phase 3 trial of rifaximin for the maintenance of he remission , patients in remission from he were treated with rifaximin 1100 mg / d ( n = 140 ) or placebo ( n = 159 ) for up to 6 months . concomitant lactulose administration was permitted during the study : 91% of patients in each group received concomitant lactulose . only 22% of patients in the rifaximin group experienced a breakthrough he episode compared with 46% of patients in the placebo group . furthermore , rifaximin significantly reduced the risk of he breakthrough by 58% compared with placebo during the 6 months of treatment ( hazard ratio ( hr ) , 0.42 ; 95% ci , 0.280.64 ; p < 0.001 ; figure 4 ) . data indicated that the number needed to treat ( nnt ) was 4 ( i.e. , for every 4 patients treated with rifaximin for 6 months , 1 episode of breakthrough he would be prevented ) . in addition , 14% of patients in the rifaximin group reported an he - related hospitalization compared with 23% of patients in the placebo group . rifaximin significantly ( p = 0.01 ) reduced the risk of he - related hospitalizations by 50% compared with placebo ( hr , 0.50 ; 95% ci , 0.290.87 ) . data indicated that the nnt was 9 ( i.e. , for every 9 patients treated with rifaximin for 6 months , 1 episode of he - related hospitalization would be prevented ) . health - related qol in the phase 3 trial was assessed using the chronic liver disease questionnaire ( cldq ) , which was administered every 4 weeks , and the time to he breakthrough recorded [ 70 , 71 ] . a significant ( p = 0.0087 to 0.0436 ) improvement with rifaximin treatment was noted in the overall cldq scores and in each domain score compared with placebo treatment , and scores were significantly ( p < 0.0001 ) lower in patients who experienced he breakthrough compared with those who remained in remission . in the phase 3 trial , rifaximin was well tolerated , with a similar incidence of aes reported in both groups . the most common aes with rifaximin and placebo were nausea ( 14.3% versus 13.2% ) , diarrhea ( 10.7% versus 13.2% ) , fatigue two cases of clostridium difficile infection ( cdi ) were reported during the double - blind portion of the trial , both in the rifaximin group . the authors noted that these 2 patients had multiple risk factors for cdi , including repeated hospitalizations during which they received multiple courses of antibiotic therapy , advanced age , and pantoprazole use . the impact of long - term rifaximin therapy on gut flora , including a risk of bacterial antibiotic resistance , is largely unknown . however , the drug appeared to have a protective effect against infections within 90 days after transplant ( p = 0.026 ) in patients treated with rifaximin for he during liver transplant candidacy and was not associated with a higher risk of multidrug - resistant bacterial infections . conventional antibiotics , neomycin and metronidazole , are also administered for the treatment of he . however , strong clinical data supporting their efficacy in the treatment of he are lacking . one randomized , double - blind study failed to demonstrate a benefit with neomycin ( n = 20 ) compared with placebo ( n = 19 ) , with no significant differences observed for time to resolution of he symptoms or for 5-day , 30-day , or 12-month mortality . two small , randomized , double - blind studies ( n = 33 and n = 45 ) and 1 randomized , unblinded trial ( n = 173 ) have suggested that neomycin and lactulose may have similar efficacy in the treatment of he [ 7476 ] . two randomized studies ( n = 35 and n = 49 ) have compared neomycin with rifaximin and suggested that they have similar efficacy in the treatment of he , although in one of the studies , patients who received rifaximin showed improvements sooner than those who received neomycin ( 3 versus 5 days , resp . ) [ 77 , 78 ] . for metronidazole , 1 small study ( n = 18 ) comparing metronidazole with neomycin suggested that both antibiotics improved mental state , reduced asterixis , and improved electroencephalogram measures . however , the risk of aes associated with neomycin and metronidazole may limit their use in patients with he and suggest that they might not be an ideal pretransplant choice of treatment . intestinal malabsorption and diarrhea have been observed with neomycin therapy and thus could impact pretransplant nutritional status of the patients . although neomycin is poorly absorbed , prolonged administration may result in cumulative systemic concentrations sufficient to increase the risk of serious aes , such as ototoxicity and nephrotoxicity . because the meld scoring system includes measures of renal dysfunction and neomycin may cause renal damage , neomycin may not be an ideal choice for patients , particularly those with high meld scores . metronidazole has been associated with peripheral neurotoxicity and requires dosing adjustments in patients with severe liver disease because of impaired drug clearance . metronidazole is a systemic antibiotic frequently administered for the treatment of cdi , and the potential risk of c. difficile resistance to metronidazole warrants judicious use of this agent . compared with nonabsorbable disaccharides , neomycin , and metronidazole , rifaximin exhibits a more favorable safety and tolerability profile [ 47 , 65 , 82 ] . rifaximin has not been associated with gastrointestinal aes such as diarrhea or nausea in clinical studies and would be unlikely to increase the risk of dehydration , weight loss , abdominal distention , malnutrition , or intestinal malabsorption in patients awaiting transplant , thereby minimizing the possible negative consequences of he therapy on patient nutritional status . a 2012 meta - analysis , incorporating data from 12 randomized controlled active comparator trials for the treatment of patients with he , assessed the efficacy and psychometric outcomes of rifaximin compared with other oral therapies ( including disaccharides and other antibiotics ) . however , more favorable effects were observed with rifaximin with regard to psychometric parameters and serum ammonia levels . a tolerability analysis , which included he prevention trial data , indicated that rifaximin was also associated with fewer adverse effects . he is a common complication of cirrhosis that substantially affects patient morbidity and mortality . furthermore , he can have a detrimental impact on posttransplant outcomes , including patient survival . data continue to emerge demonstrating the potential persistence of cognitive deficits associated with he , even after liver transplant . therefore , prevention of he in patients with cirrhosis may improve pretransplant health status and thus improve posttransplant outcomes . commonly prescribed therapies include nonabsorbable disaccharides ( e.g. , lactulose ) and nonsystemic antibiotics ( e.g. , rifaximin ) , and their various risks and benefits should be taken into consideration when deciding the most appropriate he management algorithm in patients awaiting liver transplant . further studies to evaluate currently available therapies in preventing he and improving posttransplant outcomes are warranted .

patients with cirrhosis commonly experience hepatic encephalopathy ( he ) , a condition associated with alterations in behavior , cognitive function , consciousness , and neuromuscular function of varying severity . he occurring before liver transplant can have a substantial negative impact on posttransplant outcomes , and preoperative history of he may be a predictor of posttransplant neurologic complications . even with resolution of previous episodes of overt or minimal he , some patients continue to experience cognitive deficits after transplant . because he is one of the most frequent pretransplant complications , improving patient he status before transplant may improve outcomes . current pharmacologic therapies for he , whether for the treatment of minimal or overt he or for prevention of he relapse , are primarily directed at reducing cerebral exposure to systemic levels of gut - derived toxins ( e.g. , ammonia ) . the current mainstays of he therapy are nonabsorbable disaccharides and antibiotics . the various impacts of adverse effects ( such as diarrhea , abdominal distention , and dehydration ) on patient 's health and nutritional status should be taken into consideration when deciding the most appropriate he management strategy in patients awaiting liver transplant . this paper reviews the potential consequences of pretransplant he on posttransplant outcomes and therapeutic strategies for the pretransplant management of he .

1. Introduction 2. Consequences of Pretransplant HE on Posttransplant Outcomes 3. Neurologic Complications after Transplant 4. Treatment of HE in the Pretransplant Setting 5. Summary

cirrhosis of the liver the only cure for which is liver transplant is associated with several serious complications , including ascites , spontaneous bacterial peritonitis , variceal bleeding , and hepatic encephalopathy ( he ) . guidelines established by the american association for the study of liver diseases currently recommend referring patients with cirrhosis for liver transplant when their model for end - stage liver disease ( meld ) score is 10 and their child - turcotte - pugh ( ctp ) score is 7 or when they experience their first major complication ( e.g. however , the current united network for organ sharing allocation system only uses the meld score for prioritizing adults for liver transplant . however , this scoring system does not take into account several serious complications of cirrhosis , such as he , when prioritizing patients for liver transplant . this may have negative ramifications for patient care , as the development of he may be associated with substantial morbidity , mortality , and cost . he is characterized by alterations in behavior , cognitive abilities , consciousness , and neuromuscular function . it negatively affects patient quality of life ( qol ) , and patients may be unable to drive , work , or adequately care for themselves because of its effects [ 811 ] . patients may be less compliant with all prescribed medications , and hospitalizations related to he may increase patient exposure to opportunistic infections and be associated with substantial costs . furthermore , he may be an independent predictor of mortality in patients with chronic liver disease . he occurring before liver transplant can also have a substantial negative impact on posttransplant outcomes [ 1322 ] . this paper will review the potential consequences of pretransplant he on posttransplant outcomes and therapeutic strategies for the pretransplant management of he . he may occur in patients with acute liver failure ( type a he ) , in patients with portosystemic shunting but no intrinsic hepatocellular disease ( type b he ) , or , as in the majority of he cases , in patients with cirrhosis and cirrhosis - related portosystemic shunting ( type c he ) . because he is a progressive neuropsychiatric condition , he may be graded or scored based on the severity of the clinical manifestations , which may range from subtle neurologic abnormalities in mild cases to coma in severe cases . minimal he ( sometimes referred to as covert he ) , which may occur in nearly 70% of patients with cirrhosis , is not associated with any clinical signs of brain dysfunction , but patients experience cognitive abnormalities that can lead to qol impairment [ 8 , 24 , 25 ] . unlike minimal he , overt he can manifest as a wide spectrum of symptoms that can be observed clinically , including those related to motor and neuropsychologic functions . overt he has been shown to occur in nearly half of patients with cirrhosis and , as with minimal he , also has a substantial negative impact on qol [ 710 ] . although research is ongoing , the pathogenesis of he is believed to primarily involve the exposure of the brain to elevated neurotoxin levels , particularly ammonia and other gut - derived toxins , leading to cellular morphologic changes ( e.g. many factors that can precipitate he ( e.g. , hypokalemia , infection , and gastrointestinal bleeding ) serve to increase the production of ammonia or other gut - derived toxins or to reduce toxin metabolism by the liver ( e.g. although the exact pathophysiology of he is unclear , data are accumulating to suggest that the cascade of neuropsychiatric and neuromuscular sequelae of he may have a longer term or more permanent negative impact on patients with chronic liver disease than originally suspected . neurologic complications are common following liver transplant and may include alterations in mental status , seizures , and focal motor deficits . however , neurologic complications may be observed in the long term , even 1 year after transplant [ 19 , 32 ] . of the neurologic complications , encephalopathy is most commonly observed , although the reported incidence has varied widely from 12% to 84% of patients at some point postoperatively [ 19 , 20 , 31 , 3336 ] . a variety of factors may cause neurologic complications , including encephalopathy , infection ( e.g. , sepsis ) , perioperative complications , persistence of major portosystemic shunts , and immunosuppressant - associated toxicity . neurologic complications have also been associated with a greater risk of patient mortality [ 13 , 37 ] . preoperative history of he is a significant predictor of posttransplant neurologic complications . in a prospective analysis of 84 patients with chronic liver disease who had undergone a liver transplant , the presence of an abnormal neurologic exam suggestive of he before transplant was an independent risk factor for developing in - hospital central nervous system complications after transplant ( p = 0.007 ) . in a retrospective study of 101 patients who had undergone a liver transplant , a history of he was strongly associated with neurologic complications after transplant ( univariate odds ratio ( or ) , 2.6 ; 95% confidence interval ( ci ) , 1.16.4 ; p = 0.03 ) . furthermore , using a multivariate analysis , he in the immediate preoperative period was associated with posttransplant neurologic complications ( adjusted or , 10.7 ; 95% ci , 3.829.9 ; p < 0.013 ) . data continue to accumulate suggesting that even with resolution of prior episodes of overt he , patients may continue to have cognitive deficits after transplant . patients who had undergone a liver transplant , on average 17 to 19 months previously , received a battery of cognitive tests ( e.g. , psychometric hepatic encephalopathy score ( phes ) and repeatable battery for the assessment of neuropsychological status ( rbans ; pearson education inc . , san antonio , tx ) ) to determine if the presence of he before liver transplant was associated with more substantial neurocognitive abnormalities within about 1.5 years after transplant . patients with a history of he before transplant ( n = 25 ) had significantly lower scores for 3 of 6 phes domains compared with healthy individuals ( n = 20 ) and for 2 of 6 phes domains ( attention domains ) compared with patients without he before transplant ( n = 14 ; figure 1 ) . for rbans , patients with a history of he before transplant had significantly lower scores compared with healthy individuals in total rbans score and 4 of the 5 rbans subscores ( p < 0.05 ) . although the total rbans score , immediate memory , delayed memory , and attention subscores were lower for patients with he before transplant than for patients without he before transplant , no significant differences were observed . in cross - sectional ( n = 226 ) and prospective assessments ( n = 59 ) of patients with cirrhosis , patients who had experienced overt he had greater cognitive dysfunction compared with patients without overt he . patients who had an episode of overt he had persistent impairment in cognitive function despite normalization of mental status on lactulose therapy , and the severity of impairment increased with the number of overt he episodes . thus , patients who experience overt he may have persistent and cumulative deficits in working memory , response inhibitor , and learning that are chronic , cumulative , and not readily reversible ( i.e. although not studied in patients who had eventually undergone a liver transplant , persistent cognitive impairment after overt he was also supported by a study in 106 patients with cirrhosis currently without overt he who were examined on 2 occasions within a 3-day period for the presence of mild cognitive impairment ( phes ) . among 45 patients ( 42% ) without a history of overt he and 34 patients ( 32% ) without a history of overt he but with a current diagnosis of minimal he , phes results improved significantly from the first to the second exam ( p = 0.04 and p = 0.016 , resp . ) however , there was no significant improvement in phes results at the second exam for the 27 patients ( 25% ) who had experienced at least 1 prior episode of overt he , indicating a lack of learning capacity in patients with a history of overt he . therefore , patients with a history of overt he may have persistent cognitive impairment despite having a normal mental status and , in some cases , even in the presence of normal cognitive test results ( phes ) , which further supports the hypothesis that he is not a fully reversible condition . evidence also exists for the posttransplant persistence of cognitive dysfunction or radiologic abnormalities in patients exhibiting minimal he before transplant [ 1418 ] . in a small prospective study , 14 patients with minimal he underwent liver transplant and were assessed for visuomotor function ( average time of assessment , 21 months after transplant ) . in addition , mean posttransplant results for the 14 patients with minimal he were significantly lower than for 22 age - matched healthy individuals ( p = 0.04 ) . in another study of patients with minimal he before transplant ( n = 23 ) , most assessed cognitive functions improved at 6 months after transplant , with some cognitive functions improving only 18 months after transplant ( e.g. data support the hypothesis that patients with a history of he before transplant can have more pronounced cognitive dysfunction after transplant than patients without a history of he . in addition , the mechanisms by which more pronounced posttransplant cognitive impairment occurs in patients with pretransplant histories of he are unclear , especially the lack of clearly defined variables that may play a role in short - term or long - term cognitive dysfunction . in a prospective study of 52 patients with cirrhosis who had a liver transplant ( 54% with minimal he and 0% with overt he ) , cognitive function significantly improved from pretransplant values for memory , attention , executive function , motor function , and visuospatial domains of the battery of tests administered ( p < 0.05 ) . in addition , after liver transplant , cognitive function in patients with cirrhosis of alcoholic etiology , diabetes mellitus , and prior he was more severely impaired compared with patients without these factors . posttransplant patients with alcohol - induced cirrhosis had memory decline , patients with diabetes mellitus exhibited attention impairment , and patients with histories of he had impaired motor function ( figure 2 ) . a multivariate analysis indicated that prior he , diabetes mellitus , and cirrhosis of alcoholic etiology were considered risk factors for poor cognitive function that persists after transplant . more research is necessary to gain a clearer understanding of the key demographics and disease characteristics that are involved to better identify patient subpopulations that are at the greatest risk for posttransplant neurologic complications . the development of minimal or overt he before liver transplant may affect posttransplant outcomes , and he is a probable risk factor for neuropsychiatric symptoms after transplantation . therefore , although the cause of neuropsychiatric symptoms following a liver transplant is likely multifactorial , improving patient he status before transplant may improve posttransplant outcomes . current pharmacologic therapies for he are primarily directed at reducing the systemic levels of ammonia and other toxins produced in the gastrointestinal tract , thereby reducing cerebral exposure . patients may be treated for minimal he , overt he , or the prevention of he relapse . however , most patients with minimal he do not currently receive treatment outside the context of clinical trials . in all of these cases , the mainstays of therapy are nonabsorbable disaccharides and antibiotics . nonabsorbable disaccharides , such as lactulose and lactitol ( not available in the united states ) , are metabolized in the colon by intestinal bacteria , resulting in a reduction in colonic ph . nonabsorbable disaccharides also increase the osmotic pressure of the intestinal lumen , which induces catharsis and elimination of potential sources of gut - derived toxins from the body . a 2004 meta - analysis of 22 randomized studies was conducted to evaluate the efficacy of nonabsorbable disaccharides compared with no treatment , placebo , or antibiotics in patients with acute , chronic , or minimal he . the authors concluded that there was insufficient evidence to support or contest the use of lactulose or lactitol for the treatment of he . a 2011 meta - analysis of 5 studies specifically evaluated nonabsorbable disaccharides for the treatment of minimal he and concluded that compared with placebo these agents significantly improved minimal he ( i.e. another 2011 meta - analysis of 9 studies evaluating lactulose for the treatment of minimal he confirmed that lactulose prevented the progression to overt he , compared with either placebo or no intervention . subsequent to this analysis , an open - label randomized study concluded lactulose to be effective for the primary prophylaxis of overt he in patients with cirrhosis . twenty ( 19% ) of 105 patients followed for 12 months developed an episode of overt he , six ( 11% ) in the lactulose group and 14 ( 28% ) in the nonlactulose treated group ( p = 0.02 ) . , they were associated with a higher risk of no he improvement ; p = 0.03 ) and did not have a significantly different impact on mortality . in addition , a few studies have evaluated nonabsorbable disaccharides for the prevention of he recurrence ( i.e. in 1 randomized , and unblinded , placebo - controlled study , 140 patients with cirrhosis who had recovered from a previous he episode were randomly assigned within 1 week of recovery to receive either lactulose 30 to 60 ml / d ( n = 70 ) or placebo ( n = 70 ) . adverse events ( aes ) associated with nonabsorbable disaccharides are commonly gastrointestinal - related and include abdominal pain , diarrhea , flatulence , and nausea . one additional concern with lactulose is that administration can cause abdominal distention , which may result in technical difficulties during liver transplant [ 54 , 55 ] . nutritional status before liver transplant has also been shown to correlate with posttransplant survival and is independently associated with the number of infection episodes after transplant . malnutrition , assessed by a subjective global nutritional assessment exam , was found to be an independent risk factor for the length of stay in the intensive care unit and the total number of days spent in the hospital after transplant . furthermore , alterations in specific laboratory measures ( e.g. , albumin , sodium , and potassium ) [ 5860 ] , which may be negatively impacted by gastrointestinal aes , have also been identified as risk factors for surgical complications in patients who received a liver transplant , and serum sodium levels are a prognostic factor for survival in patients awaiting liver transplant [ 61 , 62 ] . thus , gastrointestinal aes may increase patient risk , particularly for patients who are prone to malnutrition because of various comorbid variables or conditions ( e.g. it is possible that administration of nonabsorbable disaccharides such as lactulose may exacerbate pretransplant nutritional deficits , thereby contributing to poor posttransplant outcomes . antibiotics are administered to reduce systemic levels of ammonia and other gut - derived toxins by targeting gastrointestinal bacteria . rifaximin is a nonsystemic gut - selective antibiotic and more than 20 studies have evaluated rifaximin for the treatment of overt he ( see review by lawrence and klee ) or minimal he [ 6668 ] . in 2010 , rifaximin was approved by the us food and drug administration for the maintenance of overt he remission in adults . in a randomized , double - blind , phase 3 trial of rifaximin for the maintenance of he remission , patients in remission from he were treated with rifaximin 1100 mg / d ( n = 140 ) or placebo ( n = 159 ) for up to 6 months . health - related qol in the phase 3 trial was assessed using the chronic liver disease questionnaire ( cldq ) , which was administered every 4 weeks , and the time to he breakthrough recorded [ 70 , 71 ] . a significant ( p = 0.0087 to 0.0436 ) improvement with rifaximin treatment was noted in the overall cldq scores and in each domain score compared with placebo treatment , and scores were significantly ( p < 0.0001 ) lower in patients who experienced he breakthrough compared with those who remained in remission . the most common aes with rifaximin and placebo were nausea ( 14.3% versus 13.2% ) , diarrhea ( 10.7% versus 13.2% ) , fatigue two cases of clostridium difficile infection ( cdi ) were reported during the double - blind portion of the trial , both in the rifaximin group . however , the drug appeared to have a protective effect against infections within 90 days after transplant ( p = 0.026 ) in patients treated with rifaximin for he during liver transplant candidacy and was not associated with a higher risk of multidrug - resistant bacterial infections . conventional antibiotics , neomycin and metronidazole , are also administered for the treatment of he . however , strong clinical data supporting their efficacy in the treatment of he are lacking . one randomized , double - blind study failed to demonstrate a benefit with neomycin ( n = 20 ) compared with placebo ( n = 19 ) , with no significant differences observed for time to resolution of he symptoms or for 5-day , 30-day , or 12-month mortality . two small , randomized , double - blind studies ( n = 33 and n = 45 ) and 1 randomized , unblinded trial ( n = 173 ) have suggested that neomycin and lactulose may have similar efficacy in the treatment of he [ 7476 ] . two randomized studies ( n = 35 and n = 49 ) have compared neomycin with rifaximin and suggested that they have similar efficacy in the treatment of he , although in one of the studies , patients who received rifaximin showed improvements sooner than those who received neomycin ( 3 versus 5 days , resp . ) however , the risk of aes associated with neomycin and metronidazole may limit their use in patients with he and suggest that they might not be an ideal pretransplant choice of treatment . metronidazole has been associated with peripheral neurotoxicity and requires dosing adjustments in patients with severe liver disease because of impaired drug clearance . metronidazole is a systemic antibiotic frequently administered for the treatment of cdi , and the potential risk of c. difficile resistance to metronidazole warrants judicious use of this agent . compared with nonabsorbable disaccharides , neomycin , and metronidazole , rifaximin exhibits a more favorable safety and tolerability profile [ 47 , 65 , 82 ] . rifaximin has not been associated with gastrointestinal aes such as diarrhea or nausea in clinical studies and would be unlikely to increase the risk of dehydration , weight loss , abdominal distention , malnutrition , or intestinal malabsorption in patients awaiting transplant , thereby minimizing the possible negative consequences of he therapy on patient nutritional status . a 2012 meta - analysis , incorporating data from 12 randomized controlled active comparator trials for the treatment of patients with he , assessed the efficacy and psychometric outcomes of rifaximin compared with other oral therapies ( including disaccharides and other antibiotics ) . furthermore , he can have a detrimental impact on posttransplant outcomes , including patient survival . data continue to emerge demonstrating the potential persistence of cognitive deficits associated with he , even after liver transplant . therefore , prevention of he in patients with cirrhosis may improve pretransplant health status and thus improve posttransplant outcomes . commonly prescribed therapies include nonabsorbable disaccharides ( e.g. , lactulose ) and nonsystemic antibiotics ( e.g. , rifaximin ) , and their various risks and benefits should be taken into consideration when deciding the most appropriate he management algorithm in patients awaiting liver transplant .

lincidence de syphilis ottawa , en ontario , a beaucoup augment depuis 2000 , passant de six cas sur 100 000 habitants en 2003 neuf cas sur 100 000 habitants en 2007 , puis 11 cas sur 100 000 habitants en 2010 . le nombre de cas signals pendant le premier trimestre de 2010 a plus que doubl par rapport celui du premier trimestre de 2009 . en mai 2010 , le ministre de la sant et des soins de longue dure de lontario a demand laide du programme canadien dpidmiologie de terrain pour dcrire laugmentation du taux de syphilis infectieuses , en dterminer lorigine dans les rseaux sociaux et tablir les messages de prvention . les chercheurs ont collig systmatiquement les donnes de surveillance sur la syphilis entre le 1 janvier 2009 et le 15 juillet 2010 et en ont tabli les rseaux sociaux partir dun questionnaire amlior sur les rseaux sociaux . au moyen du test de kruskal - wallis et des tests du chi carr , ils ont effectu des comparaisons univaries des donnes non distribues normalement entre le groupe de surveillance amliore et les autres cas de 2009 . dix - sept des 19 cas les plus rcents ont consenti rpondre au questionnaire , qui a rvl un usage peu frquent du condom , de multiples partenaires sexuels et des relations sexuelles avec des partenaires de mme sexe . les chercheurs ont transcrit sur un graphique linformation relative aux lieux o les partenaires sexuels se sont rencontrs , ce qui a permis de relier 18 cas et 40 contacts , soit 37 % de la population de lclosion , et dassocier de nombreux individus uniques et de dyades . la dcouverte des lieux o les partenaires sexuels se sont rencontrs sest rvle une mesure indirecte efficace des activits haut risque partages avec des personnes infectes et a dmontr le potentiel dintervenir seulement dans un bar prcis et un site internet donn pour joindre une forte proportion residents of ottawa diagnosed with infectious syphilis , defined according to national case definitions ( 10 ) from january 1 , 2009 to june 15 , 2010 , were included in the investigation ( n=72 ) . electronic records were retrieved from the integrated public health information system , they were then validated and supplementary information was added to each routine surveillance record . for example , contacts known only by an e - mail and sex - partner recruitment venues had not been added into the integrated public health information system but were added before the analysis , and greatly improved the social network links . an enhanced surveillance questionnaire was developed in consultation with the oph investigative team and was pilot tested by a case manager . out of concern for oph s relationship with the community of men having sex with men , community groups were consulted and approval was obtained from oph s research ethics board . twenty syphilis cases from january 2010 to june 2010 , which represented 27.7% of all syphilis cases , were contacted up to three times using two different methods ( telephone , letter , e - mail , home visits ) . these 20 cases were the most recent of all 72 cases and , therefore , were more likely to remember details than cases from 2009 . the questionnaire elicited types of sexual partners in the past 12 months including regular , casual and anonymous partners , sex trade clients and group - sex encounters . what types of partners have you had in the last 12 months before your diagnosis ? then , there were check boxes for casual , regular and paying partners , without definitions , with a space for participants to indicate how many for each type . respondents were prompted for visited venues including public venues ( including bars ) , clubs , bath houses , spas , sex clubs , raves , circuit parties , cruising sites or pick - up joints , work , school , prisons , parks , family or friends homes , internet sites , adult book and video stores , in ottawa or in other cities , where they met their sexual partners . respondents were also asked their opinions regarding actions needed to address the increase in syphilis infections . data obtained using enhanced surveillance were combined with demographic data , syphilis staging , hiv coinfection , behavioural risk factors , the names of sexual contacts and incidental free - text fields regarding locations where partners were met were collected as part of a routine case follow - up . some data regarding exposure and it is important to note that the enhanced cases had two opportunities to provide data regarding their sex partners . all unique partners named at both interviews for the 2009 to 2010 period were incorporated into the network diagrams ; however , only the data regarding numbers of partners from the first interview were used for statistical tests to avoid ascertainment bias . the incorporation of all routinely available data , together with enhanced data to show clustering around venues for sex partner recruitment , allowed for comparison with previous studies ( 8,9 ) . descriptive and univariate analysis of the data ( , student s t and kruskal wallis tests ) were performed using microsoft office excel version 7 ( microsoft corporation , usa ) and epi info 6.04 ( centres for disease control and prevention , usa ) . where numbers allowed , all cases were compared with the most recent cases for enhanced surveillance to demonstrate that the most recent sample represented the total population of syphilis cases in most respects . pajek version 1.27 ( pajek , slovenia ) was used to construct and analyze the social networks , where lines represented sexual contact between individuals , who were represented by nodes . a network of all cases and named contacts available from routine data collection was constructed , as well as a network of all cases and contacts who frequented common venues . the numbers and sizes of components were counted in which each person was connected to at least one other place or person . the number of links from one person to another immediately adjacent were also counted , which represented the total number of sex partners named by and who named an index case ( degree ) . descriptive and univariate analysis of the data ( , student s t and kruskal wallis tests ) were performed using microsoft office excel version 7 ( microsoft corporation , usa ) and epi info 6.04 ( centres for disease control and prevention , usa ) . where numbers allowed , all cases were compared with the most recent cases for enhanced surveillance to demonstrate that the most recent sample represented the total population of syphilis cases in most respects . pajek version 1.27 ( pajek , slovenia ) was used to construct and analyze the social networks , where lines represented sexual contact between individuals , who were represented by nodes . a network of all cases and named contacts available from routine data collection was constructed , as well as a network of all cases and contacts who frequented common venues . the numbers and sizes of components were counted in which each person was connected to at least one other place or person . the number of links from one person to another immediately adjacent were also counted , which represented the total number of sex partners named by and who named an index case ( degree ) . seventy - two individuals met the infectious syphilis case definition from january 1 , 2009 to june 15 , 2010 ( table 1 ) . the epidemic curve of infectious syphilis cases during january 1 , 2009 to june 15 , 2010 was consistent with a propagated outbreak with person - to - person spread ( figure 2 ) . the majority ( 94.4% ) of reported cases almost one - half of the cases presented with secondary syphilis ( 44.4% ) and 19 ( 26.4% ) were hiv positive . five of 19 individuals were diagnosed with hiv within one year of their syphilis diagnosis , while 14 cases were diagnosed with hiv more than one year before their diagnosis of syphilis . sixty - one ( 84.7% ) cases reported a total of 321 sexual partners ranging from one to 50 partners for each case , of whom only 69 ( 21.5% ) were named . the distribution of the number of named partners ( degree ) used in the network analysis revealed a highly skewed distribution , known as a small world network , in which 90% of respondents who named contacts had one to three partners in the past 12 months , while the remainder had four to 16 named partners . cases who did not report contact information were similar to those who did , in sex and age ( mean of 46.2 and 39.6 years of age , respectively ; p=0.14 [ student s t test ] ) . during routine case follow - up , respondents reported various sex practices including infrequent condom use , sex with a same - sex partner , a homosexual partner , a new partner in the past two months and multiple partners in the past six months ( table 1 ) . seventeen of 20 ( 89% ) of the most recent cases chosen consented to be reinterviewed , two declined and one was not successfully contacted . all respondents were men , resembling the majority ( 94.4% ) of routinely reported cases and were of similar ages and syphilis stages compared with the routinely notified group ( table 1 ) . one of the enhanced surveillance respondents ( 5.9% ) was coinfected with hiv , compared with coinfection in 19 ( 26.4% ) of the routine cases ( =6.59 ; p=0.01 ) . ethnic background for the 72 reported cases was not collected ; however , the majority of the cases who responded to the enhanced surveillance questionnaire were caucasian ( 82.4% ) . the 17 cases contacted for enhanced surveillance reported a total of 81 sexual partners , although only 17 ( 21.0% ) according to name , similar to the routinely reported cases . however , they reported higher numbers of total partners than reported according to routinely notified cases ( p=0.02 [ kruskal - wallis ] ) ( table 2 ) . eleven of 17 ( 64.7% ) cases reported having regular sexual partners , 12 ( 70.6% ) reported having casual partners , 11 ( 64.7% ) reported having anonymous partners and four ( 23.5% ) reported having group - sex encounters . there was no sexual - health messaging reported at informal venues ( table 2 ) . however , there was an awareness of sexual - health messaging in bars and clubs ( four of seven responses ) and in bathhouses , spas and sex clubs ( seven of nine responses ) in ottawa and outside of ottawa ( five of seven responses ) . the proportion of respondents aware and unaware of sexual - health messaging on internet sites was evenly split . respondents suggested that oph increase education regarding symptoms , testing and transmission of syphilis , as well as the importance of condom use for oral and anal sex . they suggested targeting specific social venues frequented by the gay population such as bars , internet sites and bathhouses . the respondents also stressed the need for messaging in other venues , such as public washrooms , to include men who have sex with both women and men . advertisements directed at the general public using television , radio and magazines were also suggested to ensure all populations are aware of the messaging . raising awareness and education of family and emergency room physicians about syphilis was also identified . including the social venue information collected through routine surveillance , only 155 cases and contacts were linked by sexual intercourse in 55 components consisting of 18 single cases ; ( 11.6% of all cases and contacts ) ( figure 3 ) ; 23 dyads ( only two individuals linked ( 29.7% ) , six triads ( 11.6% ) , three tetrads ( 7.7% ) and components of six , eight , 12 and 16 each , with the largest being 19 individuals ( 12% ) ( figure 3 ) . of the 19 infectious syphilis cases coinfected with hiv , 63% were in small components of one or two cases and/or contacts . however , the potential for hiv transmission is illustrated in components eight and 14 ( marked by arrows ) in which cases who had been diagnosed with hiv were linked to hiv - negative individuals with syphilis or untested contacts . the 17 enhanced surveillance respondents reported 42 social venues , which included those already noted in the routine surveillance reports ; 16 social venues outside of ottawa and six internet sites . the most frequently reported social venue was a specific bathhouse in ottawa , named by six ( 35% ) of the respondents and followed by a popular gay internet site ( 29% ) . three different bathhouses in montreal ( quebec ) were identified as places to meet partners ( 24% ) . in addition , 16 venues in locations > 200 km away , such as florida and georgia ( usa ) and alberta were also named , comprising 38% of all venues . the above venue data were combined with the networks of 72 routine and enhanced surveillance cases and their 83 named contacts ( figure 4 ) . the network consisted of 45 components ranging in size from one to 95 nodes including 42 social venues . furthermore , the largest component , previously composed of only 19 nodes ( 12% ) , expanded three - fold to contain 18 cases and 40 contacts , representing 37% of the outbreak population ( figure 5 ) . importantly , a second case with hiv and a female contact who were previously in separate components were now included . seventy - two individuals met the infectious syphilis case definition from january 1 , 2009 to june 15 , 2010 ( table 1 ) . the epidemic curve of infectious syphilis cases during january 1 , 2009 to june 15 , 2010 was consistent with a propagated outbreak with person - to - person spread ( figure 2 ) . the majority ( 94.4% ) of reported cases almost one - half of the cases presented with secondary syphilis ( 44.4% ) and 19 ( 26.4% ) were hiv positive . five of 19 individuals were diagnosed with hiv within one year of their syphilis diagnosis , while 14 cases were diagnosed with hiv more than one year before their diagnosis of syphilis . sixty - one ( 84.7% ) cases reported a total of 321 sexual partners ranging from one to 50 partners for each case , of whom only 69 ( 21.5% ) were named . the distribution of the number of named partners ( degree ) used in the network analysis revealed a highly skewed distribution , known as a small world network , in which 90% of respondents who named contacts had one to three partners in the past 12 months , while the remainder had four to 16 named partners . cases who did not report contact information were similar to those who did , in sex and age ( mean of 46.2 and 39.6 years of age , respectively ; p=0.14 [ student s t test ] ) . during routine case follow - up , respondents reported various sex practices including infrequent condom use , sex with a same - sex partner , a homosexual partner , a new partner in the past two months and multiple partners in the past six months ( table 1 ) . seventeen of 20 ( 89% ) of the most recent cases chosen consented to be reinterviewed , two declined and one was not successfully contacted . all respondents were men , resembling the majority ( 94.4% ) of routinely reported cases and were of similar ages and syphilis stages compared with the routinely notified group ( table 1 ) . one of the enhanced surveillance respondents ( 5.9% ) was coinfected with hiv , compared with coinfection in 19 ( 26.4% ) of the routine cases ( =6.59 ; p=0.01 ) . ethnic background for the 72 reported cases was not collected ; however , the majority of the cases who responded to the enhanced surveillance questionnaire were caucasian ( 82.4% ) . the 17 cases contacted for enhanced surveillance reported a total of 81 sexual partners , although only 17 ( 21.0% ) according to name , similar to the routinely reported cases . however , they reported higher numbers of total partners than reported according to routinely notified cases ( p=0.02 [ kruskal - wallis ] ) ( table 2 ) . eleven of 17 ( 64.7% ) cases reported having regular sexual partners , 12 ( 70.6% ) reported having casual partners , 11 ( 64.7% ) reported having anonymous partners and four ( 23.5% ) reported having group - sex encounters . there was no sexual - health messaging reported at informal venues ( table 2 ) . however , there was an awareness of sexual - health messaging in bars and clubs ( four of seven responses ) and in bathhouses , spas and sex clubs ( seven of nine responses ) in ottawa and outside of ottawa ( five of seven responses ) . the proportion of respondents aware and unaware of sexual - health messaging on internet sites was evenly split . respondents suggested that oph increase education regarding symptoms , testing and transmission of syphilis , as well as the importance of condom use for oral and anal sex . they suggested targeting specific social venues frequented by the gay population such as bars , internet sites and bathhouses . the respondents also stressed the need for messaging in other venues , such as public washrooms , to include men who have sex with both women and men . advertisements directed at the general public using television , radio and magazines were also suggested to ensure all populations are aware of the messaging . raising awareness and education of family and emergency room physicians about syphilis including the social venue information collected through routine surveillance , only 155 cases and contacts were linked by sexual intercourse in 55 components consisting of 18 single cases ; ( 11.6% of all cases and contacts ) ( figure 3 ) ; 23 dyads ( only two individuals linked ( 29.7% ) , six triads ( 11.6% ) , three tetrads ( 7.7% ) and components of six , eight , 12 and 16 each , with the largest being 19 individuals ( 12% ) ( figure 3 ) . of the 19 infectious syphilis cases coinfected with hiv , 63% were in small components of one or two cases and/or contacts . however , the potential for hiv transmission is illustrated in components eight and 14 ( marked by arrows ) in which cases who had been diagnosed with hiv were linked to hiv - negative individuals with syphilis or untested contacts . the 17 enhanced surveillance respondents reported 42 social venues , which included those already noted in the routine surveillance reports ; 16 social venues outside of ottawa and six internet sites . the most frequently reported social venue was a specific bathhouse in ottawa , named by six ( 35% ) of the respondents and followed by a popular gay internet site ( 29% ) . three different bathhouses in montreal ( quebec ) were identified as places to meet partners ( 24% ) . in addition , 16 venues in locations > 200 km away , such as florida and georgia ( usa ) and alberta were also named , comprising 38% of all venues . the above venue data were combined with the networks of 72 routine and enhanced surveillance cases and their 83 named contacts ( figure 4 ) . the network consisted of 45 components ranging in size from one to 95 nodes including 42 social venues . furthermore , the largest component , previously composed of only 19 nodes ( 12% ) , expanded three - fold to contain 18 cases and 40 contacts , representing 37% of the outbreak population ( figure 5 ) . importantly , a second case with hiv and a female contact who were previously in separate components were now included . contact tracing of sex partners for individuals with infectious syphilis is universally recommended for the education and treatment of contacts ; however , this intervention is limited to known individuals in a sexual relationship . the risk for acquiring an sti is associated with the individual s entire sexual network and not solely with the number of immediate and known sexual partners ( 9,11,12 ) . in this investigation , the construction of venue - based networks identified previously unknown links between a greater number of infectious syphilis cases and their sex partners , and increased connectivity among cases and contacts . this emphasized the concept of hiv and syphilis transmission through an entire sexual network of individuals , rather than as isolated cases for whom the source is unidentified . we found that more than one - third ( 37.4% ) of the 17 cases within the past six months who answered the enhanced surveillance questions patronized a specific bathhouse in ottawa and a website . this is a similar proportion to the findings of two previous social network analyses ( 8,9 ) in which 49% and 42% of cases and contacts were linked to one local venue . social network methods are valuable in sti investigation because they include the social context in which sexual intercourse occurs and serve as a proxy measure of homophily , where patronage of places and websites indicates shared , high - risk behaviours . in practice , focusing interventions on a few commonly patronized sites for sexual - health messaging and testing may have the advantage of both precision and efficiency . conversely , we noted that the men who reported recruiting sex partners > 200 km away were significantly more likely to report higher numbers of partners than those who named venues closer to ottawa ( kruskal - wallis , p<0.001 ) . this agrees with previous findings that individuals with sex partners recruited from long distances engage in higher risk behaviours than those with local sex partners ( 13,14 ) . the combination of sex partners locally and far away is ideal in the ability to catch rarer pathogens , such as hiv and syphilis , in a wide net and then transmit them locally ( 14,15 ) . hiv transmission is two to five times more likely in couples in whom one has concomitant syphilis ( 16 ) . concomitant hiv infection in individuals with syphilis has been reported in several recent outbreaks in canada ( 1721 ) ; however , the position in sexual networks of most of the hiv positive men was peripheral , limiting the spread of hiv ( 22 ) . futhermore , there were the two hiv - positive men in the large component of the social network from whom hiv may easily spread to the rest . first , the recording of information regarding venues where cases meet partners was valuable and should continue prospectively so to better serve prevention . second , thematic analysis of suggestions for interventions identified the need for more sexual - health messaging both at specific , named gay and nongay venues . finally , an increased need for education of physicians of both gay and heterosexual populations about syphilis symptoms , testing , transmission and the need to use condoms during oral and anal sex , was cited . oph re - established public health visits to the bathhouses to provide safe - sex practice information and referrals for syphilis testing . oph also expanded syphilis awareness campaigns , in particular the syphilis is back campaign , at the 2010 pride events , and educated front line health care providers by hosting a continuing medical education event covering syphilis basics , attended by 200 primary care providers . first , only 30% of the cases during the time period of interest were followed - up for enhanced surveillance . the cases who completed the enhanced syphilis surveillance questionnaire did not represent the 72 infectious syphilis cases during the investigation period in that they had higher numbers of partners reported , higher numbers of named contacts and lower proportions of coinfection with hiv . an additional limitation was missing sexual partner data , possibly due to poor recall , anonymous partners or unwillingness to identify the partner . however , sex partners who were forgotten have been shown to be similar to those remembered with regard to the proportions subsequently testing positive ( 23 ) . finally , verification of the accuracy of the responses regarding the presence of sexual messaging was not possible . the present investigation demonstrated that inclusion of social venues in network construction increases the connectivity between cases when compared with networks based solely on cases and sexual partners to include previously unknown links . finally , we found that social network analysis provided a greater understanding of syphilis transmission in the context of the national and international sexual connections , and an opportunity to focus prevention interventions at key locations .

in ottawa , ontario , the number of syphilis cases increased fivefold from 2001 to 2006 , with a further increase reported in 2009 . through the collection of surveillance data and the contruction of social networks , the authors sought to characterize the increase in syphilis rates . this study also identified risk factors indicative of transmission and provides a record of patients views on sexual - health messaging .

HISTORIQUE : OBJECTIF : MTHODOLOGIE : RSULTATS : CONCLUSION : METHODS Data analysis RESULTS Routinely collected case data from January 1, 2010 to June 15, 2010 Enhanced surveillance data Social network data analysis DISCUSSION

lincidence de syphilis ottawa , en ontario , a beaucoup augment depuis 2000 , passant de six cas sur 100 000 habitants en 2003 neuf cas sur 100 000 habitants en 2007 , puis 11 cas sur 100 000 habitants en 2010 . le nombre de cas signals pendant le premier trimestre de 2010 a plus que doubl par rapport celui du premier trimestre de 2009 . les chercheurs ont collig systmatiquement les donnes de surveillance sur la syphilis entre le 1 janvier 2009 et le 15 juillet 2010 et en ont tabli les rseaux sociaux partir dun questionnaire amlior sur les rseaux sociaux . au moyen du test de kruskal - wallis et des tests du chi carr , ils ont effectu des comparaisons univaries des donnes non distribues normalement entre le groupe de surveillance amliore et les autres cas de 2009 . dix - sept des 19 cas les plus rcents ont consenti rpondre au questionnaire , qui a rvl un usage peu frquent du condom , de multiples partenaires sexuels et des relations sexuelles avec des partenaires de mme sexe . les chercheurs ont transcrit sur un graphique linformation relative aux lieux o les partenaires sexuels se sont rencontrs , ce qui a permis de relier 18 cas et 40 contacts , soit 37 % de la population de lclosion , et dassocier de nombreux individus uniques et de dyades . la dcouverte des lieux o les partenaires sexuels se sont rencontrs sest rvle une mesure indirecte efficace des activits haut risque partages avec des personnes infectes et a dmontr le potentiel dintervenir seulement dans un bar prcis et un site internet donn pour joindre une forte proportion residents of ottawa diagnosed with infectious syphilis , defined according to national case definitions ( 10 ) from january 1 , 2009 to june 15 , 2010 , were included in the investigation ( n=72 ) . electronic records were retrieved from the integrated public health information system , they were then validated and supplementary information was added to each routine surveillance record . for example , contacts known only by an e - mail and sex - partner recruitment venues had not been added into the integrated public health information system but were added before the analysis , and greatly improved the social network links . out of concern for oph s relationship with the community of men having sex with men , community groups were consulted and approval was obtained from oph s research ethics board . twenty syphilis cases from january 2010 to june 2010 , which represented 27.7% of all syphilis cases , were contacted up to three times using two different methods ( telephone , letter , e - mail , home visits ) . these 20 cases were the most recent of all 72 cases and , therefore , were more likely to remember details than cases from 2009 . the questionnaire elicited types of sexual partners in the past 12 months including regular , casual and anonymous partners , sex trade clients and group - sex encounters . then , there were check boxes for casual , regular and paying partners , without definitions , with a space for participants to indicate how many for each type . respondents were prompted for visited venues including public venues ( including bars ) , clubs , bath houses , spas , sex clubs , raves , circuit parties , cruising sites or pick - up joints , work , school , prisons , parks , family or friends homes , internet sites , adult book and video stores , in ottawa or in other cities , where they met their sexual partners . respondents were also asked their opinions regarding actions needed to address the increase in syphilis infections . data obtained using enhanced surveillance were combined with demographic data , syphilis staging , hiv coinfection , behavioural risk factors , the names of sexual contacts and incidental free - text fields regarding locations where partners were met were collected as part of a routine case follow - up . some data regarding exposure and it is important to note that the enhanced cases had two opportunities to provide data regarding their sex partners . the incorporation of all routinely available data , together with enhanced data to show clustering around venues for sex partner recruitment , allowed for comparison with previous studies ( 8,9 ) . descriptive and univariate analysis of the data ( , student s t and kruskal wallis tests ) were performed using microsoft office excel version 7 ( microsoft corporation , usa ) and epi info 6.04 ( centres for disease control and prevention , usa ) . where numbers allowed , all cases were compared with the most recent cases for enhanced surveillance to demonstrate that the most recent sample represented the total population of syphilis cases in most respects . pajek version 1.27 ( pajek , slovenia ) was used to construct and analyze the social networks , where lines represented sexual contact between individuals , who were represented by nodes . a network of all cases and named contacts available from routine data collection was constructed , as well as a network of all cases and contacts who frequented common venues . the numbers and sizes of components were counted in which each person was connected to at least one other place or person . the number of links from one person to another immediately adjacent were also counted , which represented the total number of sex partners named by and who named an index case ( degree ) . where numbers allowed , all cases were compared with the most recent cases for enhanced surveillance to demonstrate that the most recent sample represented the total population of syphilis cases in most respects . pajek version 1.27 ( pajek , slovenia ) was used to construct and analyze the social networks , where lines represented sexual contact between individuals , who were represented by nodes . a network of all cases and named contacts available from routine data collection was constructed , as well as a network of all cases and contacts who frequented common venues . the numbers and sizes of components were counted in which each person was connected to at least one other place or person . the number of links from one person to another immediately adjacent were also counted , which represented the total number of sex partners named by and who named an index case ( degree ) . the epidemic curve of infectious syphilis cases during january 1 , 2009 to june 15 , 2010 was consistent with a propagated outbreak with person - to - person spread ( figure 2 ) . five of 19 individuals were diagnosed with hiv within one year of their syphilis diagnosis , while 14 cases were diagnosed with hiv more than one year before their diagnosis of syphilis . sixty - one ( 84.7% ) cases reported a total of 321 sexual partners ranging from one to 50 partners for each case , of whom only 69 ( 21.5% ) were named . the distribution of the number of named partners ( degree ) used in the network analysis revealed a highly skewed distribution , known as a small world network , in which 90% of respondents who named contacts had one to three partners in the past 12 months , while the remainder had four to 16 named partners . during routine case follow - up , respondents reported various sex practices including infrequent condom use , sex with a same - sex partner , a homosexual partner , a new partner in the past two months and multiple partners in the past six months ( table 1 ) . all respondents were men , resembling the majority ( 94.4% ) of routinely reported cases and were of similar ages and syphilis stages compared with the routinely notified group ( table 1 ) . one of the enhanced surveillance respondents ( 5.9% ) was coinfected with hiv , compared with coinfection in 19 ( 26.4% ) of the routine cases ( =6.59 ; p=0.01 ) . ethnic background for the 72 reported cases was not collected ; however , the majority of the cases who responded to the enhanced surveillance questionnaire were caucasian ( 82.4% ) . the 17 cases contacted for enhanced surveillance reported a total of 81 sexual partners , although only 17 ( 21.0% ) according to name , similar to the routinely reported cases . there was no sexual - health messaging reported at informal venues ( table 2 ) . however , there was an awareness of sexual - health messaging in bars and clubs ( four of seven responses ) and in bathhouses , spas and sex clubs ( seven of nine responses ) in ottawa and outside of ottawa ( five of seven responses ) . the proportion of respondents aware and unaware of sexual - health messaging on internet sites was evenly split . respondents suggested that oph increase education regarding symptoms , testing and transmission of syphilis , as well as the importance of condom use for oral and anal sex . they suggested targeting specific social venues frequented by the gay population such as bars , internet sites and bathhouses . the respondents also stressed the need for messaging in other venues , such as public washrooms , to include men who have sex with both women and men . advertisements directed at the general public using television , radio and magazines were also suggested to ensure all populations are aware of the messaging . raising awareness and education of family and emergency room physicians about syphilis was also identified . including the social venue information collected through routine surveillance , only 155 cases and contacts were linked by sexual intercourse in 55 components consisting of 18 single cases ; ( 11.6% of all cases and contacts ) ( figure 3 ) ; 23 dyads ( only two individuals linked ( 29.7% ) , six triads ( 11.6% ) , three tetrads ( 7.7% ) and components of six , eight , 12 and 16 each , with the largest being 19 individuals ( 12% ) ( figure 3 ) . of the 19 infectious syphilis cases coinfected with hiv , 63% were in small components of one or two cases and/or contacts . however , the potential for hiv transmission is illustrated in components eight and 14 ( marked by arrows ) in which cases who had been diagnosed with hiv were linked to hiv - negative individuals with syphilis or untested contacts . the 17 enhanced surveillance respondents reported 42 social venues , which included those already noted in the routine surveillance reports ; 16 social venues outside of ottawa and six internet sites . the most frequently reported social venue was a specific bathhouse in ottawa , named by six ( 35% ) of the respondents and followed by a popular gay internet site ( 29% ) . the above venue data were combined with the networks of 72 routine and enhanced surveillance cases and their 83 named contacts ( figure 4 ) . furthermore , the largest component , previously composed of only 19 nodes ( 12% ) , expanded three - fold to contain 18 cases and 40 contacts , representing 37% of the outbreak population ( figure 5 ) . importantly , a second case with hiv and a female contact who were previously in separate components were now included . seventy - two individuals met the infectious syphilis case definition from january 1 , 2009 to june 15 , 2010 ( table 1 ) . the epidemic curve of infectious syphilis cases during january 1 , 2009 to june 15 , 2010 was consistent with a propagated outbreak with person - to - person spread ( figure 2 ) . the majority ( 94.4% ) of reported cases almost one - half of the cases presented with secondary syphilis ( 44.4% ) and 19 ( 26.4% ) were hiv positive . five of 19 individuals were diagnosed with hiv within one year of their syphilis diagnosis , while 14 cases were diagnosed with hiv more than one year before their diagnosis of syphilis . sixty - one ( 84.7% ) cases reported a total of 321 sexual partners ranging from one to 50 partners for each case , of whom only 69 ( 21.5% ) were named . the distribution of the number of named partners ( degree ) used in the network analysis revealed a highly skewed distribution , known as a small world network , in which 90% of respondents who named contacts had one to three partners in the past 12 months , while the remainder had four to 16 named partners . cases who did not report contact information were similar to those who did , in sex and age ( mean of 46.2 and 39.6 years of age , respectively ; p=0.14 [ student s t test ] ) . during routine case follow - up , respondents reported various sex practices including infrequent condom use , sex with a same - sex partner , a homosexual partner , a new partner in the past two months and multiple partners in the past six months ( table 1 ) . all respondents were men , resembling the majority ( 94.4% ) of routinely reported cases and were of similar ages and syphilis stages compared with the routinely notified group ( table 1 ) . ethnic background for the 72 reported cases was not collected ; however , the majority of the cases who responded to the enhanced surveillance questionnaire were caucasian ( 82.4% ) . the 17 cases contacted for enhanced surveillance reported a total of 81 sexual partners , although only 17 ( 21.0% ) according to name , similar to the routinely reported cases . there was no sexual - health messaging reported at informal venues ( table 2 ) . however , there was an awareness of sexual - health messaging in bars and clubs ( four of seven responses ) and in bathhouses , spas and sex clubs ( seven of nine responses ) in ottawa and outside of ottawa ( five of seven responses ) . the proportion of respondents aware and unaware of sexual - health messaging on internet sites was evenly split . respondents suggested that oph increase education regarding symptoms , testing and transmission of syphilis , as well as the importance of condom use for oral and anal sex . they suggested targeting specific social venues frequented by the gay population such as bars , internet sites and bathhouses . the respondents also stressed the need for messaging in other venues , such as public washrooms , to include men who have sex with both women and men . raising awareness and education of family and emergency room physicians about syphilis including the social venue information collected through routine surveillance , only 155 cases and contacts were linked by sexual intercourse in 55 components consisting of 18 single cases ; ( 11.6% of all cases and contacts ) ( figure 3 ) ; 23 dyads ( only two individuals linked ( 29.7% ) , six triads ( 11.6% ) , three tetrads ( 7.7% ) and components of six , eight , 12 and 16 each , with the largest being 19 individuals ( 12% ) ( figure 3 ) . of the 19 infectious syphilis cases coinfected with hiv , 63% were in small components of one or two cases and/or contacts . however , the potential for hiv transmission is illustrated in components eight and 14 ( marked by arrows ) in which cases who had been diagnosed with hiv were linked to hiv - negative individuals with syphilis or untested contacts . the 17 enhanced surveillance respondents reported 42 social venues , which included those already noted in the routine surveillance reports ; 16 social venues outside of ottawa and six internet sites . the most frequently reported social venue was a specific bathhouse in ottawa , named by six ( 35% ) of the respondents and followed by a popular gay internet site ( 29% ) . three different bathhouses in montreal ( quebec ) were identified as places to meet partners ( 24% ) . in addition , 16 venues in locations > 200 km away , such as florida and georgia ( usa ) and alberta were also named , comprising 38% of all venues . the above venue data were combined with the networks of 72 routine and enhanced surveillance cases and their 83 named contacts ( figure 4 ) . furthermore , the largest component , previously composed of only 19 nodes ( 12% ) , expanded three - fold to contain 18 cases and 40 contacts , representing 37% of the outbreak population ( figure 5 ) . importantly , a second case with hiv and a female contact who were previously in separate components were now included . contact tracing of sex partners for individuals with infectious syphilis is universally recommended for the education and treatment of contacts ; however , this intervention is limited to known individuals in a sexual relationship . the risk for acquiring an sti is associated with the individual s entire sexual network and not solely with the number of immediate and known sexual partners ( 9,11,12 ) . in this investigation , the construction of venue - based networks identified previously unknown links between a greater number of infectious syphilis cases and their sex partners , and increased connectivity among cases and contacts . we found that more than one - third ( 37.4% ) of the 17 cases within the past six months who answered the enhanced surveillance questions patronized a specific bathhouse in ottawa and a website . this is a similar proportion to the findings of two previous social network analyses ( 8,9 ) in which 49% and 42% of cases and contacts were linked to one local venue . social network methods are valuable in sti investigation because they include the social context in which sexual intercourse occurs and serve as a proxy measure of homophily , where patronage of places and websites indicates shared , high - risk behaviours . in practice , focusing interventions on a few commonly patronized sites for sexual - health messaging and testing may have the advantage of both precision and efficiency . conversely , we noted that the men who reported recruiting sex partners > 200 km away were significantly more likely to report higher numbers of partners than those who named venues closer to ottawa ( kruskal - wallis , p<0.001 ) . this agrees with previous findings that individuals with sex partners recruited from long distances engage in higher risk behaviours than those with local sex partners ( 13,14 ) . concomitant hiv infection in individuals with syphilis has been reported in several recent outbreaks in canada ( 1721 ) ; however , the position in sexual networks of most of the hiv positive men was peripheral , limiting the spread of hiv ( 22 ) . futhermore , there were the two hiv - positive men in the large component of the social network from whom hiv may easily spread to the rest . first , the recording of information regarding venues where cases meet partners was valuable and should continue prospectively so to better serve prevention . second , thematic analysis of suggestions for interventions identified the need for more sexual - health messaging both at specific , named gay and nongay venues . finally , an increased need for education of physicians of both gay and heterosexual populations about syphilis symptoms , testing , transmission and the need to use condoms during oral and anal sex , was cited . oph also expanded syphilis awareness campaigns , in particular the syphilis is back campaign , at the 2010 pride events , and educated front line health care providers by hosting a continuing medical education event covering syphilis basics , attended by 200 primary care providers . first , only 30% of the cases during the time period of interest were followed - up for enhanced surveillance . the cases who completed the enhanced syphilis surveillance questionnaire did not represent the 72 infectious syphilis cases during the investigation period in that they had higher numbers of partners reported , higher numbers of named contacts and lower proportions of coinfection with hiv . finally , verification of the accuracy of the responses regarding the presence of sexual messaging was not possible . the present investigation demonstrated that inclusion of social venues in network construction increases the connectivity between cases when compared with networks based solely on cases and sexual partners to include previously unknown links . finally , we found that social network analysis provided a greater understanding of syphilis transmission in the context of the national and international sexual connections , and an opportunity to focus prevention interventions at key locations .

local recurrent prostate cancer after primary radiotherapy is a significant clinical problem . approximately , half of all patients with high - risk prostate cancer experience a recurrence within 10 years post treatment [ 1 , 2 ] . androgen deprivation is the most common treatment for biochemical recurrence post radiotherapy , as whole gland salvage treatments are associated with significant side effects resulting in a negative impact on the quality of life [ 4 , 5 ] . new imaging techniques , like f choline positron emission tomography ( pet ) and ga - prostate specific membrane antigen ( psma ) pet - computed tomography ( ct ) [ 6 , 7 ] , enable early detection of true local prostate cancer recurrences . when registering this information with multiparametric ( mp ) magnetic resonance imaging ( mri ) , which has a higher resolution compared to pet , safe local salvage treatment is possible [ 8 , 10 , 11 ] . as primary salvage low - dose - rate brachytherapy ( ldr - bt ) has shown to be safe and feasible , one can consider to perform a second salvage treatment in a selected group of patients ( > 3-year interval between treatment , prostate - specific antigen [ psa ] doubling time > 1 year and acceptable toxicity after 1 salvage treatment ) [ 4 , 12 ] . with the added damage from the primary and salvage treatment to the normal tissue , a third treatment obviously has an increased risk of side effects . the aim of this study is to describe a 2 focal salvage 19 gy single fraction hdr - bt in 4 patients , together with data on toxicity and biochemical outcome . institutional review board approval for this analysis was obtained . between may 2015 and january 2016 , four patients with a second locally recurrent prostate cancer were treated in an outpatient setting with an mri - guided focal hdr - bt technique . the different parameters are described at time of primary treatment ( 1 ) , first salvage ( 2 ) or second salvage ( 3 ) . brachytherapy , ebrt external beam radiotherapy , imrt intensity modulated radiotherapy , fractions . all primary and secondary recurrences were pathologically proven through biopsies ( systematic trus - guided biopsies for primary recurrences and guided biopsies through fusion of diagnostic mp - mri and ultrasound images for secondary recurrences ) . staging before primary treatment was based on ( staging 1 ) digital rectal examination and transrectal ultrasound . staging of the first and second recurrences ( staging 2 and 3 ) were based on mri prior to the second salvage hdr - bt treatment , no patient received any hormonal therapy . all patients underwent choline pet - ct or ga - psma - pet - ct , to exclude regional and/or distant metastasis and a cross sectional mp - mri . the diagnostic mp - mri protocol included a t1-weighted fast gradient echo sequence ( thrive ) , t2-weighted turbo spin echo sequence ( tse ) , balanced turbo gradient echo sequence with spectral fat suppression ( btfe spair ) , diffusion weighted imaging ( dwi ) , and a dynamic contrast enhanced ( dce ) sequence . all primary and secondary recurrences were pathologically proven through biopsies ( systematic trus - guided biopsies for primary recurrences and guided biopsies through fusion of diagnostic mp - mri and ultrasound images for secondary recurrences ) . at the time of salvage treatment , psa was below 10 ng / ml , psa doubling time was one year or more , and the interval between 1 and 2 salvage treatment was 3 years or more . all patients showed gastrointestinal ( gi ) or genitourinary ( gu ) toxicity rates of 2 ( ctcae version 4.0 ) from the previous treatments . the pre - treatment mp 3 t mri ( sequences described above ) was used to delineate the prostate , gross tumor volume ( gtv ) , and surrounding organs at risk ( oar ) . as opposed to the first salvage prostate brachytherapy , no margins around the gtv were applied to reduce the risk of inducing toxicity . a pre - plan was made to estimate whether the salvage procedure would be feasible with regards to the risk of severe toxicity . the pre - plan was considered feasible if dose of oar was below the prescription ( d1cc rectum / bladder < 12 gy , d10% urethra < 17.7 gy ) , independent of any overlap with the first or second treatment . all patients were treated with a single fraction of 19 gy in a shielded room , equipped with a 1.5 t mr scanner that was suited for salvage mri - guided hdr - bt . on the day of treatment , patients received spinal anesthesia on the mri table and a urinary catheter was inserted to optimize urethra visualization . with the patient in lithotomy position , the regions of interest of the diagnostic 3 t mp - mri were registered ( using rigid fusion ) with the intraoperative ultrasound . next , brachytherapy catheters were inserted into the target area ( gtv ) under ultrasound guidance with a transperineal approach . subsequently , the ultrasound equipment was removed and an intra - operative 1.5 t mri was made for treatment planning . the intra - operative mri consisted of a transversal t2-weighted turbo spin echo ( tse ) images and 2 series of 3d balanced turbo gradient echo images , one with spair ( spectral attenuated inversion recovery ) and one with spir ( spectral presaturation with inversion recovery ) . next , an intra - operative simulation of dose distribution to the gtv and oar was made . if constraints for the oar were met , patients received a single fraction of 19 gy to > 95% of the gtv . however , if the dose constraints for the oar were exceeded , the dose to the gtv was reduced . as no literature on second salvage dose prescription is available , dose constraints based on holly et al . were used , for rectum and bladder a d1cc < 12 gy , for urethra a d10% < 17.7 gy . also , post - implant mri sequences ( same sequences as the intra - operative mri ) were performed to assess dose differences caused by intra - operative catheter shifts . toxicity was reported according to the common toxicity criteria of adverse events ( ctcae ) version 4 . follow - up , toxicity assessment , and psa measurements were performed at 1 month and every 3 months during the first year after treatment , every 6 months during the second year after treatment , and annually thereafter . institutional review board approval for this analysis was obtained . between may 2015 and january 2016 , four patients with a second locally recurrent prostate cancer were treated in an outpatient setting with an mri - guided focal hdr - bt technique . the different parameters are described at time of primary treatment ( 1 ) , first salvage ( 2 ) or second salvage ( 3 ) . brachytherapy , ebrt external beam radiotherapy , imrt intensity modulated radiotherapy , fractions . all primary and secondary recurrences were pathologically proven through biopsies ( systematic trus - guided biopsies for primary recurrences and guided biopsies through fusion of diagnostic mp - mri and ultrasound images for secondary recurrences ) . staging before primary treatment was based on ( staging 1 ) digital rectal examination and transrectal ultrasound . staging of the first and second recurrences ( staging 2 and 3 ) were based on mri prior to the second salvage hdr - bt treatment , no patient received any hormonal therapy . all patients underwent choline pet - ct or ga - psma - pet - ct , to exclude regional and/or distant metastasis and a cross sectional mp - mri . the diagnostic mp - mri protocol included a t1-weighted fast gradient echo sequence ( thrive ) , t2-weighted turbo spin echo sequence ( tse ) , balanced turbo gradient echo sequence with spectral fat suppression ( btfe spair ) , diffusion weighted imaging ( dwi ) , and a dynamic contrast enhanced ( dce ) sequence . all primary and secondary recurrences were pathologically proven through biopsies ( systematic trus - guided biopsies for primary recurrences and guided biopsies through fusion of diagnostic mp - mri and ultrasound images for secondary recurrences ) . at the time of salvage treatment , psa was below 10 ng / ml , psa doubling time was one year or more , and the interval between 1 and 2 salvage treatment was 3 years or more . all patients showed gastrointestinal ( gi ) or genitourinary ( gu ) toxicity rates of 2 ( ctcae version 4.0 ) from the previous treatments . the pre - treatment mp 3 t mri ( sequences described above ) was used to delineate the prostate , gross tumor volume ( gtv ) , and surrounding organs at risk ( oar ) . as opposed to the first salvage prostate brachytherapy , no margins around the gtv were applied to reduce the risk of inducing toxicity . a pre - plan was made to estimate whether the salvage procedure would be feasible with regards to the risk of severe toxicity . the pre - plan was considered feasible if dose of oar was below the prescription ( d1cc rectum / bladder < 12 gy , d10% urethra < 17.7 gy ) , independent of any overlap with the first or second treatment . all patients were treated with a single fraction of 19 gy in a shielded room , equipped with a 1.5 t mr scanner that was suited for salvage mri - guided hdr - bt . on the day of treatment , patients received spinal anesthesia on the mri table and a urinary catheter was inserted to optimize urethra visualization . with the patient in lithotomy position , the regions of interest of the diagnostic 3 t mp - mri were registered ( using rigid fusion ) with the intraoperative ultrasound . next , brachytherapy catheters were inserted into the target area ( gtv ) under ultrasound guidance with a transperineal approach . subsequently , the ultrasound equipment was removed and an intra - operative 1.5 t mri was made for treatment planning . the intra - operative mri consisted of a transversal t2-weighted turbo spin echo ( tse ) images and 2 series of 3d balanced turbo gradient echo images , one with spair ( spectral attenuated inversion recovery ) and one with spir ( spectral presaturation with inversion recovery ) . next , an intra - operative simulation of dose distribution to the gtv and oar was made . if constraints for the oar were met , patients received a single fraction of 19 gy to > 95% of the gtv . however , if the dose constraints for the oar were exceeded , the dose to the gtv was reduced . as no literature on second salvage dose prescription is available , dose constraints based on holly et al . were used , for rectum and bladder a d1cc < 12 gy , for urethra a d10% < 17.7 gy . also , post - implant mri sequences ( same sequences as the intra - operative mri ) were performed to assess dose differences caused by intra - operative catheter shifts . toxicity was reported according to the common toxicity criteria of adverse events ( ctcae ) version 4 . follow - up , toxicity assessment , and psa measurements were performed at 1 month and every 3 months during the first year after treatment , every 6 months during the second year after treatment , and annually thereafter . the median follow - up time for patients treated with a second focal salvage hdr - bt was 12 months ( range , 6 - 15 months ) ( table 1 ) . the age of the treated patients ranged from 63 to 72 years . at time of primary treatment , two patients had a t1c , one patient a t2 , and one patient a t3a disease . three patients received i brachytherapy , and one patient external beam radiation therapy ( ebrt ) as primary treatment . the median time to the first relapse was 7 years , with a range of 5 - 8 years . at time of first salvage treatment , all patients had local recurrences within the prostate . for the first salvage treatment , all patients were treated with focal salvage i brachytherapy . median time to second salvage was 4 years ( range , 3 - 6 years ) . at the time of secondary salvage treatment , all 4 patients had locally recurrent prostatic cancer . however , for one patient there was seminal vesicle extension ( it3b ) . at time of secondary salvage treatment , median psa was 3.75 ng / ml ( range , 3.0 - 5.3 ng / ml ) . prostate volumes ranged from 25.7 - 47.0 cc , gtv ranged from 1.9 to 6.6 cc . for 3 of the 4 patients , the d95 gtv was lower ( 15.5 gy ) because the gtv was in close proximity to the rectal wall . this structure had already received a high cumulative dose from earlier radiation treatments . the constraints for rectum , bladder , and urethra gray , gtv gross tumor volume , d95 gtv dose in 95% of the gtv , d1cc dose in 1 cc of the bladder / rectum , d10% dose in 10% of the urethra volume at baseline ( before second salvage treatment ) , 2 patients had gi grade 1 toxicity , and 2 patients had gi grade 0 toxicity . one patient had grade 1 gu toxicity , and 3 patients grade 0 gu toxicity . regarding erectile dysfunction ( ed ) , one patient had grade 0 , one grade 1 , one grade 2 , and one grade 3 toxicity . all baseline toxicity scores prior to second focal salvage hdr - bt did not worsen during follow - up , except for one patient who developed grade 1 ed after treatment . for both gu and gi toxicity , prostate - specific antigen curves of all 4 patients are shown in figure 1 . for 3 patients , two patients had a biochemical recurrence as per the phoenix definition ( psa rise of 2 ng / ml above nadir ) . in one of these patients , a lymph node metastasis was detected during follow - up using ga - psma . in the other two patients , so far no local recurrences or distant metastasis have been detected . in the one patient who did not receive the prescribed gtv dose of 19 gy the median follow - up time for patients treated with a second focal salvage hdr - bt was 12 months ( range , 6 - 15 months ) ( table 1 ) . the age of the treated patients ranged from 63 to 72 years . at time of primary treatment , two patients had a t1c , one patient a t2 , and one patient a t3a disease . three patients received i brachytherapy , and one patient external beam radiation therapy ( ebrt ) as primary treatment . the median time to the first relapse was 7 years , with a range of 5 - 8 years . at time of first salvage treatment , all patients had local recurrences within the prostate . for the first salvage treatment , all patients were treated with focal salvage i brachytherapy . median time to second salvage was 4 years ( range , 3 - 6 years ) . at the time of secondary salvage treatment , all 4 patients had locally recurrent prostatic cancer . however , for one patient there was seminal vesicle extension ( it3b ) . at time of secondary salvage treatment , median psa was 3.75 ng / ml ( range , 3.0 - 5.3 ng / ml ) . prostate volumes ranged from 25.7 - 47.0 cc , gtv ranged from 1.9 to 6.6 cc . for 3 of the 4 patients , the d95 gtv was lower ( 15.5 gy ) because the gtv was in close proximity to the rectal wall . this structure had already received a high cumulative dose from earlier radiation treatments . the constraints for rectum , bladder , and urethra gray , gtv gross tumor volume , d95 gtv dose in 95% of the gtv , d1cc dose in 1 cc of the bladder / rectum , d10% dose in 10% of the urethra volume at baseline ( before second salvage treatment ) , 2 patients had gi grade 1 toxicity , and 2 patients had gi grade 0 toxicity . one patient had grade 1 gu toxicity , and 3 patients grade 0 gu toxicity . regarding erectile dysfunction ( ed ) , one patient had grade 0 , one grade 1 , one grade 2 , and one grade 3 toxicity . all baseline toxicity scores prior to second focal salvage hdr - bt did not worsen during follow - up , except for one patient who developed grade 1 ed after treatment . for both gu and gi toxicity , prostate - specific antigen curves of all 4 patients are shown in figure 1 . for 3 patients , two patients had a biochemical recurrence as per the phoenix definition ( psa rise of 2 ng / ml above nadir ) . in one of these patients , a lymph node metastasis was detected during follow - up using ga - psma . in the other two patients , so far no local recurrences or distant metastasis have been detected . in the one patient who did not receive the prescribed gtv dose of 19 gy this study shows that a second salvage treatment is feasible with regards to acute toxicity . however , median follow - up is limited to 12 months and late toxicity may be underestimated . to our knowledge , there is no literature regarding secondary salvage treatments performed by other treatment modalities . ga - psma scans or choline pet scans are considered to have a high sensitivity to detect a local recurrence [ 6 , 7 ] . the mp - mri has optimal soft tissue contrast , and also has a high negative and positive predictive value for clinically significant prostate cancer [ 9 , 14 ] . beside these aspects , the mp - mri has superior resolution when compared to the ga - psma scan . a high resolution is of great importance when delineating the target area ( gtv ) and the oar in preparation for focal salvage hdr - bt . furthermore , there is high conformity between the area of recurrence and the treated primary index lesion . by reducing the rectum and bladder dose through focal salvage brachytherapy , it can be expected that toxicity will decrease when compared to whole gland salvage treatments [ 5 , 10 ] . as results for salvage modalities for both tumor control and toxicity are promising , a second salvage treatment can be considered in a highly selected group of patients with locally recurrent prostate cancer . in the same light as the first salvage treatment , the goal of a second salvage treatment is to further postpone androgen deprivation therapy , with its known deterioration on the quality of life [ 4 , 17 , 18 ] . the current availability of ga - psma scans also plays a key role in the selection of true locally recurrent prostate cancer in patients with a low psa . this allows for earlier treatment of these patients with possible smaller chance of metastasis from the index lesion . prognostic factors are known for outcome after first ( whole - gland ) salvage treatment . in multivariable analysis , disease - free survival interval ( dfsi ) after primary therapy and pre - salvage psa - doubling time were predictors of biochemical failure . these factors may be used for second salvage treatment , since the literature does not describe predictors for outcomes in this scenario . in this study , no margin was applied to the gtv , as rectum and bladder already received radiation twice . it is not known whether there was microscopic spread around the gtv , but such a microscopic spread seems likely . with a margin of 0 mm , we still expect some dose to be delivered to the first few mms around the gtv . as the area of microscopic spread will need less dose compared to the gtv due to a lower tumor density , we agreed on reducing the ctv and ptv margin to 0 mm around the gtv . longer follow - up is necessary to determine if prostate cancer will recur adjacent , or perhaps even within the gtv after second salvage treatment . for the 1 salvage ldr - bt treatment , dose restrictions for bladder , urethra , and rectum have been published to minimize the risk of late severe toxicity [ 20 , 21 ] , namely a bladder d2cc < 70 gy , a urethral v100 < 0.4 cc and a rectal d0.1cc < 160 gy . unfortunately , no such restrictions are available for salvage hdr - bt . as an accurate spatial dose delivery in salvage treatments is essential , i seeds have the tendency to migrate from their original position and this phenomenon may have a negative effect on the intended dose delivery to the gtv . a shift in catheters in focal hdr - bt will also contribute to a suboptimal dose to the gtv , especially since in bt there is a steep dose gradient . with self - anchoring catheters , there is minimal displacement of the catheter tips , and it seems that these shifts have a minimal impact on dosimetry . an advantage of hdr - bt compared to ldr - bt is that the dwell positions and times of the source can be regulated . with hdr - bt , more homogeneous dose to the gtv can be delivered and it is often possible to better modulate the dose around the adjacent oar when compared to ldr - bt . there is a need for second salvage hdr - bt dose restriction values , since it is questionable whether the first salvage dose restriction parameters can be used for a second salvage treatment . in the current situation , this will be difficult to accomplish , as events ( grade 3 gu and gi toxicity ) and number of treated patients are low . furthermore , determination of the cumulative dose may be difficult , since a different treatment modality was used as primary treatment . ( for rectum and bladder d1cc < 12 gy , for urethra d10% < 17.7 gy ) may be considered in clinical practice for both 1 and 2 salvage hdr - bt . the area of recurrence for the four 2 salvage patients described in this study was 1 infield and 3 out of field . for each patient , the infield or out of field recurrences the infield recurrence after the primary salvage treatment may have been caused by inadequate spacing of the i seeds and/or by an aggressive tumor . the out of field recurrences may be explained by the fact that psma scans were not yet available at the time when patients underwent a primary salvage treatment . out of field recurrences treated with a 2 salvage hdr - bt will most probably have less impact on the oar , since the accumulated dose for the specific oar will generally be lower than with an infield recurrence . perhaps in or out of field 2 recurrences can be taken into account when contemplating a 2 salvage hdr - bt , and it may be an important selection criteria in future predictive models . in 2 of 4 patients , there was a biochemical recurrence ( phoenix criteria ) during follow - up ( figure 1 ) . although follow - up is still limited , it can not be concluded whether patient selection had been appropriate . one patient showed a single nodal distant metastasis on a psma scan after biochemical recurrence , which was not present on the pre - treatment psma scan . one patient did not receive the prescribed dose to the gtv , and also shows psa increase after treatment , although biochemical recurrence according to the phoenix definition has not been reached . the increasing psa in two of the patients may also be explained by a psa bounce , as the median follow - up in this study is only 12 months . in the study of mehta et al . , psa bounce was noticed after an average of 9 months . there is no literature that describes psa bounce after a 2 salvage treatment . concluding , focal second salvage hdr - bt for locally recurrent prostate cancer is feasible in a highly selected group of patients , with a secondary local recurrence , minimal gi and gu toxicity , low psa values at time of second salvage , and an interval of more than 3 years after earlier treatments . this approach may be used in the future to postpone hormonal therapy and positively influence the quality of life for patients with a 2 locally recurrent prostate cancer .

purposesalvage treatments for localized radiorecurrent prostate cancer can be performed safely when a focal and image guided approach is used . due to the low toxicity , the opportunity exists to investigate a second salvage treatment when a second locally recurrent prostate cancer occurs . here , we describe a second salvage treatment procedure of 4 patients.material and methodsfour patients with a pathologically proven second local recurrence were treated in an outpatient magnetic resonance imaging ( mri)-guided setting with a single fraction of 19 gy focal high - dose - rate brachytherapy ( hdr - bt ) . delineation was performed using choline - pet - ct or a 68ga - psma pet in combination with multiparametric 3 tesla mri in all four patients . toxicity was measured using common toxicity criteria for adverse events ( ctcae ) version 4.0.resultswith a median follow - up of 12 months ( range , 6 - 15 ) , there were 2 patients with biochemical recurrence as defined by the phoenix - definition . there were no patients with grade 3 or more toxicity . in all second salvage hdr - bt treatments , the constraints for rectum , bladder , and urethra were met . median treatment volume ( gtv ) was 4.8 cc ( range , 1.9 - 6.6 cc ) . a median of 8 catheters ( range , 6 - 9 ) were used , and the median dose to the treatment volume ( gtv ) was a d95 : 19.3 gy ( sd 15.5 - 19.4 gy).conclusionssecond focal salvage mri - guided hdr - bt for a select group of patients with a second locally recurrent prostate cancer is feasible . there was no grade 3 or more acute toxicity for these four patients .

Purpose Material and methods Patients Treatment procedures Endpoints and follow-up Results Baseline characteristics Dosimetry Toxicity Biochemical outcomes Discussion Conclusions Disclosure

local recurrent prostate cancer after primary radiotherapy is a significant clinical problem . approximately , half of all patients with high - risk prostate cancer experience a recurrence within 10 years post treatment [ 1 , 2 ] . new imaging techniques , like f choline positron emission tomography ( pet ) and ga - prostate specific membrane antigen ( psma ) pet - computed tomography ( ct ) [ 6 , 7 ] , enable early detection of true local prostate cancer recurrences . when registering this information with multiparametric ( mp ) magnetic resonance imaging ( mri ) , which has a higher resolution compared to pet , safe local salvage treatment is possible [ 8 , 10 , 11 ] . as primary salvage low - dose - rate brachytherapy ( ldr - bt ) has shown to be safe and feasible , one can consider to perform a second salvage treatment in a selected group of patients ( > 3-year interval between treatment , prostate - specific antigen [ psa ] doubling time > 1 year and acceptable toxicity after 1 salvage treatment ) [ 4 , 12 ] . with the added damage from the primary and salvage treatment to the normal tissue , a third treatment obviously has an increased risk of side effects . the aim of this study is to describe a 2 focal salvage 19 gy single fraction hdr - bt in 4 patients , together with data on toxicity and biochemical outcome . between may 2015 and january 2016 , four patients with a second locally recurrent prostate cancer were treated in an outpatient setting with an mri - guided focal hdr - bt technique . the different parameters are described at time of primary treatment ( 1 ) , first salvage ( 2 ) or second salvage ( 3 ) . all primary and secondary recurrences were pathologically proven through biopsies ( systematic trus - guided biopsies for primary recurrences and guided biopsies through fusion of diagnostic mp - mri and ultrasound images for secondary recurrences ) . staging of the first and second recurrences ( staging 2 and 3 ) were based on mri prior to the second salvage hdr - bt treatment , no patient received any hormonal therapy . all patients underwent choline pet - ct or ga - psma - pet - ct , to exclude regional and/or distant metastasis and a cross sectional mp - mri . the diagnostic mp - mri protocol included a t1-weighted fast gradient echo sequence ( thrive ) , t2-weighted turbo spin echo sequence ( tse ) , balanced turbo gradient echo sequence with spectral fat suppression ( btfe spair ) , diffusion weighted imaging ( dwi ) , and a dynamic contrast enhanced ( dce ) sequence . all primary and secondary recurrences were pathologically proven through biopsies ( systematic trus - guided biopsies for primary recurrences and guided biopsies through fusion of diagnostic mp - mri and ultrasound images for secondary recurrences ) . at the time of salvage treatment , psa was below 10 ng / ml , psa doubling time was one year or more , and the interval between 1 and 2 salvage treatment was 3 years or more . all patients showed gastrointestinal ( gi ) or genitourinary ( gu ) toxicity rates of 2 ( ctcae version 4.0 ) from the previous treatments . the pre - treatment mp 3 t mri ( sequences described above ) was used to delineate the prostate , gross tumor volume ( gtv ) , and surrounding organs at risk ( oar ) . a pre - plan was made to estimate whether the salvage procedure would be feasible with regards to the risk of severe toxicity . all patients were treated with a single fraction of 19 gy in a shielded room , equipped with a 1.5 t mr scanner that was suited for salvage mri - guided hdr - bt . with the patient in lithotomy position , the regions of interest of the diagnostic 3 t mp - mri were registered ( using rigid fusion ) with the intraoperative ultrasound . next , brachytherapy catheters were inserted into the target area ( gtv ) under ultrasound guidance with a transperineal approach . subsequently , the ultrasound equipment was removed and an intra - operative 1.5 t mri was made for treatment planning . if constraints for the oar were met , patients received a single fraction of 19 gy to > 95% of the gtv . however , if the dose constraints for the oar were exceeded , the dose to the gtv was reduced . were used , for rectum and bladder a d1cc < 12 gy , for urethra a d10% < 17.7 gy . also , post - implant mri sequences ( same sequences as the intra - operative mri ) were performed to assess dose differences caused by intra - operative catheter shifts . toxicity was reported according to the common toxicity criteria of adverse events ( ctcae ) version 4 . follow - up , toxicity assessment , and psa measurements were performed at 1 month and every 3 months during the first year after treatment , every 6 months during the second year after treatment , and annually thereafter . between may 2015 and january 2016 , four patients with a second locally recurrent prostate cancer were treated in an outpatient setting with an mri - guided focal hdr - bt technique . the different parameters are described at time of primary treatment ( 1 ) , first salvage ( 2 ) or second salvage ( 3 ) . all primary and secondary recurrences were pathologically proven through biopsies ( systematic trus - guided biopsies for primary recurrences and guided biopsies through fusion of diagnostic mp - mri and ultrasound images for secondary recurrences ) . staging of the first and second recurrences ( staging 2 and 3 ) were based on mri prior to the second salvage hdr - bt treatment , no patient received any hormonal therapy . all patients underwent choline pet - ct or ga - psma - pet - ct , to exclude regional and/or distant metastasis and a cross sectional mp - mri . the diagnostic mp - mri protocol included a t1-weighted fast gradient echo sequence ( thrive ) , t2-weighted turbo spin echo sequence ( tse ) , balanced turbo gradient echo sequence with spectral fat suppression ( btfe spair ) , diffusion weighted imaging ( dwi ) , and a dynamic contrast enhanced ( dce ) sequence . all primary and secondary recurrences were pathologically proven through biopsies ( systematic trus - guided biopsies for primary recurrences and guided biopsies through fusion of diagnostic mp - mri and ultrasound images for secondary recurrences ) . at the time of salvage treatment , psa was below 10 ng / ml , psa doubling time was one year or more , and the interval between 1 and 2 salvage treatment was 3 years or more . all patients showed gastrointestinal ( gi ) or genitourinary ( gu ) toxicity rates of 2 ( ctcae version 4.0 ) from the previous treatments . the pre - treatment mp 3 t mri ( sequences described above ) was used to delineate the prostate , gross tumor volume ( gtv ) , and surrounding organs at risk ( oar ) . a pre - plan was made to estimate whether the salvage procedure would be feasible with regards to the risk of severe toxicity . the pre - plan was considered feasible if dose of oar was below the prescription ( d1cc rectum / bladder < 12 gy , d10% urethra < 17.7 gy ) , independent of any overlap with the first or second treatment . all patients were treated with a single fraction of 19 gy in a shielded room , equipped with a 1.5 t mr scanner that was suited for salvage mri - guided hdr - bt . with the patient in lithotomy position , the regions of interest of the diagnostic 3 t mp - mri were registered ( using rigid fusion ) with the intraoperative ultrasound . next , brachytherapy catheters were inserted into the target area ( gtv ) under ultrasound guidance with a transperineal approach . next , an intra - operative simulation of dose distribution to the gtv and oar was made . if constraints for the oar were met , patients received a single fraction of 19 gy to > 95% of the gtv . however , if the dose constraints for the oar were exceeded , the dose to the gtv was reduced . were used , for rectum and bladder a d1cc < 12 gy , for urethra a d10% < 17.7 gy . toxicity was reported according to the common toxicity criteria of adverse events ( ctcae ) version 4 . follow - up , toxicity assessment , and psa measurements were performed at 1 month and every 3 months during the first year after treatment , every 6 months during the second year after treatment , and annually thereafter . the median follow - up time for patients treated with a second focal salvage hdr - bt was 12 months ( range , 6 - 15 months ) ( table 1 ) . three patients received i brachytherapy , and one patient external beam radiation therapy ( ebrt ) as primary treatment . the median time to the first relapse was 7 years , with a range of 5 - 8 years . at time of first salvage treatment , all patients had local recurrences within the prostate . for the first salvage treatment , all patients were treated with focal salvage i brachytherapy . median time to second salvage was 4 years ( range , 3 - 6 years ) . at the time of secondary salvage treatment , all 4 patients had locally recurrent prostatic cancer . however , for one patient there was seminal vesicle extension ( it3b ) . at time of secondary salvage treatment , median psa was 3.75 ng / ml ( range , 3.0 - 5.3 ng / ml ) . prostate volumes ranged from 25.7 - 47.0 cc , gtv ranged from 1.9 to 6.6 cc . for 3 of the 4 patients , the d95 gtv was lower ( 15.5 gy ) because the gtv was in close proximity to the rectal wall . the constraints for rectum , bladder , and urethra gray , gtv gross tumor volume , d95 gtv dose in 95% of the gtv , d1cc dose in 1 cc of the bladder / rectum , d10% dose in 10% of the urethra volume at baseline ( before second salvage treatment ) , 2 patients had gi grade 1 toxicity , and 2 patients had gi grade 0 toxicity . one patient had grade 1 gu toxicity , and 3 patients grade 0 gu toxicity . regarding erectile dysfunction ( ed ) , one patient had grade 0 , one grade 1 , one grade 2 , and one grade 3 toxicity . all baseline toxicity scores prior to second focal salvage hdr - bt did not worsen during follow - up , except for one patient who developed grade 1 ed after treatment . for both gu and gi toxicity , prostate - specific antigen curves of all 4 patients are shown in figure 1 . for 3 patients , two patients had a biochemical recurrence as per the phoenix definition ( psa rise of 2 ng / ml above nadir ) . in one of these patients , a lymph node metastasis was detected during follow - up using ga - psma . in the one patient who did not receive the prescribed gtv dose of 19 gy the median follow - up time for patients treated with a second focal salvage hdr - bt was 12 months ( range , 6 - 15 months ) ( table 1 ) . three patients received i brachytherapy , and one patient external beam radiation therapy ( ebrt ) as primary treatment . the median time to the first relapse was 7 years , with a range of 5 - 8 years . for the first salvage treatment , all patients were treated with focal salvage i brachytherapy . median time to second salvage was 4 years ( range , 3 - 6 years ) . at the time of secondary salvage treatment , all 4 patients had locally recurrent prostatic cancer . at time of secondary salvage treatment , median psa was 3.75 ng / ml ( range , 3.0 - 5.3 ng / ml ) . prostate volumes ranged from 25.7 - 47.0 cc , gtv ranged from 1.9 to 6.6 cc . for 3 of the 4 patients , the d95 gtv was lower ( 15.5 gy ) because the gtv was in close proximity to the rectal wall . the constraints for rectum , bladder , and urethra gray , gtv gross tumor volume , d95 gtv dose in 95% of the gtv , d1cc dose in 1 cc of the bladder / rectum , d10% dose in 10% of the urethra volume at baseline ( before second salvage treatment ) , 2 patients had gi grade 1 toxicity , and 2 patients had gi grade 0 toxicity . one patient had grade 1 gu toxicity , and 3 patients grade 0 gu toxicity . regarding erectile dysfunction ( ed ) , one patient had grade 0 , one grade 1 , one grade 2 , and one grade 3 toxicity . all baseline toxicity scores prior to second focal salvage hdr - bt did not worsen during follow - up , except for one patient who developed grade 1 ed after treatment . for both gu and gi toxicity , prostate - specific antigen curves of all 4 patients are shown in figure 1 . for 3 patients , two patients had a biochemical recurrence as per the phoenix definition ( psa rise of 2 ng / ml above nadir ) . in one of these patients , a lymph node metastasis was detected during follow - up using ga - psma . in the one patient who did not receive the prescribed gtv dose of 19 gy this study shows that a second salvage treatment is feasible with regards to acute toxicity . however , median follow - up is limited to 12 months and late toxicity may be underestimated . ga - psma scans or choline pet scans are considered to have a high sensitivity to detect a local recurrence [ 6 , 7 ] . the mp - mri has optimal soft tissue contrast , and also has a high negative and positive predictive value for clinically significant prostate cancer [ 9 , 14 ] . beside these aspects , the mp - mri has superior resolution when compared to the ga - psma scan . a high resolution is of great importance when delineating the target area ( gtv ) and the oar in preparation for focal salvage hdr - bt . furthermore , there is high conformity between the area of recurrence and the treated primary index lesion . by reducing the rectum and bladder dose through focal salvage brachytherapy , it can be expected that toxicity will decrease when compared to whole gland salvage treatments [ 5 , 10 ] . as results for salvage modalities for both tumor control and toxicity are promising , a second salvage treatment can be considered in a highly selected group of patients with locally recurrent prostate cancer . in the same light as the first salvage treatment , the goal of a second salvage treatment is to further postpone androgen deprivation therapy , with its known deterioration on the quality of life [ 4 , 17 , 18 ] . the current availability of ga - psma scans also plays a key role in the selection of true locally recurrent prostate cancer in patients with a low psa . these factors may be used for second salvage treatment , since the literature does not describe predictors for outcomes in this scenario . with a margin of 0 mm , we still expect some dose to be delivered to the first few mms around the gtv . as the area of microscopic spread will need less dose compared to the gtv due to a lower tumor density , we agreed on reducing the ctv and ptv margin to 0 mm around the gtv . longer follow - up is necessary to determine if prostate cancer will recur adjacent , or perhaps even within the gtv after second salvage treatment . for the 1 salvage ldr - bt treatment , dose restrictions for bladder , urethra , and rectum have been published to minimize the risk of late severe toxicity [ 20 , 21 ] , namely a bladder d2cc < 70 gy , a urethral v100 < 0.4 cc and a rectal d0.1cc < 160 gy . unfortunately , no such restrictions are available for salvage hdr - bt . as an accurate spatial dose delivery in salvage treatments is essential , i seeds have the tendency to migrate from their original position and this phenomenon may have a negative effect on the intended dose delivery to the gtv . a shift in catheters in focal hdr - bt will also contribute to a suboptimal dose to the gtv , especially since in bt there is a steep dose gradient . with self - anchoring catheters , there is minimal displacement of the catheter tips , and it seems that these shifts have a minimal impact on dosimetry . an advantage of hdr - bt compared to ldr - bt is that the dwell positions and times of the source can be regulated . with hdr - bt , more homogeneous dose to the gtv can be delivered and it is often possible to better modulate the dose around the adjacent oar when compared to ldr - bt . there is a need for second salvage hdr - bt dose restriction values , since it is questionable whether the first salvage dose restriction parameters can be used for a second salvage treatment . in the current situation , this will be difficult to accomplish , as events ( grade 3 gu and gi toxicity ) and number of treated patients are low . ( for rectum and bladder d1cc < 12 gy , for urethra d10% < 17.7 gy ) may be considered in clinical practice for both 1 and 2 salvage hdr - bt . for each patient , the infield or out of field recurrences the infield recurrence after the primary salvage treatment may have been caused by inadequate spacing of the i seeds and/or by an aggressive tumor . the out of field recurrences may be explained by the fact that psma scans were not yet available at the time when patients underwent a primary salvage treatment . out of field recurrences treated with a 2 salvage hdr - bt will most probably have less impact on the oar , since the accumulated dose for the specific oar will generally be lower than with an infield recurrence . perhaps in or out of field 2 recurrences can be taken into account when contemplating a 2 salvage hdr - bt , and it may be an important selection criteria in future predictive models . in 2 of 4 patients , there was a biochemical recurrence ( phoenix criteria ) during follow - up ( figure 1 ) . although follow - up is still limited , it can not be concluded whether patient selection had been appropriate . one patient showed a single nodal distant metastasis on a psma scan after biochemical recurrence , which was not present on the pre - treatment psma scan . one patient did not receive the prescribed dose to the gtv , and also shows psa increase after treatment , although biochemical recurrence according to the phoenix definition has not been reached . the increasing psa in two of the patients may also be explained by a psa bounce , as the median follow - up in this study is only 12 months . there is no literature that describes psa bounce after a 2 salvage treatment . concluding , focal second salvage hdr - bt for locally recurrent prostate cancer is feasible in a highly selected group of patients , with a secondary local recurrence , minimal gi and gu toxicity , low psa values at time of second salvage , and an interval of more than 3 years after earlier treatments . this approach may be used in the future to postpone hormonal therapy and positively influence the quality of life for patients with a 2 locally recurrent prostate cancer .

plant samples of p. poilanei collected in vietnam at different times and locations were extracted with meoh and then partitioned with n - hexane and chcl3 . when the cytotoxic chcl3 partitions were subjected to chromatographic separation guided by inhibitory activity against the ht-29 cell line , two new [ phyllanthusmins d ( 1 ) and e ( 2 ) ] and four known [ phyllanthusmins a ( 6 ) , b ( 3 ) , and c ( 4 ) and cleistanthin b ( 5 ) ] arylnaphthalene lignan lactones were purified . the various plant collections and the compounds isolated from each collection are summarized in table 1 . initial collection of the combined leaves , twigs , flowers , and fruits of p. poilanei . second collection of the combined leaves , twigs , flowers , and fruits of p. poilanei . second collection of the stems of p. poilanei . large collection of the combined leaves , twigs , and stems of p. poilanei . located in khanh hoa province , vietnam . located in hatinh province , vietnam . compound 1 was isolated as a new compound in the form of colorless fine needles , mp 210211 c . a sodiated molecular ion peak at m / z 619.1444 ( calcd 619.1422 ) observed in the hresims in conjunction with c nmr spectroscopic data corresponded to a molecular formula of c30h28o13 . the uv ( max 260 nm ) and ir [ max 3446 ( hydroxy ) , 1747 ( -lactone ) , 1619 , 1507 , and 1481 ( aromatic ) cm ] spectra showed the absorption characteristics of an arylnaphthalene lignan lactone . the h nmr data of 1 ( table 2 ) exhibited resonances for two substituted aromatic rings at h 6.81 , 6.83 , 6.97 , 7.09 , and 7.94 , a lactone methylene group at h 5.47 and 5.56 , a methylenedioxy group at h 6.05 and 6.10 , two methoxy groups at h 3.81 and 4.03 , two acetyl groups at h 2.14 and 2.23 , and proton resonances for a sugar moiety in the range h 3.604.99 . analogous resonances consistent with the presence of these functionalities appeared in the c nmr data of 1 ( table 2 ) . the lactone moiety was located at the c-8 and c-8 positions , as supported by the hmbc correlations ( figure 1 ) between h-9/c-7 , c-8 , c-8 , and c-9 ( figure 1 ) . the methylenedioxy group could be located at the c-3 and c-4 positions , as indicated by the hmbc correlations between these methylene protons and c-3 and c-4. two methoxy groups were assigned at the c-4 and c-5 positions from the hmbc correlations between these methoxy groups and c-4 and c-5 . the sugar unit was assigned to the c-7 position , as supported by the hmbc correlation between h-1 and c-7 . two acetyl groups were placed at the c-3 and c-4 positions of the sugar residue , as indicated by the hmbc correlations between the h-3 and h-4 resonances and the acetyl carbonyl groups . the resonances at 3.52 for h-3 and at 3.71 for h-4 appearing in the h nmr data of 4 ( table s1 , supporting information ) were deshielded to 4.99 ( h-3 ) and 5.30 ( h-4 ) in the h nmr spectrum of 1 , due to the electron - withdrawing effects that resulted from the acetyl carbonyl groups linked at the c-3 and c-4 positions . this was also supported by the molecular weight of 596 da of 1 , or 42 atomic mass units more than that of 3 , representing the presence of a diacetylglycosyl residue in 1 rather than a monoacetylglycosyl unit as in 3 . thus , compound 1 was proposed as an o - acetyl analogue of the known compounds phyllanthusmins b ( 3 ) and c ( 4 ) , with both being characterized from phyllanthus oligospermus in a previous study . cosy ( , h h ) , key hmbc ( , h c ) , and selected noesy ( , h h ) correlations of 1 . measured in cdcl3 and assignments of chemical shifts are based on the analysis of 1d and 2d nmr spectra . the overlapped signals were assigned from h h cosy , hsqc , and hmbc spectra without designating multiplicity . ch3 , ch2 , ch , and c multiplicities were determined by dept 90 , dept 135 , and hsqc experiments . data ( ) measured at 100.6 mhz and referenced to the solvent residual peak at 77.16 . data ( ) measured at 400.1 mhz and referenced to the solvent residual peak at 7.26 . data ( ) measured at 150.9 mhz and referenced to the solvent residual peak at 77.16 . data ( ) measured at 600.2 mhz and referenced to the solvent residual peak at 7.26 . the unusual value may result from the restricted rotation of the d ring . comparison of the nmr data of compound 1 with those of phyllanthusmins b ( 3 ) and c ( 4 ) ( table 2 and tables s1 and s2 , supporting information ) showed that these compounds displayed closely similar nmr resonances for the diphyllin aglycone unit but different resonances for their saccharide portions . an l - arabinopyranosyl residue of 1 could be proposed based on the several lines of evidence that follow . first , both the noesy correlations and the specific rotation value of 1 were consistent with those of the known compound 3 , as reported and isolated in the present study . second , the noesy correlations and specific rotation value of 1 were consistent with those determined for 4 ( phyllanthusmin c ) in this investigation . the latter compound , when isolated from p. poilanei , showed a specific rotation value { [ ]d 8 ( c 0.1 , chcl3 ) } that coincided with a synthetic version of compound 4 { [ ]d 8 ( c 1 , chcl3)}. finally , both 1 and 4 were acetylated to form the same compound , 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl)]diphyllin ( 7 ) , which exhibited the same specific rotation value of [ ]d 12 ( c 0.1 , chcl3 ) , and were closely comparable to the same compound synthesized from diphyllin and l - arabinose { [ ]d 13 ( c 0.3 , chcl3)}. the h-1 doublet at h 4.86 with a coupling constant of 7.6 hz indicated the presence of an anomeric proton in an axial orientation in 1 . the noesy correlations between h-1 and h-3 and h-3 and h-4 suggested that h-1 , h-3 , and aco-4 are all axial ( figure 1 ) . thus , the structure of this new compound ( phyllanthusmin d , 1 ) was assigned as 7-o-[(3,4-di - o - acetyl)--l - arabinopyranosyl]-4,5-dimethoxy-3,4-methylenedioxy-2,7-cycloligna-7,7-dieno-9,9-lactone , or 7-o-[(3,4-di - o - acetyl)--l - arabinopyranosyl]diphyllin . this was confirmed by analysis of its single - crystal x - ray diffraction data . as shown in figure 2 , this compound existed as an atropisomeric mixture because of hindered rotation of ring d about the c-1c-7 bond , which resulted in duplication of the c-8 resonance at c 131.42 and 131.43 ( table 2 ) and an optical activity indicated by its ecd spectrum ( figure s17 , supporting information ) . the same hindered rotation would likely be observed in 35 and other arylnaphthalene lignan lactones , as indicated by the duplicated resonances in their nmr spectra ( tables s1 , s2 , and s4 , supporting information ) and optical activity shown in their ecd spectra . these atropisomers interconvert slowly , hence permitting the two diastereomers observed in the nmr spectra , but the rotation barrier is too small for the individual diastereomers to be isolated at room temperature . ortep plots for the molecular structure of 1 drawn with 50% probability displacement ellipsoids ( oxygen atoms are red , carbon atoms are blue , and the small white circles represent hydrogen atoms , which are drawn with an artificial radius ) . the similar uv and ir spectra to those of 1 indicated that 2 is also an arylnaphthalene lignan lactone . the molecular formula of c28h26o12 deduced from c nmr data and a sodiated molecular ion peak at m / z 577.1319 ( calcd 577.1316 ) observed in the hresims and the similar nmr data to those of 1 demonstrated that this compound is a diphyllin monoacetylarabinoside , a regioisomer of phyllanthusmin b ( 3 ) . comparison of the h and c nmr data of 2 with those of 3 showed that the resonances for h-3 and c-3 of 2 were deshielded , but those for h-4 and c-4 were shielded ( table 2 and tables s1 and s2 , supporting information ) . this indicated that the acetyl group is attached to c-3 in 2 rather than to c-4 in 3 , as supported by the hmbc correlation between the h-3 and the acetyl carbonyl carbon resonance . a doublet at h 4.84 showing a coupling constant of 6.0 hz displayed in the h nmr spectrum of 2 supported the presence of the anomeric proton in an axial orientation . the noesy correlations between h-1 and h-5ax and h-3 and h-4 and h-5ax suggested that h-1 , h-3 , and oh-4 are all axial ( figure s19 , supporting information ) . although sample limitations precluded hydrolysis of 2 to yield the sugar unit or acetylation of 2 into 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl)]diphyllin ( 7 ) to determine its absolute configuration , it may be assumed that this compound has the same absolute configuration as that of 1 , 3 , and 4 , from the consistent noesy correlations ( figure s19 , supporting information ) and specific rotation values { [ ]d 3 ( c 0.1 , chcl3 ) for 1 , [ ]d 4 ( c 0.1 , chcl3 ) for 2 , [ ]d 6 ( c 0.1 , chcl3 ) for 3 , and [ ]d 8 ( c 0.1 , chcl3 ) for 4 ( supporting information)}. therefore , the structure of 2 ( phyllanthusmin e ) was defined as 7-o-[(3-o - acetyl)--l - arabinopyranosyl]-4,5-dimethoxy-3,4-methylenedioxy-2,7-cycloligna-7,7-dieno-9,9-lactone , or 7-o-[(3-o - acetyl)--l - arabinopyranosyl]diphyllin . altogether , six arylnaphthalene lignan lactones were characterized from the different parts of p. poilanei collected in different locations in vietnam . as summarized in table 1 , the most cytotoxic isolate , phyllanthusmin d ( 1 ) , was isolated from all parts of p. poilanei , and this compound can be regarded as a major cytotoxic principle of p. poilanei . the known arylnaphthalene lignan lactones isolated from p. poilanei were identified by analysis of their spectroscopic data and comparison of these data with literature values , and full assignments of their h and c nmr spectroscopic data are listed in tables s1s4 ( supporting information ) . to partially discern the effects of both the arabinose unit and the acetyl group in the mediation of cytotoxicity of the diphyllin lignans , 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl]diphyllin ( 7 ) was produced by acetylation of 1 and 4 using a standard method and was identified by its molecular formula of c32h30o14 , as determined by hresims and comparison of its spectroscopic data ( supporting information ) with those reported for a synthetic sample . the aglycone diphyllin ( 8) was generated by hydrolysis of phyllanthusmin c ( 4 ) , and its structure was determined by comparison of its spectroscopic data with reference values . all arylnaphthalene lignans isolated from p. poilanei in the present study and their semisynthetic analogues were evaluated for their cytotoxicity against ht-29 human colon cancer cells , using paclitaxel and etoposide as the positive controls ( table 3 ) . compounds 14 , 7 , and 8 were found to be cytotoxic , of which 1 and 7 were the most potently active , with ic50 values of 170 and 110 nm , respectively , but compounds 5 , 6 , and etoposide were inactive . inspection of the lignan structures and their cytotoxicity showed that compounds containing more lipophilic acetyl groups exhibit higher potency , so the presence of one or more acetyl groups linked to the arabinose residue improved the resultant cytotoxicity . compounds 2 and 3 exhibited the same activity , indicating that the acetyl group linked to c-3 contributed to this activity equally when linked to c-4. phyllanthusmin c ( 4 ) showed a higher cytotoxicity than diphyllin ( 8) and cleistanthin b ( 5 ) , implying that the -l - arabinose unit at c-7 played an important role in generating this effect and was more active than a -d - glucose unit in mediating compound cytotoxicity toward ht-29 cells . diphyllin ( 8) was active , but phyllanthusmin a ( 6 ) was inactive , showing that the c-4 and c-5 methoxy groups and the c-7 hydroxy group are all required for diphyllin to mediate its cytotoxicity . the new compound phyllanthusmin d ( 1 ) showed a much greater ic50 value than etoposide , indicating that this compound is more potently cytotoxic against ht-29 cells . ic50 values were calculated using nonlinear regression analysis with measurements performed in triplicate and representative of two independent experiments in which the values generally agreed within 10% investigation of the cytotoxic arylnaphthalene lignans reported in previous studies showed that most active compounds are analogues of diphyllin or diphyllin glycosides . a methylated diphyllin derivative , justicidin a , was highly cytotoxic , but its close analogue , chinensinaphthol methyl ether , did not show such activity , indicating the importance of the substituents on the a and d rings of these compounds . cleistanthoside a tetraacetate showed potent cytotoxicity with ig50 values in the nanomolar range , but its precursor , cleistanthoside a , was inactive , suggesting the importance of acetylation of the glycose unit of cleistanthoside a. the structure cytotoxicity relationships of arylnaphthalene lignan lactones observed from the present study are consistent with observations made from these previous studies . the two cytotoxic isolates 1 and 4 were tested for their cytotoxicity toward ccd-112con human normal colon cells using a previous protocol . both compounds were found to be noncytotoxic toward this cell line ( table 3 ) , indicating some selectivity for ht-29 human colon cancer cells . the new cytotoxic compound , phyllanthusmin d ( 1 , ic50 , 170 nm ) , isolated from p. poilanei , was tested further in an in vivo hollow fiber assay for its possible antitumor efficacy . immunodeficient ncr nu / nu mice implanted with ht-29 human colon cancer cells placed in hollow fibers were treated once daily by 1 at doses of 5.0 , 10.0 , 15.0 , or 20.0 mg / kg , or the vehicle control , or paclitaxel ( 5 mg / kg ) , by ip injection for 4 days . the results showed that the values from the treatment of 1 at 10.0 , 15.0 , or 20.0 mg / kg ( ip ) were all statistically significantly different from those at a dose of 5 mg / kg ( ip ) , and they showed a dose - dependent tendency ( figure 3 ) . effect of phyllanthusmin d ( 1 ) on the growth of human colon cancer ht-29 cells implanted in ncr nu / nu mice tested in an in vivo hollow fiber assay . mice were treated with the indicated doses of 1 once daily by intraperitoneal injection from day 3 to day 6 after implantation of the ht-29 cells facilitated in hollow fibers . on day 7 , the mice were sacrificed , and fibers were retrieved and analyzed ( experimental section ) . the results are shown as the average percentage cell growth relative to control [ columns , mean in each group ( n = 6 for the control group and n = 3 for the treatment group ) ; bars , se ; * * p 0.05 and * * * p 0.01 for significant differences from the 5 mg / kg ( 1 ) treatment ] . the enzyme dna topoisomerase is an established molecular target of etoposide , on which this compound acts to form dna double - strand breaks via stabilization of the intermediate topo ii - dna covalent complex to initiate the cell death pathway . in the present study , several diphyllin arabinosides , including phyllanthusmins c ( 4 ) and d ( 1 ) and 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl)]diphyllin ( 7 ) , together with etoposide , were tested for their ability to inhibit dna topo ii ( figure 4 ) , using a method reported previously . consistent with a previous report that etoposide showed topo ii inhibitory activity , this agent exhibited similar activity in the present study ( figure 4 ) . however , arylnaphthalene lignan lactones investigated herein neither inhibited topo ii-mediated dna strand passage / catalytic activity ( conversion of supercoiled dna to relaxed dna ) nor induced topo ii-mediated dna cleavage ( linearized double - strand dna ) compared to the control , indicating that these substances are not topo ii inhibitors . previous reports have documented that some diphyllin glycosides inhibited topo ii , but other close analogues of these compounds did not . this indicates that the glycosidic moiety of these lignans plays a key role in topo ii inhibition . some specific diphyllin analogues might exert their cytotoxicity through a mechanism of action different from that of etoposide , as supported by additional chemical and biological studies for an arylnaphthalene lignan lactone , daurinol , which did not induce the formation of open circular and linear dna that originated from the topoisomerase - dna cleavable intermediate . evaluation of arylnaphthalene lignan lactones { phyllanthusmins c ( 4 ) and d ( 1 ) and 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl)]diphyllin ( 7 ) ] from p. poilanei for activity as topoisomerase ii ( topo ii ) inhibitors . topo ii - dna covalent complexes induced by test samples and etoposide were trapped by rapidly denaturing the complexed enzyme with sodium dodecyl sulfate ( sds ) , digesting the enzyme , and releasing the cleaved dna as linear dna . the formation of linear dna was detected by separating the sds - treated reaction products using ethidium bromide gel electrophoresis and quantified by accounting for the relationship between fluorescence and relative band intensity for open circular ( oc ) , linear ( lnr ) , supercoiled ( sc ) , and relaxed ( rlx ) configurations of dna ( experimental section ) . apoptosis , or programmed cell death , occurs during normal cellular differentiation and the development of multicellular organisms . to remain malignant , cancer cells must evade apoptosis to avoid elimination , and many anticancer agents induce cancer cell apoptosis . a previous study showed that an 8-day treatment of etoposide induced ht-29 human colon cancer cell apoptosis , but shorter term treatment with this compound did not show this activity . after ht-29 cells were treated with compound 1 or etoposide at different concentrations , annexin v flow cytometry was performed following a previous protocol . treatment of ht-29 cells with 1 or 5 m phyllanthusmin d ( 1 ) for 72 h resulted in 28.2% or 30.3% of ht-29 cells undergoing early apoptosis , respectively , while the analogous values for the vehicle control or 1 or 5 m etoposide treatments were 3.9% , 12.9% , and 12.5% , respectively ( figure 5 ) . also , 1 induced 27.3% ( at 1 m ) and 38.0% ( at 5 m ) ht-29 cell apoptosis at the late stage , while the vehicle control or 1 or 5 m etoposide treatments induced 8.6% , 19.8% , or 25.3% ht-29 cell apoptosis at this stage , respectively ( figure 5 ) . these results indicated that compound 1 showed a higher potency than etoposide in inducing ht-29 cell apoptosis . ht-29 cells were treated with 1 or 5 m phyllanthusmin d ( 1 ) , 1 or 5 m etoposide , or the vehicle control for 72 h , followed by an annexin v staining method ( experimental section ) . lower left quadrant : percentage of viable cells ; lower right quadrant : percentage of apoptotic cells ; upper left quadrant : percentage of necrotic cells ; upper right quadrant : percentage of late - stage apoptotic cells or dead cells . caspase-3 , a key effector of programmed cell death and a well - known anticancer drug target , is activated only during cell apoptosis and contributes fundamentally to this process . following a previous procedure , both 1 and etoposide were tested for their caspase-3 activation in ht-29 cells ( figure 6 ) . after 24 h incubation , phyllanthusmin d ( 1 ) induced activation of caspase-3 at as low a dose as 1 m , as well as at 5 and 10 m . in contrast , under the same experimental conditions , etoposide did not induce caspase-3 activation , which is consistent with the known resistance of ht-29 cells to etoposide . these results again indicate a fundamental difference in the mechanism(s ) of action of these agents . ht-29 cells were incubated with phyllanthusmin d ( 1 ) and etoposide with different concentrations for 24 h , and caspase-3-like activity was determined by western blotting using rabbit monoclonal cleaved caspase-3 ( asp175 ) antibody . the data shown are a representative blot from three independent experiments with similar results ( experimental section ) . h and c , dept , hsqc , hmbc , noesy , and cosy nmr spectra were recorded at room temperature on bruker avance drx-400 , drx-600 , or drx-800 mhz nmr spectrometers . esims and hresims were measured on an lct - tof or a q - tof mass spectrometer in the positive - ion mode . column chromatography was conducted using silica gel ( 65 250 or 230 400 mesh , sorbent technologies , atlanta , ga , usa ) . analytical thin - layer chromatography ( tlc ) was performed on precoated silica gel 60 f254 plates ( sorbent technologies ) . sephadex lh-20 was purchased from amersham biosciences , uppsala , sweden . for visualization of tlc plates , all procedures were carried out using anhydrous solvents purchased from commercial sources and employed without further purification . reagents for chemical synthesis were purchased from sigma except where indicated , and reactions were monitored by tlc using precoated silica gel plates . crystallographic data were collected through the service crystallography at advanced light source ( scrals ) program at the small - crystal crystallography beamline 11.3.1 at the advanced light source ( als ) , lawrence berkeley national laboratory , with a bruker apexii ccd detector ( bruker analytical x - ray instruments , inc . , initial collections of separate samples of the combined leaves , twigs , flowers , and fruits ( acquisition number a06024 ) and the stems ( acquisition number a06025 ) of phyllanthus poilanei were collected from a shrub at the road transect from suoi cat village to hon ba peak ( 1207.873 n ; 10601.532 e ) , dinh khanh district , khanh hoa province , vietnam , in november 2004 , by d.d.s . a voucher herbarium specimen ( dds 13619 ) representing this collection was identified as phyllanthus poilanei beille by d.d.s . and deposited at the john g. searle herbarium of the field museum of natural history , chicago , il , usa , under the accession number fm-2256257 . second collections of separate samples of the combined leaves , twigs , flowers , and fruits ( acquisition number a06473 ) and the stems ( acquisition number a06474 ) of p. poilanei were obtained from a liana - like shrub in the forest occurring at the south end of kego lake , across from mui tru ranger station ( 1806.530 n ; 10600.891 e ) , kego nature reserve , cam xuyen district , hatinh province , vietnam , in december 2008 , by d.d.s . a voucher herbarium specimen ( dds 14308 ) representing this collection was identified as phyllanthus poilanei beille by d.d.s . and deposited at the john g. searle herbarium of the field museum of natural history , chicago , il , under the accession number fm-2287526 . a larger sample of the combined leaves , twigs , and stems ( acquisition number aa06024 ) of p. poilanei was collected from a liana in the hon ba mountain region , 25 km from soi cat on a peak along roadside forest ( 1206.745 n ; 10858.80 e ) , dinh khanh district , khan hoa province , vietnam , in august 2011 , by d.d.s . . a voucher herbarium specimen ( dds 14886 ) representing this collection was identified as phyllanthus poilanei beille by d.d.s . and deposited at the john g. searle herbarium of the field museum of natural history , chicago , il , under the accession number fm-2300873 . the milled air - dried leaves , twigs , flowers , and fruits of p. poilanei ( sample a06024 , 2000 g ) were extracted with meoh ( 7 l 6 ) at room temperature . the solvent was evaporated in vacuo , and the dried meoh extract ( 170.0 g , 8.5% ) was resuspended in 10% h2o in meoh ( 1000 ml ) and partitioned with n - hexane ( 700 ml 2 and 500 ml ) to yield an n - hexane - soluble residue ( d1 , 22.4 g , 1.1% ) . the aqueous meoh layer was then partitioned with chcl3 ( 800 , 700 , and 600 ml ) to afford a chcl3-soluble extract ( d2 , 3.0 g , 0.15% ) , which was washed with a 1% aqueous solution of nacl , to partially remove tannins . the chcl3-soluble extract exhibited cytotoxicity toward the ht-29 cell line ( ic50 < 5.0 g / ml ) . both the n - hexane- and water - soluble extracts were inactive in the bioassay system used . the chcl3-soluble extract ( 2.8 g ) was subjected to silica gel column chromatography ( 2.5 45 cm ) , eluted with a gradient of n - hexane acetone . eluates were pooled by tlc analysis to give 13 combined fractions ( d2f1d2f13 ) . of these , d2f4d2f6 ( ic50 < 2 g / ml ) were combined and further chromatographed over a silica gel column ( 2.5 20 cm ) , eluted with a gradient of n - hexane fractions d2f11 and d2f12 ( ic50 < 5 g / ml ) were combined and further chromatographed over a silica gel column ( 2.5 20 cm ) , eluted with a gradient of n - hexane subfraction d2f4f2 was chromatographed over silica gel , with a gradient of n - hexane acetone , and purified by separation over a sephadex lh-20 column , eluted with ch2cl2meoh ( 1:1 ) , affording phyllanthusmin d ( 1 , 20 mg ) . the combined subfractions d2f4f3d2f4f5 were separated by silica gel chromatography , eluted with n - hexane acetone ( 3:1 ) , and purified by passage over a sephadex lh-20 column , eluted with a mixture of ch2cl2meoh ( 1:1 ) , to afford phyllanthusmin a ( 6 , 2.0 mg ) , phyllanthusmin b ( 3 , 1.0 mg ) , and phyllanthusmin e ( 2 , 1.5 mg ) . fractions d2f11f2d2f11f4 were combined and chromatographed over silica gel , eluted by n - hexane acetone ( 2:1 ) , and purified by separation over a sephadex lh-20 column , using ch2cl2meoh ( 1:1 ) for elution , affording phyllanthusmin c ( 4 , 7.0 mg ) . the milled air - dried stems of p. poilanei ( sample a06025 , 580 g ) were extracted with meoh ( 3 l 4 and then 2 the solvent was evaporated in vacuo , and the dried meoh extract ( 47.4 g , 8.2% ) was resuspended in 10% h2o in meoh ( 500 ml ) and partitioned with n - hexane ( 500 , 300 , 200 ml ) , to yield an n - hexane - soluble residue ( d1 , 1.4 g , 0.24% ) . the aqueous meoh layer was partitioned with chcl3 ( 500 , 300 , and 300 ml ) to afford a chcl3-soluble extract ( d2 , 2.0 g , 0.34% ) , which was followed by washing with a 1% aqueous solution of nacl , to partially remove tannins . the chcl3-soluble extract exhibited cytotoxicity toward the ht-29 cell line ( ic50 < 5.0 g / ml ) . both the n - hexane- and water - soluble extracts were inactive in the bioassay system used . the chcl3-soluble extract ( 1.8 g ) was subjected to silica gel column chromatography ( 2.5 45 cm ) , eluted with a gradient of n - hexane acetone . fractions were pooled by tlc analysis to give 13 combined fractions ( d2f1d2f13 ) . of these , d2f4d2f6 ( ic50 < 2 g / ml ) were combined , further chromatographed over a silica gel column , eluted with a gradient of n - hexane acetone , and purified by separation over a sephadex lh-20 column , eluted with ch2cl2meoh ( 1:1 ) , affording phyllanthusmin d ( 1 , 7.0 mg ) . the milled air - dried combined leaves , twigs , flowers , and fruits of p. poilanei ( sample a06473 , 851 g ) were extracted with meoh ( 3 the solvent was evaporated in vacuo , and the dried meoh extract ( 96 g , 11.3% ) was resuspended in 10% h2o in meoh ( 500 ml ) and partitioned with n - hexane ( 500 , 300 , 200 ml ) , to yield an n - hexane - soluble residue ( d1 , 10.2 g , 1.2% ) . the aqueous meoh layer was then partitioned with chcl3 ( 500 , 300 , and 300 ml ) to afford a chcl3-soluble extract ( d2 , 3.0 g , 0.35% ) , which was followed by washing with a 1% aqueous solution of nacl , to partially remove tannins . the chcl3-soluble extract exhibited cytotoxicity toward the ht-29 cell line ( ic50 < 10.0 g / ml ) . both the n - hexane- and water - soluble extracts were inactive in the bioassay system used . the chcl3-soluble extract ( 2.8 g ) was subjected to silica gel column chromatography ( 2.5 45 cm ) , eluted with a gradient of n - hexane acetone . fractions were pooled by tlc analysis to give 11 combined fractions ( d2f1d2f11 ) . of these , fractions d2f8 and d2f9 ( ic50 < 5.0 g / ml ) were combined , further chromatographed over a silica gel column ( 2.5 20 cm ) , eluted with a gradient of n - hexane acetone , and purified by separation over a sephadex lh-20 column , eluted with ch2cl2meoh ( 1:1 ) , affording phyllanthusmins c ( 4 , 2.0 mg ) and d ( 1 , 3.0 mg ) . the milled air - dried stems of p. poilanei ( sample a06474 , 517 g ) were extracted with meoh ( 2 l 6 ) at room temperature . the solvent was evaporated in vacuo , and the dried meoh extract ( 75 g , 14.5% ) was resuspended in 10% h2o in meoh ( 600 ml ) and partitioned with n - hexane ( 500 , 400 , and then 300 ml ) , to yield an n - hexane - soluble residue ( d1 , 1.0 g , 0.2% ) . the aqueous meoh layer was then partitioned with chcl3 ( 500 , 300 , and 300 ml ) to afford a chcl3-soluble extract ( d2 , 2.0 g , 0.38% ) , which was followed by washing with a 1% aqueous solution of nacl , to partially remove tannins . the chcl3-soluble extract exhibited cytotoxicity toward the ht-29 cell line ( ic50 < 10.0 g / ml ) . both the n - hexane- and water - soluble extracts were inactive in the bioassay system used . the chcl3-soluble extract ( 1.8 g ) was subjected to silica gel column chromatography ( 2.5 45 cm ) , eluted with a gradient of n - hexane acetone . fractions were pooled by tlc analysis to give 11 combined fractions ( d2f1d2f11 ) . of these , fraction d2f10 ( ic50 < 5.0 g / ml ) was chromatographed over a silica gel column , eluted with a gradient of n - hexane acetone , and purified by separation over a sephadex lh-20 column , eluted with ch2cl2meoh ( 1:1 ) , to afford phyllanthusmin d ( 1 , 2.0 mg ) . in an attempt to accumulate a greater quantity of phyllanthusmin d ( 1 ) for in vivo biological evaluation , a larger re - collection of the combined leaves , twigs , and stems of p. poilanei was made . the milled air - dried combined leaves , twigs , and stems of this sample ( aa06024 , 3200 g ) were extracted with meoh ( 7 l 6 ) at room temperature . the solvent was evaporated in vacuo , and the dried meoh extract ( 278.0 g , 8.7% ) was resuspended in 10% h2o in meoh ( 1000 ml ) and partitioned with n - hexane ( 800 ml 3 and 500 ml 3 ) , to yield an n - hexane - soluble residue ( d1 , 27.0 g , 0.84% ) . the aqueous meoh layer was partitioned with chcl3 ( 800 ml 3 and 500 ml 3 ) to afford a chcl3-soluble extract ( d2 , 8.5 g , 0.27% ) , which was followed by washing with a 1% aqueous solution of nacl , to partially remove tannins . the aqueous meoh layer was further partitioned with etoac ( 800 ml 3 and 500 ml 3 ) to afford an etoac - soluble extract ( d3 , 10.0 g , 0.31% ) , which was also washed with a 1% aqueous solution of nacl . the chcl3-soluble extract exhibited cytotoxicity toward the ht-29 cell line ( ic50 < 5.0 g / ml ) . however , all of the n - hexane- , etoac- , and water - soluble extracts were inactive in the bioassay system used . the chcl3-soluble extract ( 8.0 g ) was subjected to silica gel column chromatography ( 4.5 45 cm ) , eluted with a gradient of n - hexane acetone . fractions were pooled by tlc analysis to give 11 combined fractions ( d2f1d2f11 ) . of these , d2f4d2f6 ( ic50 < 5 g / ml ) were combined and further chromatographed over a silica gel column ( 2.5 20 cm ) , eluted with a gradient of n - hexane acetone , to yield phyllanthusmins b ( 3 , 1.0 mg ) , d ( 1 , 10.5 mg ) , and e ( 2 , 1.0 mg ) . fraction d2f8 was separated by silica gel chromatography , eluted with n - hexane acetone ( 2:1 ) , and purified by passage over a sephadex lh-20 column , eluted with a mixture of ch2cl2meoh ( 1:1 ) , to afford phyllanthusmin c ( 4 , 9.5 mg ) . to isolate the polar analogues of 4 , the etoac - soluble extract ( 9.0 g ) was subjected to silica gel column chromatography ( 4.5 45 cm ) , eluted with a gradient of ch2cl2meoh . fractions were pooled by tlc analysis to give five combined fractions ( d3f1d3f5 ) . of these , d3f1 and d3f2 were combined , further chromatographed over a silica gel column ( 2.5 20 cm ) , eluted with a gradient of ch2cl2meoh , and purified by passage over a sephadex lh-20 column , eluted with a mixture of ch2cl2meoh ( 1:1 ) , to afford cleistanthin b ( 5 , 1.5 mg ) . colorless , fine needles ( n - hexane acetone ) , showing a blue color under uv light at 365 nm ; mp 210211 c ; [ ]d 3 ( c 0.1 , chcl3 ) ; uv ( meoh ) max ( log ) 260 ( 4.54 ) nm ; ecd ( meoh , nm ) max ( ) 292 ( 3.65 ) ; ir ( dried film ) max 3446 , 1747 , 1619 , 1507 , 1481 , 767 cm ; h and c nmr data , see table 2 ; positive - ion hresims m / z 619.1444 , calcd for c30h28o13na , 619.1422 . amorphous , colorless powder showing a blue color under uv light at 365 nm ; [ ]d 4 ( c 0.1 , chcl3 ) ; uv ( meoh ) max ( log ) 260 ( 4.58 ) nm ; ecd ( meoh , nm ) max ( ) 296 ( 4.15 ) ; ir ( dried film ) max 3419 , 1738 , 1622 , 1506 , 1481 , 770 cm ; h and c nmr data , see table 2 ; positive - ion hresims m / z 577.1319 , calcd for c28h26o12na , 577.1316 . intensity data for a small , colorless needle ( mp 210211 c ; molecular formula c30h28o13 , mw = 596.52 , hexagonal , space group p6122 , a = 21.4292(6 ) , c = 21.4162(6 ) , v = 8517.0(4 ) , z = 12 , density(calculated ) = 1.396 mg / m , size 0.01 0.01 0.20 mm ) from 1 were collected at 150 k on a d8 goniostat equipped with a bruker apexii ccd detector at beamline 11.3.1 using synchrotron radiation tuned to = 1.2399 at the advanced light source at lawrence berkeley national laboratory . for data collection , frames were measured for a duration of 1 s for low - angle data and 4 s for high - angle data at 0.3 intervals of with a maximum 2 value of around 91. the data frames were collected using the program apex2 and processed using the program saint within apex2 , and the data were corrected for absorption and beam corrections based on the multiscan technique as implemented in sadabs . full - matrix least - squares refinements based on f were performed in shelxl-97 , as incorporated in the wingx package . it was necessary to apply distance restraints for this group along with restraints on the anisotropic displacement parameters ( simu and delu ) . for each methyl group , the hydrogen atoms were added at calculated positions using a riding model with u(h ) = 1.5ueq(bonded carbon atom ) . the torsion angle , which defines the orientation of the methyl group about the c c or o c bond , was refined . the hydroxy group hydrogen atom bonded to o(8 ) was refined isotropically and is involved in an intermolecular hydrogen bond with atom o(2 ) . the rest of the hydrogen atoms were included in the model at calculated positions using a riding model with u(h ) = 1.2ueq(bonded atom ) . the final refinement cycle was based on 4507 intensities , 191 restraints , and 478 variables and resulted in agreement factors of r1(f ) = 0.050 and wr2(f ) = 0.089 . for the subset of data with i the final difference electron density map contains maximum and minimum peak heights of 0.12 and 0.16 e / . the cif file of the x - ray data of 1 has been deposited in the cambridge crystallographic data centre ( deposition no . ccdc 981532 ) . to a dried 25 ml flask equipped with a magnetic stirrer , containing 3.0 mg of phyllanthusmin d ( 1 ) , after the mixture was stirred at 40 c for 1 h , it was cooled to room temperature . then , 5 ml of chcl3 was transferred into the flask , and the solution was extracted with distilled h2o . the organic layer was washed with distilled h2o and evaporated at reduced pressure . acetone ( 5:1 1:1 ) , to afford 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl)]diphyllin { 7 ( 1.0 mg , [ ]d 12 ( c 0.1 , chcl3)}. using the same protocol , 5.0 mg of phyllanthusmin c ( 4 ) was reacted with 10 l of acetic anhydride and 2 ml of pyridine at 60 c for 1 h and yielded 1.0 mg of 7 { [ ]d 12 ( c 0.1 , chcl3)}. these values are very close to that of { [ ]d 13 ( c 0.3 , chcl3 ) } reported for synthetic 7 . to a dried 25 ml flask equipped with a water condenser and magnetic stirrer containing phyllanthusmin c ( 4 , 5.0 mg dissolved in 1 ml of meoh ) , was transferred 5 ml of 37% hydrochloric acid ( hcl ) into the flask . after the mixture was stirred at 70 c for 30 min , the mixture was cooled to room temperature and diluted by 0.1 n naoh to ph 7.0 . then , 5 ml of chcl3 was transferred into the flask , and the solution was extracted with distilled h2o . the residue was separated by silica gel column chromatography , using n - hexane acetone ( 3:1 ) , to afford diphyllin ( 8 , 1.5 mg ; data obtained are shown in the supporting information ) . the cytotoxicity of the test compounds was screened against ht-29 cells by a previously reported procedure . paclitaxel and etoposide were used as positive controls . following a previous procedure and the method for screening cytotoxicity toward ht-29 cells mentioned above , the cytotoxicity of the samples was screened against ccd-112con normal human colon cells . the hollow fiber assay is an excellent method for evaluating the potential of natural products for activity in vivo . the human colon cancer cell line ht-29 was used to evaluate 1 using procedures previously described in detail by our group . eight- to nine - week - old immunodeficient ncr nu / nu mice were purchased from the jackson laboratory ( bar harbor , me , usa ) and housed in microisolation cages at room temperature and a relative humidity of 5060% under 12:12 h light all animal work was approved by university of illinois at chicago animal care and use committee ( protocol number 13 - 057 ) , and the mice were treated in accordance with the institutional guidelines for animal care . phyllanthusmin d ( 1 ) was dissolved initially in dmso and subsequently diluted with cremophor . the mice were injected ip once daily for 4 days with 1 or the positive control ( paclitaxel ) . animals showed no signs of toxicity even at the highest concentration of 1 , and all the remaining mice were sacrificed on day 7 . the fibers were retrieved , and viable cell mass was evaluated by a modified mtt [ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ] assay . the percentage of the net growth for the cells in each treatment group was calculated by subtracting the day 0 absorbance from the day 7 absorbance and dividing this difference by the net growth in the vehicle control ( minus the value between day 7 and day 0 ) . data were compared by student s t test , and a p value less than 0.05 was considered statistically significant ( figure 3 ) . topo ii - dna covalent complexes induced by topo ii poisons such as etoposide may be trapped by rapidly denaturing the complexed enzyme with sodium dodecyl sulfate ( sds ) , digesting the enzyme , and releasing the cleaved dna as linear dna . the formation of linear dna was detected by separating the sds - treated reaction products using ethidium bromide gel electrophoresis by a modification of a previously described procedure . in this system , topo ii - mediated catalytic conversion of supercoiled pbr322 dna to the relaxed form of plasmid dna can also be observed . a 20 l cleavage assay reaction mixture contained 250 ng of topo ii protein , 160 ng of pbr322 plasmid dna ( neb , ipswich , ma , usa ) , 1.0 mm atp in assay buffer [ 10 mm tris - hcl ( ph 7.5 ) , 50 mm kcl , 50 mm nacl , 0.1 mm edta , 5 mm mgcl2 , 2.5% glycerol ] , and 100 m test compounds or dmso solvent , as indicated . assay buffer ( 17 l ) and test compound / dmso ( 1 l ) were mixed and allowed to sit at room temperature for 30 min , after which 2 l of topo ii was added to initiate the reaction . tubes were incubated at 37 c for 15 min and then quenched with 1% ( v / v ) sds10 mm disodium edta200 mm nacl . the mixture was treated subsequently with 0.77 mg / ml proteinase k ( sigma ) at 55 c for 60 min to digest the protein , and dna bands were separated by electrophoresis ( 18 h at 2 v / cm ) on an agarose gel ( 1.3% w / v ) containing 0.7 g / ml ethidium bromide in tae buffer ph 8.0 ( 40 mm tris base , 0.114% ( v / v ) glacial acetic acid , 2 mm edta ) . the dna in the gel was imaged by its fluorescence on a chemi - doc xrs+ imager ( bio - rad , hercules , ca , usa ) . linear dna was quantified by accounting for the relationship between fluorescence and relative band intensity for open circular , linear ( lnr ) , supercoiled , and relaxed configurations of dna , then calculating the percent of lnr from the total dna content in each lane . results are shown for etoposide , phyllanthusmins c ( 4 ) and d ( 1 ) , and 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl)]diphyllin ( 7 ) in replicate experiments performed on separate days ( figure 4 ) . as described in previous studies , ht-29 cells were treated with the vehicle control or etoposide ( 1 or 5 m ) or 1 ( 1 or 5 m ) for 72 h. the cells were washed with annexin v binding buffer , centrifuged at 300 g for 10 min , and suspended ( 1 10 ) in 100 l of 1 annexin v binding buffer . then , 10 l of annexin v - apc was added to the suspension . after the suspension was mixed and incubated in a dark room at room temperature for 15 min , the cells were centrifuged , and the cell pellet was resuspended in 500 l of 1 annexin v binding buffer . then , 5 g / ml of 7-aad solution was added in the suspension , and flow cytometry was conducted immediately . after a 24 h treatment , ht-29 cells were harvested , washed once with ice - cold pbs , and lysed ( 10 cells / ml lysis buffer ) in hypertonic buffer { 1% np-40 , 10 mm hepes ( ph 7.5 ) , 0.5 m nacl , 10% glycerol supplemented with protease and phosphatase inhibitors [ ( 1 mm phenylmethylsulfonylfluoride , 1 mm na3vo4 , 50 mm naf , 10 mm -glycerol phosphate , 1 mm edta ] , and protease inhibitor cocktail tablet ( roche applied science , indianapolis , in , usa]}. cell lysates , adding 4 or 2 laemmli buffer ( bio - rad ) by supplementing with 2.5% -mercaptoethanol to give 1 sds sample buffer , were boiled for 5 min and subjected to immunoblot analysis , as described previously . protein samples were resolved on 415% sds - page ( bio - rad ) , and immunoblot analysis was performed using antibodies against the indicated signaling molecules . the antibodies used were rabbit monoclonal cleaved caspase-3 ( asp175 ) ( cell signaling technology , beverly , ma , usa ) and goat polyclonal -actin ( santa cruz biotechnology , santa cruz , ca , usa ) .

two new ( 1 and 2 ) and four known arylnaphthalene lignan lactones ( 36 ) were isolated from different plant parts of phyllanthus poilanei collected in vietnam , with two further known analogues ( 7 and 8) being prepared from phyllanthusmin c ( 4 ) . the structures of the new compounds were determined by interpretation of their spectroscopic data and by chemical methods , and the structure of phyllanthusmin d ( 1 ) was confirmed by single - crystal x - ray diffraction analysis . several of these arylnaphthalene lignan lactones were cytotoxic toward ht-29 human colon cancer cells , with compounds 1 and 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl)]diphyllin ( 7 ) found to be the most potent , exhibiting ic50 values of 170 and 110 nm , respectively . compound 1 showed activity when tested in an in vivo hollow fiber assay using ht-29 cells implanted in immunodeficient ncr nu / nu mice . mechanistic studies showed that this compound mediated its cytotoxic effects by inducing tumor cell apoptosis through activation of caspase-3 , but it did not inhibit dna topoisomerase ii activity .

Results and Discussion Experimental Section

when the cytotoxic chcl3 partitions were subjected to chromatographic separation guided by inhibitory activity against the ht-29 cell line , two new [ phyllanthusmins d ( 1 ) and e ( 2 ) ] and four known [ phyllanthusmins a ( 6 ) , b ( 3 ) , and c ( 4 ) and cleistanthin b ( 5 ) ] arylnaphthalene lignan lactones were purified . thus , compound 1 was proposed as an o - acetyl analogue of the known compounds phyllanthusmins b ( 3 ) and c ( 4 ) , with both being characterized from phyllanthus oligospermus in a previous study . comparison of the nmr data of compound 1 with those of phyllanthusmins b ( 3 ) and c ( 4 ) ( table 2 and tables s1 and s2 , supporting information ) showed that these compounds displayed closely similar nmr resonances for the diphyllin aglycone unit but different resonances for their saccharide portions . finally , both 1 and 4 were acetylated to form the same compound , 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl)]diphyllin ( 7 ) , which exhibited the same specific rotation value of [ ]d 12 ( c 0.1 , chcl3 ) , and were closely comparable to the same compound synthesized from diphyllin and l - arabinose { [ ]d 13 ( c 0.3 , chcl3)}. thus , the structure of this new compound ( phyllanthusmin d , 1 ) was assigned as 7-o-[(3,4-di - o - acetyl)--l - arabinopyranosyl]-4,5-dimethoxy-3,4-methylenedioxy-2,7-cycloligna-7,7-dieno-9,9-lactone , or 7-o-[(3,4-di - o - acetyl)--l - arabinopyranosyl]diphyllin . this was confirmed by analysis of its single - crystal x - ray diffraction data . the same hindered rotation would likely be observed in 35 and other arylnaphthalene lignan lactones , as indicated by the duplicated resonances in their nmr spectra ( tables s1 , s2 , and s4 , supporting information ) and optical activity shown in their ecd spectra . the molecular formula of c28h26o12 deduced from c nmr data and a sodiated molecular ion peak at m / z 577.1319 ( calcd 577.1316 ) observed in the hresims and the similar nmr data to those of 1 demonstrated that this compound is a diphyllin monoacetylarabinoside , a regioisomer of phyllanthusmin b ( 3 ) . although sample limitations precluded hydrolysis of 2 to yield the sugar unit or acetylation of 2 into 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl)]diphyllin ( 7 ) to determine its absolute configuration , it may be assumed that this compound has the same absolute configuration as that of 1 , 3 , and 4 , from the consistent noesy correlations ( figure s19 , supporting information ) and specific rotation values { [ ]d 3 ( c 0.1 , chcl3 ) for 1 , [ ]d 4 ( c 0.1 , chcl3 ) for 2 , [ ]d 6 ( c 0.1 , chcl3 ) for 3 , and [ ]d 8 ( c 0.1 , chcl3 ) for 4 ( supporting information)}. therefore , the structure of 2 ( phyllanthusmin e ) was defined as 7-o-[(3-o - acetyl)--l - arabinopyranosyl]-4,5-dimethoxy-3,4-methylenedioxy-2,7-cycloligna-7,7-dieno-9,9-lactone , or 7-o-[(3-o - acetyl)--l - arabinopyranosyl]diphyllin . altogether , six arylnaphthalene lignan lactones were characterized from the different parts of p. poilanei collected in different locations in vietnam . as summarized in table 1 , the most cytotoxic isolate , phyllanthusmin d ( 1 ) , was isolated from all parts of p. poilanei , and this compound can be regarded as a major cytotoxic principle of p. poilanei . the known arylnaphthalene lignan lactones isolated from p. poilanei were identified by analysis of their spectroscopic data and comparison of these data with literature values , and full assignments of their h and c nmr spectroscopic data are listed in tables s1s4 ( supporting information ) . to partially discern the effects of both the arabinose unit and the acetyl group in the mediation of cytotoxicity of the diphyllin lignans , 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl]diphyllin ( 7 ) was produced by acetylation of 1 and 4 using a standard method and was identified by its molecular formula of c32h30o14 , as determined by hresims and comparison of its spectroscopic data ( supporting information ) with those reported for a synthetic sample . the aglycone diphyllin ( 8) was generated by hydrolysis of phyllanthusmin c ( 4 ) , and its structure was determined by comparison of its spectroscopic data with reference values . all arylnaphthalene lignans isolated from p. poilanei in the present study and their semisynthetic analogues were evaluated for their cytotoxicity against ht-29 human colon cancer cells , using paclitaxel and etoposide as the positive controls ( table 3 ) . compounds 14 , 7 , and 8 were found to be cytotoxic , of which 1 and 7 were the most potently active , with ic50 values of 170 and 110 nm , respectively , but compounds 5 , 6 , and etoposide were inactive . phyllanthusmin c ( 4 ) showed a higher cytotoxicity than diphyllin ( 8) and cleistanthin b ( 5 ) , implying that the -l - arabinose unit at c-7 played an important role in generating this effect and was more active than a -d - glucose unit in mediating compound cytotoxicity toward ht-29 cells . the new compound phyllanthusmin d ( 1 ) showed a much greater ic50 value than etoposide , indicating that this compound is more potently cytotoxic against ht-29 cells . cleistanthoside a tetraacetate showed potent cytotoxicity with ig50 values in the nanomolar range , but its precursor , cleistanthoside a , was inactive , suggesting the importance of acetylation of the glycose unit of cleistanthoside a. the structure cytotoxicity relationships of arylnaphthalene lignan lactones observed from the present study are consistent with observations made from these previous studies . both compounds were found to be noncytotoxic toward this cell line ( table 3 ) , indicating some selectivity for ht-29 human colon cancer cells . the new cytotoxic compound , phyllanthusmin d ( 1 , ic50 , 170 nm ) , isolated from p. poilanei , was tested further in an in vivo hollow fiber assay for its possible antitumor efficacy . immunodeficient ncr nu / nu mice implanted with ht-29 human colon cancer cells placed in hollow fibers were treated once daily by 1 at doses of 5.0 , 10.0 , 15.0 , or 20.0 mg / kg , or the vehicle control , or paclitaxel ( 5 mg / kg ) , by ip injection for 4 days . effect of phyllanthusmin d ( 1 ) on the growth of human colon cancer ht-29 cells implanted in ncr nu / nu mice tested in an in vivo hollow fiber assay . in the present study , several diphyllin arabinosides , including phyllanthusmins c ( 4 ) and d ( 1 ) and 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl)]diphyllin ( 7 ) , together with etoposide , were tested for their ability to inhibit dna topo ii ( figure 4 ) , using a method reported previously . evaluation of arylnaphthalene lignan lactones { phyllanthusmins c ( 4 ) and d ( 1 ) and 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl)]diphyllin ( 7 ) ] from p. poilanei for activity as topoisomerase ii ( topo ii ) inhibitors . a previous study showed that an 8-day treatment of etoposide induced ht-29 human colon cancer cell apoptosis , but shorter term treatment with this compound did not show this activity . treatment of ht-29 cells with 1 or 5 m phyllanthusmin d ( 1 ) for 72 h resulted in 28.2% or 30.3% of ht-29 cells undergoing early apoptosis , respectively , while the analogous values for the vehicle control or 1 or 5 m etoposide treatments were 3.9% , 12.9% , and 12.5% , respectively ( figure 5 ) . ht-29 cells were treated with 1 or 5 m phyllanthusmin d ( 1 ) , 1 or 5 m etoposide , or the vehicle control for 72 h , followed by an annexin v staining method ( experimental section ) . after 24 h incubation , phyllanthusmin d ( 1 ) induced activation of caspase-3 at as low a dose as 1 m , as well as at 5 and 10 m . ht-29 cells were incubated with phyllanthusmin d ( 1 ) and etoposide with different concentrations for 24 h , and caspase-3-like activity was determined by western blotting using rabbit monoclonal cleaved caspase-3 ( asp175 ) antibody . , initial collections of separate samples of the combined leaves , twigs , flowers , and fruits ( acquisition number a06024 ) and the stems ( acquisition number a06025 ) of phyllanthus poilanei were collected from a shrub at the road transect from suoi cat village to hon ba peak ( 1207.873 n ; 10601.532 e ) , dinh khanh district , khanh hoa province , vietnam , in november 2004 , by d.d.s . second collections of separate samples of the combined leaves , twigs , flowers , and fruits ( acquisition number a06473 ) and the stems ( acquisition number a06474 ) of p. poilanei were obtained from a liana - like shrub in the forest occurring at the south end of kego lake , across from mui tru ranger station ( 1806.530 n ; 10600.891 e ) , kego nature reserve , cam xuyen district , hatinh province , vietnam , in december 2008 , by d.d.s . of these , d2f4d2f6 ( ic50 < 2 g / ml ) were combined and further chromatographed over a silica gel column ( 2.5 20 cm ) , eluted with a gradient of n - hexane fractions d2f11 and d2f12 ( ic50 < 5 g / ml ) were combined and further chromatographed over a silica gel column ( 2.5 20 cm ) , eluted with a gradient of n - hexane subfraction d2f4f2 was chromatographed over silica gel , with a gradient of n - hexane acetone , and purified by separation over a sephadex lh-20 column , eluted with ch2cl2meoh ( 1:1 ) , affording phyllanthusmin d ( 1 , 20 mg ) . the milled air - dried stems of p. poilanei ( sample a06025 , 580 g ) were extracted with meoh ( 3 l 4 and then 2 the solvent was evaporated in vacuo , and the dried meoh extract ( 47.4 g , 8.2% ) was resuspended in 10% h2o in meoh ( 500 ml ) and partitioned with n - hexane ( 500 , 300 , 200 ml ) , to yield an n - hexane - soluble residue ( d1 , 1.4 g , 0.24% ) . of these , d2f4d2f6 ( ic50 < 2 g / ml ) were combined , further chromatographed over a silica gel column , eluted with a gradient of n - hexane acetone , and purified by separation over a sephadex lh-20 column , eluted with ch2cl2meoh ( 1:1 ) , affording phyllanthusmin d ( 1 , 7.0 mg ) . the milled air - dried combined leaves , twigs , flowers , and fruits of p. poilanei ( sample a06473 , 851 g ) were extracted with meoh ( 3 the solvent was evaporated in vacuo , and the dried meoh extract ( 96 g , 11.3% ) was resuspended in 10% h2o in meoh ( 500 ml ) and partitioned with n - hexane ( 500 , 300 , 200 ml ) , to yield an n - hexane - soluble residue ( d1 , 10.2 g , 1.2% ) . of these , fractions d2f8 and d2f9 ( ic50 < 5.0 g / ml ) were combined , further chromatographed over a silica gel column ( 2.5 20 cm ) , eluted with a gradient of n - hexane acetone , and purified by separation over a sephadex lh-20 column , eluted with ch2cl2meoh ( 1:1 ) , affording phyllanthusmins c ( 4 , 2.0 mg ) and d ( 1 , 3.0 mg ) . of these , fraction d2f10 ( ic50 < 5.0 g / ml ) was chromatographed over a silica gel column , eluted with a gradient of n - hexane acetone , and purified by separation over a sephadex lh-20 column , eluted with ch2cl2meoh ( 1:1 ) , to afford phyllanthusmin d ( 1 , 2.0 mg ) . in an attempt to accumulate a greater quantity of phyllanthusmin d ( 1 ) for in vivo biological evaluation , a larger re - collection of the combined leaves , twigs , and stems of p. poilanei was made . of these , d2f4d2f6 ( ic50 < 5 g / ml ) were combined and further chromatographed over a silica gel column ( 2.5 20 cm ) , eluted with a gradient of n - hexane acetone , to yield phyllanthusmins b ( 3 , 1.0 mg ) , d ( 1 , 10.5 mg ) , and e ( 2 , 1.0 mg ) . to a dried 25 ml flask equipped with a magnetic stirrer , containing 3.0 mg of phyllanthusmin d ( 1 ) , after the mixture was stirred at 40 c for 1 h , it was cooled to room temperature . acetone ( 5:1 1:1 ) , to afford 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl)]diphyllin { 7 ( 1.0 mg , [ ]d 12 ( c 0.1 , chcl3)}. using the same protocol , 5.0 mg of phyllanthusmin c ( 4 ) was reacted with 10 l of acetic anhydride and 2 ml of pyridine at 60 c for 1 h and yielded 1.0 mg of 7 { [ ]d 12 ( c 0.1 , chcl3)}. following a previous procedure and the method for screening cytotoxicity toward ht-29 cells mentioned above , the cytotoxicity of the samples was screened against ccd-112con normal human colon cells . eight- to nine - week - old immunodeficient ncr nu / nu mice were purchased from the jackson laboratory ( bar harbor , me , usa ) and housed in microisolation cages at room temperature and a relative humidity of 5060% under 12:12 h light all animal work was approved by university of illinois at chicago animal care and use committee ( protocol number 13 - 057 ) , and the mice were treated in accordance with the institutional guidelines for animal care . phyllanthusmin d ( 1 ) was dissolved initially in dmso and subsequently diluted with cremophor . results are shown for etoposide , phyllanthusmins c ( 4 ) and d ( 1 ) , and 7-o-[(2,3,4-tri - o - acetyl)--l - arabinopyranosyl)]diphyllin ( 7 ) in replicate experiments performed on separate days ( figure 4 ) .